Chapter 1 - Introduction

1.1 Epigenetic modifications and gene silencing

The fact that a multicellular organism is able to undergo cellular differentiation, even though every cell contains the same genetic material, indicates the existence of an extra layer of phenotype-determining factors. Cellular differentiation is associated with the development of different patterns of gene expression in individual cells or groups of cells, and the way in which these patterns are established involves epigenetics. Epigenetics refers to the process by which transcriptional states are changed in a relatively permanent way in the absence of DNA mutation. Epigenetic gene silencing or activation results from modifications to the gene in question either by changing the methylation state of the DNA or by modifying the proteins that package the DNA. There is increasing evidence that in some cases RNA molecules are also involved. Epigenetic modifications are generally erased and reset between generations in order to provide the necessary pluripotent starting state from which to begin development in the next generation.

1.1.1 DNA methylation

DNA methylation is the best studied epigenetic modification. In mammals DNA methylation is always associated with cytosine residues, generally occurring at CpG dinucleotides in a symmetrical fashion, where the cytosines on both strands are methylated. Methylation of cytosine involves the transfer of a methyl group from an

S-adenosyl-L-methionine to the 5-position of cytosine, resulting in the modified base

5-methylcytosine (Jeltsch, 2002) (Figure 1.1). Estimates of methylation levels at

CpGs in mammals range from 50-90% (Gruenbaum et al., 1981; Jeltsch, 2002). DNA

hypermethylation at promoters usually correlates with transcriptional repression and gene silencing.

cytosine 5-methylcytosine NH NH2 2

CH3 N DNA methyltransferase N

O N O N H H

HO NH 2 HO NH2

O + O S S O O CH N N 3 N N

NH2 NH HO HO 2 OH N OH N N N

S-adenosyl-L-methionine S-adenosyl-L-homocysteine

Figure 1.1 Conversion of cytosine into 5-methylcytosine. A DNA methyltransferase enzyme transfers a methyl group (blue circle) from S-adenosyl-L-methionine to the 5-position of cytosine, producing 5-methylcytosine.

CpG dinucleotides tend to be clustered in specific regions termed CpG islands. These are generally associated with the promoter regions of genes and tend to be hypomethylated and transcriptionally active. The remainder of the genome is CpG sparse and this is thought to be due to the frequent transition of 5-methylcytosine to thymine via spontaneous deamination (Duncan and Miller, 1980). The CpGs in the islands are thought to be protected from deamination because they are not methylated.

DNA methylation patterns are maintained mitotically by DNA methyltransferase 1

(Dnmt1). This enzyme recognises hemi-methylated DNA substrates following DNA

replication, and copies the methylation state onto the newly synthesized strand

(Leonhardt et al., 1992). De novo methylation is performed by the two methyltransferases Dnmt3a and Dnmt3b, which are required for the establishment of

DNA methylation in early development (Okano et al., 1999). All methyltransferases are essential for normal mammalian development. Homozygous knockouts of Dnmt1 show embryonic lethality around mid-gestation (Li et al., 1992). Dnmt3a and Dnmt3b show an overlap in function during early embryogenesis; homozygous knockouts of

Dnmt3a die within a few weeks of birth, homozygous knockouts of Dnmt3b develop normally until approximately 9.5 days post coitum (dpc) but later show developmental defects and embryonic lethality, compound homozygous knockouts of

Dnmt3a and Dnmt3b die before 11.5 dpc with more severe embryonic defects than either single Dnmt3a or Dnmt3b homozygous knockouts (Okano et al., 1999).

A large proportion of DNA methylation in mammals is associated with retrotransposons and other transposable elements, and so it has been suggested that

DNA methylation is involved in genome defence (Yoder et al., 1997). Transposable elements are very common in the mammalian genome, with estimates predicting up to

45% of the genome being transposable element derived (Jordan et al., 2003), although much of this is non-functional due to mutation. Some elements however remain transcriptionally active and some can even undergo retrotransposition. Integration of transposable elements can result in the production of transposon-derived transcripts and subsequent proteins that could be deleterious to the host. Transposable elements can take over control of endogenous genes by donating cis-regulatory sites, leading to aberrant expression or the production of mutant transcripts (Druker et al., 2004;

Hatada et al., 2003; Ukai et al., 2003). As a result, in a wide variety of organisms

transposable elements become heavily methylated once they integrate into the host genome, and this is associated with transcriptional silencing (Cambareri et al., 1996;

Lavie et al., 2005). In confirmation of this fact, mouse embryos deficient in Dnmt1 show increased retroviral transcriptional activity (Gaudet et al., 2004; Walsh et al.,

1998).

While DNA methylation is an important epigenetic process in development and gene silencing, there continues to be much debate about whether it is causative or a consequence of the silencing. There is increased awareness that changes to the chromatin packaging the DNA also plays a significant role.

1.1.2 Histone modifications

Post-translational modifications to histone proteins are associated with changes to chromatin structure, resulting in transcriptionally active or inactive chromatin states.

These modifications involve covalent additions of small molecules to the amino- terminal tail of the histone protein.

Histone acetylation

Acetylation of histones occurs at lysine residues, predominantly on histones H3 and

H4. Acetylation is associated with a transcriptionally active chromatin configuration.

When the lysine residues are acetylated the overall positive charge of the histone protein is neutralised, resulting in a weakening of charge interactions with the DNA molecule (Allfrey, 1966). Histone acetylation also influences histone-histone and histone-regulatory protein interactions (Luger and Richmond, 1998). All these

interactions lead to an open chromatin configuration that is more accessible to transcription factors.

Histone acetylation is performed by histone acetyltransferases (HATS) of which there are several families. The main HATS in the mouse are grouped in the GNAT (GCN5- related N-acetyltransferases) family and the MYST (named after its founding members: MOZ, Ybf2/Sas3, Sas2, Tip60) family, although there are other HATS such as Crebbp (CREB-binding protein) and Ncoa1 (nuclear receptor coactivator 1)

(Marmorstein and Roth, 2001; Thomas et al., 2007). Histone deacetylation is performed by histone deacetylases (HDACS), of which 11 (Hdac1-11) have been identified in the mouse. Having both acetyltransferases and deacetylases present means that the acetylation of histones is in constant flux and can be tightly regulated.

Histone methylation

Methylation of histones occurs at lysine residues of histones H3 and H4. There is no simple correlation between histone methylation and transcriptional activity, since the latter is dependent upon which lysine residue is methylated and whether that residue is mono-, di- or tri-methylated.

A good example of the specificity associated with individual lysine residues and their methylation state is the behaviour of H3Lys4. Di-methylation of H3Lys4 is associated with both active and inactive euchromatic regions, while tri-methylation is present exclusively at active genes. In the case of H3Lys9 however, both di- and tri- methylation are associated with transcriptionally inactive heterochromatin, with the

methyl moiety on H3Lys9 able to recruit heterochromatin protein 1 (HP1) (Bannister et al., 2001; Lachner et al., 2001).

Both histone methyltransferases (HMTases) and demethylases have been identified in mammals. Histone methyltransferases are SET (Su(var)3-9, Enhancer-of-zeste,

Trithorax) domain-containing proteins that show different specificities for different lysine residues. For example, Suv39h1 and h2 appear to only methylate H3Lys9, whereas G9a can methylate H3Lys9 and H3Lys27 (Tachibana et al., 2001). Histone demethylases have only recently been discovered. Lysine-specific demethylase 1

(LSD1) is a histone demethylase, and inhibition of this protein leads to increased levels of H3Lys4 methylation and increased gene activity (Huang et al., 2007). Other demethylases include the JMJD2 subfamily (part of the Jumonji C domain-containing family), which consists of four unique demethylases that have different activities.

JMJD2A and C demethylate di- and tri-methylated H3Lys9 and H3Lys36, whereas

JMJD2D demethylates mono-, di- and tri-methylated H3Lys9 (Shin and Janknecht,

2007).

Other histone modifications

Apart from histone acetylation and methylation, there are several other covalent modifications that can occur to histone tails. Phosphorylation of H3Ser10 has been linked to chromatin condensation and possibly gene expression (Johansen and

Johansen, 2006). Ubiquitination of histone H2A is associated with the transcriptionally silent XY body (a specialised chromatin territory containing the transcriptionally repressed sex chromosomes) in males and the inactive X in females

(Baarends et al., 2005). Sumoylation of histone H4 is thought to repress transcription

through the recruitment of HDACS and HP1 (Shiio and Eisenman, 2003). Although there are a wide variety of histone modifications, it is the combined status of the histone proteins that determines the accessibility of the chromatin structure to transcriptional machinery and hence determines gene activity (Li, 2002).

1.1.3 RNA-mediated gene silencing

Not all epigenetic control is related to direct modification of DNA and its associated histones. Small RNA molecules are also known to play a role (Michalak, 2006).

RNA interference (RNAi) is a mechanism by which gene expression is controlled by small RNA molecules, at either the transcriptional or post-transcriptional level (Figure

1.2). At the post-transcriptional level, a double stranded RNA (dsRNA) molecule is cut into short interfering RNAs (siRNAs) by the enzyme Dicer, which then bind homologous mRNA molecules, targeting the mRNA for degradation. siRNAs are also known to be involved in repressing transcription at chromosomal regions. By combining with companion proteins, specific DNA sequences are targeted based on homology to the siRNAs. Recruitment of other chromatin-modifying proteins ensues, resulting in gene silencing (Hannon, 2002). The transcriptional repression by siRNAs is best characterised in plants, and its presence in mammalian systems remains controversial.

MicroRNAs (miRNAs) participate in the same RNAi pathway that is used by siRNAs, but they originate from longer intergenic transcripts. Like siRNAs, miRNAs can repress mRNA at a post-transcriptional level. Most animal miRNAs only partially pair with their target mRNA molecule, and in these cases translational repression

precludes mRNA degradation, following which the mRNA is targeted to processing bodies (P bodies) in which the mRNA is stored or degraded (Pillai et al., 2005; Pillai et al., 2007).

mRNA degradation AAA

siRNAs siRNAs

dsRNA siRNAs or cleavage miRNAs by Transcriptional transcription Dicer repression

miRNAs

DNA AAA

Translational P body repression

AAA

mRNA storage and degradation

Figure 1.2 RNAi mechanism of gene silencing. Both siRNA and miRNA molecules participate in the RNAi pathway. dsRNA is transcribed from DNA and is then cleaved by the enzyme Dicer producing siRNA/miRNA molecules. siRNA molecules can either cause degradation of homologous mRNA molecules or transcriptional repression by interacting with homologous DNA regions. Animal miRNA molecules generally only show partial pairing with homologous mRNA molecules, which leads to translational repression and subsequent storage and degradation of the mRNA in P bodies.

There are also other types of small RNA molecules that have been discovered. For example, small nucleolar RNAs (snoRNAs) are mostly intron-derived RNAs and have been implicated in epigenetic imprinting and the processing of mRNAs (Bachellerie

et al., 2002; Cavaille et al., 2002). There are also many other non-coding RNAs that have yet to be classified.

1.1.4 Epigenetic reprogramming

In mammals there are two developmental stages at which epigenetic marks are known to be erased and reset (Dean et al., 2003). This process is termed epigenetic reprogramming, and is generally regarded as an almost genome-wide event.

The first of the epigenetic reprogramming events occurs during early embryogenesis

(Figure 1.3). Immediately following fertilization there is a rapid genome-wide demethylation of the paternally derived genome. This has been observed in the mouse at a global level by immunofluorescence using antibodies against 5-methylcytosine

(Mayer et al., 2000; Santos et al., 2002), as well as by bisulfite sequencing of some single copy regions in the genome (Blewitt et al., 2006; Oswald et al., 2000). Because this demethylation occurs before any cell divisions, we can infer that active demethylation must be occurring. How this active demethylation is achieved is unknown, but it probably involves either direct or indirect demethylation; i.e. the removal of the methyl group or the excision and repair of the entire 5-methylcytosine

(Morgan et al., 2005). Several enzymes, such as methyl-binding domain proteins 2 and 4 (Mbd2 and 4) and the cytidine deaminases Activation-induced cytidine deaminase (AID) and apolipoprotein B editing complex 1 (Apobec1), have been implicated in active demethylation (Morgan et al., 2005; Santos et al., 2002).

After this active demethylation event, passive demethylation is likely to occur as the embryo goes through its early rounds of cell division. Passive demethylation relies on

DNA replication “diluting” the remaining methylation state amongst a larger set of cells. The newly replicated DNA cannot be methylated at this stage because Dnmt1 is excluded from the nuclei of these early embryonic cells (Carlson et al., 1992; Howell et al., 2001). The maternal set of chromosomes appears to undergo only passive demethylation, which does not commence until the first round of cell division after the formation of the zygote. Re-establishment of methylation begins after the morula stage of the embryo at around 4.5 dpc, presumably by the de novo methyltransferases

Dnmt3a and 3b (Okano et al., 1999; Santos et al., 2002).

All embryonic cells Germ cells only

High Methylation

Low

Fertilisation ~4.5 dpc ~10.5 dpc ~18.5 dpc

Figure 1.3 Epigenetic reprogramming in the mouse embryo with respect to DNA methylation. Adapted from Dean et al. 2003. Reprogramming during early embryonic development (yellow background) and gametogenesis (green background). Methylation levels over developmental time is shown for imprinted genes (black line), paternal genes (blue line) and maternal genes (red line).

Despite the fact that most of the epigenome is erased and re-established during early development, it has become apparent that there are some loci at which epigenetic state is at least partially resistant to erasure, allowing for the transmission of the epigenetic state across multiple generations. Imprinted genes (see section 1.2.2), for example, appear to be exempt from the reprogramming events seen in the somatic tissue of the

early embryo (Olek and Walter, 1997; Reik and Walter, 2001; Tremblay et al., 1997).

These genes are resistant to both active and passive demethylation. Intracisternal A particles (IAPs), a type of retrotransposon, also appear to be resistant to DNA methylation reprogramming during active demethylation in early embryogenesis (Kim et al., 2004; Lane et al., 2003). However, other repetitive sequences such as Line 1 elements do undergo reprogramming. It is possible that IAPs and imprinted genes share sequence elements or epigenetic modifications that protect them from demethylation during this period, but the exact nature of these protective marks remain unknown (Lane et al., 2003). IAPs do undergo passive demethylation up to the blastocyst stage, but only to a limited extent.

The second reprogramming event occurs between 10.5 and 13.5 dpc of embryogenesis, but only in the primordial germ cells (PGCs) (Figure 1.3). All other somatic cell lines retain the methylation established earlier in development. During the reprogramming in primordial germ cells, demethylation occurs not only at single- copy genes, but also at imprinted genes and IAPs. This demethylation is likely to be active because too few DNA replication events occur in this period to produce the observed levels of demethylation, and also because Dnmt1 is present throughout

(Hajkova et al., 2002). De novo methylation of the gametes to establish their final methylation state occurs between 15.5 and 18.5 dpc. This is how imprinted genes are able to acquire their new imprint. The reprogramming that occurs in the PGCs is poorly understood, mainly due to the difficulty in obtaining purified PGCs.

It is important to realise that at present these two reprogramming events have been studied almost exclusively at the level of DNA methylation. Both events involve a

global demethylation, followed by de novo methylation, generating a new set of methylation patterns. However, we must remember that DNA methylation is not the only epigenetic mark. Currently, studies are being carried out to see whether histone modification changes correlate closely with the DNA methylation changes at these developmental stages. These studies have been difficult due to the small number of cells available for analysis at early developmental time points. While DNA methylation can be studied in small populations of cells with relative ease, analysis of chromatin requires much larger numbers of cells.

1.2 The role of epigenetic gene silencing in mammals

1.2.1 X-inactivation

X-inactivation is a classic example of epigenetic regulation of gene expression in mammals. Female mammals have two X chromosomes, and one X chromosome is inactivated during early embryonic development, in order to achieve dosage compensation with males. Embryonic tissues undergo random X-inactivation, resulting in cells expressing either the maternal or paternal X chromosome. Once the inactivation occurs, the “chosen” inactive X remains the inactive X in all daughter cells. On the other hand the extra-embryonic tissues show parental imprinting with respect to X-inactivation, and it is the paternal X chromosome (Xp) that is always silenced (Takagi and Sasaki, 1975; West et al., 1977).

Transcriptional inactivation of the Xp appears to commence very early in development (by the four- to eight-cell stage) due to the rapid accumulation of Xist

(inactive X specific transcripts) RNA, and by the 32-cell stage X-inactivation has

silenced the Xp in almost all cells (Okamoto et al., 2004). This inactivation is later reversed in the cells of the inner cell mass, so that random X-inactivation can occur.

Recent studies suggest that the Xp, which is known to be transcriptionally inactivated during spermatogenesis (Solari, 1974; Turner, 2007), may retain this inactivation through to fertilisation and early development (Namekawa et al., 2006). Therefore the inactive X in the next generation may in fact be directly descended from the inactive spermatid X.

X-inactivation involves the non-coding RNA Xist, which is transcribed only from the

X-chromosome that is to be inactivated. The Xist RNA coats the X-chromosome to be inactivated, initiating inactivation at a region known as the X-inactivation centre. The

Xist RNA recruits HMTases, which methylate H3Lys27, initiating inactivation of the entire chromosome (Plath et al., 2003; Wang et al., 2001a). Other modifications, such as methylation at H3Lys9, occur slightly later (Okamoto et al., 2004).

Gene promoter regions on the inactive X generally become hypermethylated, which is consistent with the silencing of gene expression. However, this hypermethylation does not occur along the entire length of the inactive X chromosome. In fact the inactive X appears to be globally hypomethylated compared with the active X (Weber et al.,

2005). Chromosomal-wide analysis of heterochromatin-related histone code on the inactive X also revealed that it might be quite heterogeneous and gene-specific

(Valley et al., 2006). These results imply that the mechanism of X-inactivation is more complex than first thought, and that different silencing mechanisms may be involved at individual X chromosome loci.

1.2.2 Imprinting

Imprinting in mammals refers to the silencing of one allele of a gene in a parent-of- origin specific manner, resulting in monoallelic expression. Because of genomic imprinting, parthenogenic mammalian offspring generally display abnormal development, because both the maternal and paternal genes are required to produce a complete set of normally expressing genes. Oocytes containing two haploid sets of maternal genome can produce viable offspring if certain imprinted genes are artificially induced to mimic normal expression levels (Kono et al., 2004).

Many imprinted genes are associated with growth and development, which has led to the idea that imprinting arose because of parental conflict over the size of the offspring (Moore and Haig, 1991). This theory postulates that imprinted paternally expressed genes have a bias towards increasing the size and overall fitness of offspring at the cost of the mother‟s health. Imprinted maternally expressed genes have the opposite effect, with a bias towards smaller offspring, which is in the best interest of the mother. However, the finding that imprinting is present in non-placental organisms and also in genes unrelated to growth suggests that imprinting arose for other reasons (Joanis and Lloyd, 2002; Lloyd, 2000).

The regulation of imprinted genes is largely dependent on methylation marks at imprinting control regions (ICRs), which differ between the maternal and paternal alleles (Delaval and Feil, 2004). These marks are established during the embryological development of germ cells following the second reprogramming event, as discussed previously. Once established they are retained through the first reprogramming event in the early embryo in the next generation.

1.3 Metastable epialleles

1.3.1 Metastable epialleles in mice

Some loci are known to have labile epigenetic states, and can display both variegating and variably expressed phenotypes. Variegation is the differential expression of a particular gene amongst cells of the same cell type within an individual. Variable expressivity refers to differential expression amongst a group of isogenic (genetically identical) individuals. These loci have been termed metastable epialleles (Rakyan et al., 2002).

Two of the best studied metastable epialleles in the mouse are the agouti viable yellow

(Avy) allele (Perry et al., 1994), and the axin fused (AxinFu) allele (Belyaev et al.,

1981; Reed, 1937). In both cases, isogenic individuals display variable expressivity.

In the case of Avy, isogenic mice display coat colours that range from normal agouti

(dark-brown) through a spectrum of intermediate mottled phenotypes to yellow

(Figure 1.4a). In the case of AxinFu, isogenic mice display a kinky tail phenotype that varies from normal (no tail-kink) through to severe tail kinks (Figure 1.4b). Avy also displays variegation of the phenotype within each individual mouse, and hence many mice are mottled.

At both Avy and AxinFu, the variability correlates with differential methylation at an

IAP retrotransposon at the relevant locus, agouti or axin, which affects the transcriptional activity of the entire locus (Morgan et al., 1999; Rakyan et al., 2003).

Hypermethylation at a cryptic promoter within the 5‟ long terminal repeat (LTR) of the IAP is associated with transcriptional silencing and a normal (pseudo wildtype)

phenotype. Hypomethylation results in active transcription, which is associated with the aberrant phenotypes.

(a) (b)

Figure 1.4 Avy and AxinFu phenotypes. (a) Mice carrying the Avy allele. Each mouse is isogenic and yet there is a range of coat colour phenotypes observed; yellow through mottled through agouti (termed pseudoagouti). (b) Mice carrying the AxinFu allele. Each mouse is isogenic, but one has a kinky tail while the other does not. In both cases the variable expressivity correlates with differential methylation at the cryptic promoter of an IAP retrotransposon inserted at the relevant locus; agouti or axin. Mice are littermates and age matched.

1.3.2 Transgenerational epigenetic inheritance

The direct transmission of epigenetic states to offspring is referred to as transgenerational, or meiotic, epigenetic inheritance. This should not be confused with mitotic epigenetic inheritance, which concerns the maintenance of the epigenetic state within an individual through multiple rounds of mitotic cell division. Meiotic epigenetic inheritance involves the passage of the epigenetic marks through the germline to subsequent generations.

Although originally discovered in plants, transgenerational epigenetic inheritance appears to occur in a wide range of organisms. There is now considerable evidence that transgenerational epigenetic inheritance does occur at a small number of alleles in mice, despite the fact that there are genome-wide epigenetic reprogramming events

during both gametogenesis and embryogenesis. The strongest evidence for transgenerational epigenetic inheritance at genes other than imprinted loci comes from studies of metastable epialleles.

Transgenerational epigenetic inheritance has been shown to occur at both the Avy and

AxinFu alleles. The Avy allele displays transgenerational epigenetic inheritance following maternal transmission of the allele, but not following paternal transmission

(Morgan et al., 1999). AxinFu shows epigenetic inheritance when transmitted both maternally and paternally (Rakyan et al., 2003). It is puzzling that different processes appear to be operating at these two alleles, since they both result from IAP retrotransposon insertions. Recent data suggests that the differences are associated with the strain background of the zygote. Avy mice are maintained in the C57BL/6J background, while AxinFu mice are maintained in the 129P4/RrRk background.

Crosses between Avy/a C57 sires and AxinFu/+ 129 dams displayed transgenerational epigenetic inheritance of the Avy allele following paternal transmission (which was not observed in the pure C57 background). Also, the reciprocal cross with AxinFu/+ 129 sires and Avy/a C57 dams (paternal transmission of AxinFu) showed no inheritance of the AxinFu allele (inheritance was observed in the pure 129 background). This suggests that C57 fertilised eggs completely erase epigenetic marks on paternally expressed alleles, but that 129 fertilized eggs do not (Rakyan et al., 2003).

Although the variability associated with these loci correlates with differential methylation, analysis of the Avy allele at various time points in development suggests that methylation is not the epigenetic mark inherited across generations. We know that while the Avy allele is resistant to DNA methylation reprogramming in primordial

germ cells, both the maternal and paternal Avy alleles undergo DNA methylation reprogramming in early development, as is the case for the majority of the genome.

Following maternal transmission, in particular, there is a complete clearing of DNA methylation, so methylation alone cannot be the inherited mark in this case (Blewitt et al., 2006).

It is possible that histone modifications allow for the inheritance of epigenetic information across generations. In support of this idea Avy mice have been crossed with mice that are haplo-insufficient for Mel18 (also known as Pcgf2; polycomb group ring finger 2), a polycomb group protein (Blewitt et al., 2006). Polycomb group proteins are repressive chromatin modifiers thought to be involved in transgenerational epigenetic inheritance in Drosophila. When an Avy dam and a

Mel18-/+ sire (both in a C57 background) are mated, epigenetic inheritance is observed in both wild type and Mel18-/+ offspring, which is what we would expect.

Surprisingly, however, epigenetic inheritance was also observed with the reciprocal cross, i.e. following paternal inheritance of the Avy allele and maternal Mel18 haplo- insufficiency. Since the paternal genome has been completely demethylated by 6 h post-fertilization, this finding lends weight to the idea that DNA methylation is not the inherited mark and suggests the involvement of chromatin state.

It is not entirely surprising that DNA methylation is not the inherited mark in mammals, since we know that epigenetic marks can be inherited during meiosis in fission yeast, which do not methylate their DNA at all (Grewal and Klar, 1996). On the other hand, DNA methylation appears to play a role in the inheritance of epigenetic states in plants (Takeda and Paszkowski, 2006).

1.4 Screening for modifiers of epigenetic gene silencing in

Drosophila

Finding the genes involved in epigenetic gene silencing is of critical importance to understanding the regulatory mechanisms required for the correct establishment and reprogramming of the epigenome. One way of identifying new genes is via phenotype-driven random mutagenesis.

One of the major strengths of random mutagenesis is that it will identify novel genes in any process. Random mutagenesis is therefore referred to as forward genetics; one identifies the phenotype associated with the mutation first and then finds the underlying gene. Furthermore, random mutagenesis by chemical treatment such as N- ethyl-N-nitrosourea (ENU) can produce subtle mutant alleles (hypomorphs or hypermorphs), which may provide additional insight into biological pathways over and above that obtained from null alleles.

The majority of random mutagenesis screens for genes involved in epigenetic silencing have been performed in Drosophila. Many of these screens have relied on position effect variegation (PEV) as a read out of changes to epigenetic gene silencing. PEV refers to the variable expression of a euchromatic gene region when it is placed next to a heterochromatic genomic region. The most widely used model of

PEV is an inversion that places the white locus next to a heterochromatic region,

In(1)wm4, resulting in a variegating red/white eye colour (Figure 1.5). Silencing of the white gene is indicated by a white eye colour phenotype. Mutations in genes that decrease the number of white patches are referred to as suppressors of variegation,

Su(var). Conversely, enhancers of variegation, E(var), refer to those mutations that increase the white eye colour phenotype. Various independent mutagenesis screens on flies with this translocation have identified at least 380 dominant mutants, both suppressors and enhancers of PEV, which represent around 150 unique genes (Schotta et al., 2003). A partial list of modifiers of PEV identified in Drosophila is shown in

Table 1.1.

(a) w heterochromatin

(b) (c) (d)

In(1)wm4 Su(var) E(var)

Figure 1.5 Position effect rearrangement, In(1)wm4. Adapted from Schotta et al. 2003. (a) A chromosomal inversion places the white gene next to heterochromatin. (b) Variegation of the white gene in eye of the In(1)wm4 fly. Silencing of the white gene results in a white eye colour phenotype. (c) Mutating a suppressor of variegation, Su(var), results in loss of silencing. (d) Mutating an enhancer of variegation, E(var), results in increased silencing.

Table 1.1 Modifiers of position effect variegation identified in Drosophila.

Effect on Mouse Gene Classification References PEV homologue (Grienenberger et al., chm suppressor histone acetyltransferase Myst2 2002; Miotto et al., 2006) crm suppressor Polycomb group (Yamamoto et al., 1997) Cramp1l forkhead/winged-helix Dom suppressor (Strodicke et al., 2000) class cell cycle transcription E2f1, 2, 3 E2f enhancer (Seum et al., 1996) factor and 6 Epc1 and E(Pc) suppressor novel chromatin protein (Sinclair et al., 1998) Epc2 (Czermin et al., 2002; Jones and Gelbart, 1993; trithorax homology / E(z) suppressor Laible et al., 1997; Ezh2 histone methyltransferase LaJeunesse and Shearn, 1996) HP1 / chromo domain- Cbx1, 3 and suppressor (Eissenberg et al., 1990) Su(var)205 containing protein 5 (Jin et al., 1999; Jin et al., histone serine/threonine JIL-1 suppressor 2000; Lerach et al., 2006; ? kinase Wang et al., 2001b) methyl-CpG binding MBD-like suppressor (Marhold et al., 2004) Mbd2 domain M(2)21AB / methionine (Larsson et al., 1996; Mat1a and suppressor Su(z)5 adenosyltransferase Persson, 1976) Mat2a (Dorn et al., 1993; zbtb20 and mod(mdg4) enhancer BTB domain protein Gerasimova et al., 1995) ZBTB45 mod suppressor RRM-containing domain (Garzino et al., 1992)

mus209 / proliferating cell nuclear suppressor (Henderson et al., 1994) Pcna PCNA antigen Pp1-87B / Ppp1ca and suppressor protein phosphatase (Baksa et al., 1993) Su(var)3-6 Ppp1cc ATPase associated with the histone rept suppressor (Qi et al., 2006) Ruvbl2 acetyltransferase TIP60 complex Hdac1 and Rpd3 suppressor histone deacetelyase (De Rubertis et al., 1996) 2 (Newman et al., 2002; NAD-dependent histone Sir2 suppressor Rosenberg and Parkhurst, Sirt1 deacetylase 2002) zinc finger-containing Su(var)3-7 suppressor (Cleard et al., 1995) ? protein chromo domain / histone Su(var)3-9 suppressor (Tschiersch et al., 1994) ? methyltransferase transcription factor - (Gerasimova et al., 1995; Su(Hw) bidirectional insulator region binding ? Parkhurst et al., 1988) protein zinc finger-containing Trl enhancer (Farkas et al., 1994) ? protein

The Drosophila PEV screens are examples of sensitised mutagenesis screens. Such screens are reliant on the presence of an existing genetic variant that acts as a reporter locus. For example, the white locus mutant with variegating eye colour will show changes in the level of variegation in the presence of a mutation in a gene involved in epigenetic gene silencing. This enables the detection of mutations that could be

“invisible” in the wildtype strain.

Until recently, mutagenesis screens in the mouse were difficult due to the genetic complexity associated with a mammalian organism. Experiments were limited to genotype-driven screens, where the gene was already known and could be selectively targeted for mutation (usually via knockout technologies). However, the complete sequencing of the mouse genome has provided an opportunity to conduct extensive random phenotype-driven mutagenesis screens in the mouse.

1.5 An ENU screen for modifiers of epigenetic gene silencing in the mouse

Our laboratory has initiated a mutagenesis screen for modifiers of epigenetic gene silencing in the mouse, similar to that carried out in Drosophila. We have carried out the screen on mice expressing a GFP transgene in erythrocytes, i.e. it is a sensitised screen (Blewitt et al., 2005). This mouse line, maintained in the FVB/NJ strain, and called Line3, shows a variegating phenotype with respect to expression of the GFP transgene in peripheral blood, with approximately 55% of erythrocytes expressing the transgene (Preis et al., 2003). Variegated expression of transgenes in mice has been studied for many years, and the underlying gene silencing mechanisms are very

similar to those involved in PEV (Martin and Whitelaw, 1996). Flow cytometry is used to detect fluorescence from the GFP transgene, which allows for fast and reproducible phenotyping (Figure 1.6). As with the Drosophila PEV screens, the screen provides an opportunity to detect both suppressors and enhancers of variegation.

100

80 Wildtype FVB

60 Counts 40 M1 FVB Line3 20

1 100 101 102 103 104 GFP Fluorescence

Figure 1.6 Histogram of GFP fluorescence analysed by flow cytometry for FVB Line3 and wildtype FVB mice. The flow cytometry profiles of peripheral blood of four FVB Line3 mice and four wildtype FVB mice are overlayed. These profiles are representative of those seen in all Line3 and wildtype mice. The x-axis is an arbitrary fluorescence scale and the y- axis indicates the number of cells counted. A gate (M1) is used to determine which cells are fluorescing, and is set to exclude 99.9% of autofluorescing cells. The overlay shows that the profiles are reproducible from mouse to mouse within the inbred strain.

ENU produces point mutations in spermatogonial stem cells at a theoretical density of one mutation per gene in every 175-655 gametes (Hitotsumachi et al., 1985; Weber et al., 2000). Offspring of ENU-treated Line3 males can be phenotyped for mutations that change the expression of the GFP transgene, and which are therefore affecting epigenetic gene silencing, using flow cytometry. To date we have recovered 12 dominant and seven recessive mutant mouse lines from these screens, of which most

have been investigated to some extent (see Table 1.2). These mutants are named

Mommes (Modifiers of murine metastable epialleles), with a „D‟ indicating a dominant mutation, and an „R‟ a recessive mutation. The screen has already proven to be productive, with the identification of a number of interesting genes that influence both expression of the GFP transgene and other alleles sensitive to epigenetic state

(Blewitt et al., 2005; Chong et al., 2007; and data not shown). Some of the genes identified are already known to be involved in epigenetic mechanisms, and as such they act as a positive control for the screen.

Table 1.2 Summary of Momme mutants to date. These data have been collected from a number of members of the Whitelaw Laboratory. It includes results previously published in Blewitt et al., submitted and Chong et., 2007, while the rest remains unpublished. Mommes discussed in this thesis are highlighted.

Semi-dominant / Effect on Linkage (Chromosome Name Gene Recessive transgene and interval size) MommeD1 semi-dominant suppressor Chr 17 - 2.4 Mbp Smchd1 MommeD2 semi-dominant suppressor Chr 9 - 1.5 Mbp Dnmt1 MommeD3 semi-dominant suppressor Chr 11 - 6 Mbp MommeD4 semi-dominant enhancer Chr 8 - 5.2 Mbp Snf2h MommeD5 semi-dominant enhancer Chr 4 - 1.8 Mbp MommeD6 MommeD7 semi-dominant suppressor Chr 7 - 0.5 Mbp MommeD8 semi-dominant enhancer Chr 18 - 2.5 Mbp MommeD9 MommeD10 semi-dominant enhancer Chr 5 - 3.5 Mbp WSTF MommeD11 semi-dominant enhancer Chr 4 - 3 Mbp MommeD12 MommeR1 recessive suppressor Chr 10 - 38 Mbp Foxo3a MommeR2 recessive suppressor Chr 7 - 2.5 Mbp Uble1b MommeR3 recessive enhancer Chr 17 - 1.7 Mbp

1.6 Aims of this thesis

A clearer understanding of the complex nature of epigenetic gene silencing and its role in development can only come about by identifying more of the genes involved and characterising their function. The primary aim of my thesis was to characterise two mutant lines, MommeD6 and MommeD9, and to identify the underlying genes.

My hope was that I would identify novel modifiers that would add to our knowledge of epigenetic gene regulation, and provide new mouse models to study processes such as transgenerational epigenetic inheritance.

A further aim of my studies was to create a congenic C57 strain containing the GFP transgene from the FVB Line3 background. The reason for creating this strain was to improve future mutagenesis screens. It gave me an opportunity to observe the behaviour of the GFP transgene in a different strain background, which it was hoped would lead to the identification of strain-specific modifiers of gene silencing.

Chapter 2 - Materials and methods

2.1 Materials

2.1.1 Chemicals and reagents

All chemicals and reagents are of molecular biology grade unless otherwise specified.

The chemicals and reagents are listed below alphabetically alongside their suppliers.

3-[N-morphino]propanesulfonic acid (MOPS) Sigma-Aldrich Company, MO, USA 3‟,3‟‟,5‟,5‟‟-tetrabromophenolsulfonephthlein Sigma-Aldrich (bromophenol blue) acetic acid (glacial) VWR International Ltd, UK agarose, SeaKem® LE Cambrex Bio Science Rockland Inc, ME, USA agarose, Certified Low Range Ultra Bio-Rad Laboratories Inc, CA, USA albumin bovine serum (BSA), fraction V powder Sigma-Aldrich boric acid Ajax Finechem, NSW, Australia chloroform Ajax Finechem deoxycytidine 5‟-[α-32P] triphosphate (α-32P Amersham Biosciences dCTP), 10 mCi/mL deoxynucleoside triphosphates (dATP, dCTP, Astral Scientific, NSW, dGTP, dTTP) Australia diethyl pyrocarbonate (DEPC) Sigma-Aldrich

di-sodium hydrogen citrate .1,5H2O Riedel-de Haën,Germany ™ DNA ladder mix (GeneRuler ) Fermentas Inc, USA ethanol Ajax Finechem ethidium bromide Sigma-Aldrich ethylenediaminetetraacetic acid disodium salt Sigma-Aldrich (EDTA), 0.5 M solution ExpressHyb™ hybridisation solution Clontech Laboratories Inc., CA, USA Ficoll®-400 Sigma-Aldrich formaldehyde, 37% solution Sigma-Aldrich formamide Sigma-Aldrich

glycerol Ajax Finechem isoflurane Laser Animal Health, QLD, Australia isopropanol Ajax Finechem phenol (pH 7.9) Amresco, OH, USA sodium acetate, 3 M solution Sigma-Aldrich sodium chloride Merck, Germany sodium dodecyl sulfate (lauryl sulfate sodium Invitrogen Corporation, CA, salt) (SDS), 10% solution USA sodium hydroxide Ajax Finechem tris-hydroxymethyl-methylamine (Tris base) Ajax Finechem tri-sodium citrate Ajax Finechem xylene cyanol Sigma-Aldrich yeast tRNA Invitrogen

2.1.2 Buffers and solutions

All buffers and solutions were made using Milli-Q water (MQW) unless otherwise stated.

DNA lysis solution (low EDTA) 50 mM Tris (pH 8.0), 10 mM EDTA, 100 mM NaCl, 1% (w/v) SDS Ficoll stop mix 50% glycerol, 75 mM EDTA (pH 8.0), 6% (w/v) Ficoll-400, 0.09% (w/v) bromophenol blue, 0.09% (w/v) xylene cyanol 5x MOPS 0.1 M MOPS (pH 7.0), 5 mM EDTA, 25 mM sodium acetate, 45 mM NaOH. Made up with DEPC-treated MQW. Osmosol (flow cytometry buffer) 180 meq/L sodium, 153 meq/L chloride, 5.1 meq/L potassium, 1.0 meq/L EDTA. (Lab Aids Pty Ltd, NSW, Australia) phenol:chloroform 1:1 phenol:chloroform phosphate buffered saline (PBS) 0.01 M phosphate buffer (pH 7.4), 2.7 mM KCl, 0.137 M NaCl (Sigma-Aldrich) RNA loading buffer 50% glycerol, 1 mM EDTA (pH 8.0), 0.4% (w/v) bromophenol blue. Made with

DEPC-treated MQW. RNA precipitation solution 1.2 M NaCl, 0.8 M di-sodium hydrogen citrate.1,5H2O. Made with DEPC-treated MQW. RNA sample buffer 65% (v/v) formamide, 8.3% (v/v) formaldehyde, 0.65x MOPS buffer. Made with DEPC-treated MQW. 20x SSC 0.3 M tri-sodium citrate, 3 M NaCl (pH 7.2) 50x TAE 2 M Tris base, 1 M glacial acetic acid, 50 mM EDTA (pH 8.0) 5x TBE 0.45 M Tris base, 0.45 M boric acid, 10 mM EDTA (pH 8.0) TE (100x concentrated) 1 M Tris-HCl, 0.1 mM EDTA (pH 8.0) (Sigma-Aldrich)

2.1.3 Enzymes

All enzymes were used as per the conditions specified by the manufacturer.

Proteinase K from Tritirachium album (EC 3.4.21.64) Astral Scientific RNase from bovine pancreas (crystalline ribonuclease, Astral Scientific EC 3.1.27.5) Taq DNA polymerase from Thermus aquaticus BioLine Ltd, UK (deoxynucleoside-triphosphate: DNA deoxynucleotidyl transferase, EC 2.7.7.7) Type II restriction endonucleases: AlwNI, BamHI, New England Biolabs BglII, BsrI, BtsI, DdeI, HindIII, NsiI, PstI, XbaI (type (NEB), MA, USA II site-specific deoxyribonucleases, EC 3.1.21.4)

2.1.4 Oligonucleotides

All oligonucleotides used in this study were provided by Sigma-Genosys (Castle Hill,

NSW, Australia). Lists of oligonucleotides used are provided in appropriate places throughout this thesis.

2.1.5 Mouse strains and housing conditions

Ethical approval for the use of transgenic animals in this project was obtained from the Animal Ethics Committees at the University of Sydney and QIMR.

Animals used were from the following Mus musculus strains:

1) C57BL/6J mice carrying the relevant coat colour allele: a or Avy. The Avy allele has been backcrossed into this strain for over 30 generations, and maintained as an inbred strain (Oak Ridge National Laboratory, Oak Ridge, TN, USA).

2) FVB/NJ mice (ARC, Perth, Australia).

3) Line3 transgenic mice, produced in FVB/NJ mice by Jost Preis in the Whitelaw laboratory (Preis et al., 2003).

The mice were housed at a temperature of 21-23°C with a 12 hour light/dark cycle and had unlimited access to water and mouse pellets (In Molecular and Microbial

Biosciences animal house: Y.S. Feeds Mouse Breeder 602 - Y.S. Feeds, Young,

NSW, Australia; In QIMR animal house: Rat and Mouse Nuts - Norco Pty Ltd.,

Rocklea, Brisbane, Queensland, Australia). Male and female mice were housed in separate cages unless being used for breeding experiments. Mice were weaned 21 days after birth.

2.2 General methods

2.2.1 Isolation of genomic DNA

Genomic DNA was isolated from tails. To perform tail biopsies, mice were anaesthetised with isoflurane in a bell chamber until sedated and a 1.0 cm section of

tail was removed. Proteolytic digestion was achieved by adding 500 µL of DNA lysis solution containing proteinase K at 1 mg/mL and incubating at 55°C overnight.

RNase was added to approximately 50 mg/mL and the sample was incubated at 37°C for 30 min. Organic extractions were performed sequentially in equal volumes of phenol, phenol:chloroform and chloroform, by vigorous shaking at room temperature for 5 min, centrifugation at 12000 g for 10 min and discarding the organic phase after each extraction. To precipitate high molecular weight chromosomal DNA, an equal volume of isopropanol was added to the final aqueous phase and the tube gently mixed. DNA was spooled onto a glass rod and allowed to air-dry. DNA was dissolved in 200 µL TE.

2.2.2 Restriction endonuclease digestion of DNA

Restriction endonuclease digestion of DNA was performed in the buffers supplied and under the conditions specified by the manufacturer. Typically, restriction endonucleases were used at a concentration of 3-10 U per µg of DNA. Digestion was generally performed at 37°C for 1-2 h.

2.2.3 Agarose gel electrophoresis

Electrophoretic resolution of DNA fragments was performed in agarose gels. Gels were composed of 2% (w/v) agarose in 1x TAE and contained ethidium bromide at a concentration of 0.5 µg/mL. Samples were loaded with Ficoll stop mix at approximately 15% (v/v). Electrophoresis was carried out in 1x TAE at 8 V/cm until the dye front had migrated a distance appropriate for the size of the DNA fragment

being resolved. Gels were photographed under UV transillumination using a Bio-Rad

Gel Imaging System (Bio-Rad).

2.2.4 Avy genotyping using the polymerase chain reaction

Genotyping for the Avy allele was performed using a multiplex PCR. PCR was carried out in a Bio-Rad DNAEngine Peltier thermal cycler (Bio-Rad). Each reaction contained approximately 100 ng of genomic DNA, 1 U of BIOTAQ™ DNA polymerase (Bioline), 500 nM each of dATP, dCTP, dGTP and dTTP, and 1x

™ BIOTAQ PCR Reaction Buffer. MgCl2 was supplemented to 2 mM and BSA to 0.4

µg/µL. After an initial denaturation at 94°C for 3 min, 32 cycles were performed of: denaturation (94°C, 30 s), annealing (50°C, 60 s) and elongation (72°C, 90 s). A final elongation at 72°C for 4 min was included. The Agouti 5‟ and Agouti 3‟ primers produce a 295 bp agouti-specific product, from the Avy, A and a alleles. The IAP-F and Agouti 3‟ primers produce a 413 bp Avy-specific product. This allowed for the discrimination of Avy/a and A/a mice (Rakyan et al., 2003). The oligonucleotides used are shown in Table 2.1.

Table 2.1 Oligonucleotides used for Avy genotyping.

2.2.5 Avy phenotyping

All Avy mice were assessed at weaning for their coat colour phenotype. They were subdivided into seven subcategories based on the percentage of yellow in their coat; Y

(100% yellow), mY (95-100% yellow), YM (75-95% yellow), M (25-75% yellow),

ΨM (5-25% yellow), mΨ (0-5% yellow) and Ψ (0% yellow). For the purposes of statistical analysis, subgroups were combined into three categories; yellow (Y, mY,

YM), mottled (M, ΨM) and pseudoagouti (mΨ, Ψ).

2.2.6 Embryonic dissections

On the specified day after the detection of a vaginal plug, pregnant females were sacrificed. Females were dissected and uterine horns removed and washed in PBS.

Embryos were removed from the uterine horns and washed in PBS. Embryo images were captured with a Leica DFC camera (Leica Microsystems).

2.2.7 Sequencing of PCR products

Each reaction contained approximately 100 ng of genomic DNA, 1 U of BIOTAQ™

DNA polymerase (Bioline), 500 nM each of dATP, dCTP, dGTP and dTTP, and 1x

™ BIOTAQ PCR Reaction Buffer. MgCl2 was supplemented to 1.5 mM and BSA to

0.4 µg/µL. After an initial denaturation at 94°C for 3 min, 35 cycles were performed of: denaturation (94°C, 30 s), annealing (variable temperature; 53-64°C, 30 s) and elongation (72°C, 30 s). A final elongation at 72°C for 5 min was included.

PCR reactions were cleaned up using the Agencourt® AMPure® PCR Purification kit

(Agencourt Bioscience) according to the manufacturer‟s instructions and resuspended

in 40 µL of MQW. The recovered product was quantified using a Generuler™ DNA ladder mix. Approximately 1-2 ng per 100 bp of PCR product was used in ABI Big

Dye 3.1 (Applied Biosystems) sequencing reactions with 800 nM of forward or reverse primer, 0.6 µL of Dye Terminator and 4.2 µL of 5x reaction buffer in a final volume of 12 µL. Sequencing reactions underwent thermocycling in the following conditions: 96°C for 1 min, followed by 25 cycles of 96°C for 10 s, 50°C for 5 s,

60°C for 3 min. Samples were then cleaned up by adding 72 µL of 70% isopropanol, vortexing and incubating for 15 min at room temperature, followed by centrifugation at 12000 g for 30 min at room temperature. The supernatant was removed and samples were washed in 300 µL of 70% isopropanol and centrifuged for 10 min. The supernatant was removed and samples were air-dried at room temperature for 1 h. The precipitated pellet was sent to the sequencing facility at QIMR.

2.2.8 Total RNA extraction

RNA was isolated from spleens. Mice were sacrificed at the appropriate age and their spleens were removed. Each spleen was cut in half, and each half was placed in a separate Eppendorf tube and snap frozen in liquid nitrogen. Total cellular RNA was extracted using TRI® reagent (Sigma-Aldrich) according to the manufacturer‟s instructions. Briefly, half a spleen was homogenised in 1 mL of TRI® reagent and then stored at room temperature for 5 min. 0.2 mL of chloroform was added to the samples, they were shaken vigorously for 15 s, rested at room temperature for 15 min, and then centrifuged at 12000 g for 15 min at 4°C. The top aqueous phase was transferred to a fresh microcentrifuge tube containing 250 µL of 100% isopropanol and 250 µL of RNA precipitation solution. The samples were stored at room temperature for 10 min and then centrifuged at high speed for 15 min at 4°C. The

samples were washed with 75% ethanol, air-dried at room temperature and resuspended in 200 µL of 55°C DEPC-treated TE. Samples were stored at -80°C.

2.2.9 PolyA+ mRNA isolation

PolyA+ mRNA was extracted using the PolyATract® mRNA isolation system

(Promega Corporation) according to the manufacturer‟s instructions. Briefly, 0.1-1.0 mg of total RNA, in a final volume of 500 µL, was heated at 65°C for 10 min. 3 µL of

Biotinylated-Oligo(dT) probe and 13 µL of 20x SSC was added to the RNA, gently mixed and incubated at room temperature until cooled. Meanwhile, the Streptavidin-

Paramagnetic Particles (SA-PMPs) were washed three times with 300 µL of 0.5x SSC and resuspended in 100 µL of 0.5x SSC. The entire contents of the annealing reaction was added to the washed SA-PMPs and incubated at room temperature for 10 min with gentle mixing by inversion every 1-2 min. The SA-PMPs were washed four times with 300 µL of 0.1x SSC. The polyA+ mRNA was eluted twice, first with 100

µL and then 150 µL of RNase-free water. The polyA+ mRNA was precipitated overnight at 4°C by the addition of 20 ng of yeast tRNA, 25 µL of 3 M sodium acetate and 250 µL of 100% isopropanol. The polyA+ mRNA was centrifuged at

12000 g for 15 min at 4°C, washed with 200 µL of 75% ethanol, air-dried at room temperature for 10 min and finally resuspended in 12 µL of DEPC-treated TE.

Samples were stored at -80°C. The concentration of PolyA+ mRNA samples was determined using a Nanodrop spectrophotometer.

2.2.10 Northern blotting and hybridisation

Gel electrophoresis and Northern blot transfer of RNA

All solutions were made with DEPC-treated MQW. Approximately 500 ng of each

PolyA+ mRNA sample was denatured by adding 23 µL of RNA sample buffer, heating at 65°C for 15 min, immediately chilling on ice and adding 3 µL of RNA loading buffer. The entire sample was loaded on a 1.2% agarose gel made up with 1x

MOPS and containing a final concentration of 6.7% (v/v) formaldehyde. The gel was run in 1x MOPS buffer at 7 V/cm for 2-3 h. The gel was soaked in 0.05 M NaOH for

20 min, followed by 20x SSC for 50 min. The RNA was capillary blotted to a pre-wet

Hybond™-XL nylon membrane (Amersham Biosciences) with 20x SSC transfer solution overnight. The RNA was cross-linked to the membrane using a GS Gene

Linker UV chamber (Bio-Rad) at 80 mJ, and the membrane was rinsed in 6x SSC.

Preparation of radiolabelled DNA probes

DNA fragments to be radiolabelled for use as probes were purified from agarose gels.

For all probes, approximately 15 ng of DNA was radiolabelled with [α-32P] dCTP

(3000 Ci/mmol) by random primer labelling using Ready-To-Go DNA Labelling

Beads (-dCTP) (Amersham Biosciences) according to the manufacturer‟s instructions.

Labelled DNA was purified from unincorporated deoxyribonucleotides by column chromatography through a NICK® Sephadex® G-50 column (Amersham Biosciences) according to the manufacturer‟s instructions, except that fractions were eluted with

200 µL of TE. Immediately prior to use, the fraction containing the labelled probe was denatured by heating at 100 °C for 2 min and immediately cooled on ice.

Table 2.2 Radiolabelled DNA probes used in Northern analysis.

Hybridisation and washing of membrane

The membrane was pre-hybridised in ExpressHyb™ for 30 min at 68°C, before hybridisation at 68°C overnight with freshly denatured, radiolabelled GFP probe

(Table 2.2) in a hybridisation bottle (Amersham Biosciences) in a hybridisation oven

(Amersham Biosciences). Non-specifically hybridised probe was washed from the membrane with three washes of 2x SSC / 0.05% (w/v) SDS at room temperature for

13 min and two washes of 0.1x SSC / 0.1% (w/v) SDS at 50°C for 20 min. The membrane was exposed to a PhosphorImager® storage phosphor screen (Molecular

Dynamics) overnight. The signal arising from the radiolabelled probe was visualised using Special Performance PhosphorImager® hardware and ImageQuant version 5.1 software (Molecular Dynamics). To assess the relative quantities of RNA in each sample, the membrane was rehybridised with a radiolabelled GAPDH probe (Table

2.2), as above, after removing the GFP probe by agitating the membrane in boiling

(but cooling down) 0.1% (w/v) SDS for 15 min, and then rinsing it in 2x SSC.

2.2.11 Haematological analysis

To obtain blood for haematology, three week old mice were anaesthetised with isoflurane and the tail tip removed. 30 µL of peripheral blood was collected from the

® tail directly into a VACUETTE tube containing K2 EDTA (Greiner Bio-One). 25 µL

of blood was diluted with 25 µL of PBS and full blood counts were measured using a

Coulter Ac.T diff™ (Beckman Coulter, CA, USA).

2.2.12 Urinalysis

To obtain urine for urinalysis, mice were placed in specially prepared cages with a metal wire grid raised off the cage bottom. Fresh plastic was laid on the base of the cage below the grid for each mouse. The mouse was left in the cage until it urinated.

Multistix® urinalysis strips (Bayer) were used to test for glucose in the urine following the manufacturer‟s instructions.

2.2.13 Flow cytometry

Blood collection from three week old mice

To obtain blood for flow cytometry, three week old mice were anaesthetised with isoflurane and the tail tip removed (~1 mm). A drop of blood from the tail was collected into 1 mL of Osmosol and inverted to mix.

Two-channel flow cytometry

Either a FACSCalibur or FACScan flow cytometer (both Becton Dickinson, NJ,

USA) was used to detect and quantify levels of cellular fluorescence. Two channels were used with different excitation wavelengths. The first channel (488 nm) was used to detect fluorescence from the transgene, whereas the second channel (550 nm) was used when required to gauge the autofluorescence of cells. This approach achieves greater accuracy in distinguishing GFP expressing and non-expressing cell populations (Rasko et al., 1999). Analysis of data produced from flow cytometry was

analysed using Cell Quest Pro software (Becton Dickinson) using conservative gates set to exclude 99.9% of wildtype non-transgenic, autofluorescing erythrocytes.

Histograms shown depict only the GFP fluorescence channel (488 nm).

2.2.14 Mapping of Momme mutations

Microsatellite markers

Microsatellite markers spaced evenly throughout the genome (75 for MommeD6 and

36 for MommeD9) that differed in size between FVB/NJ and C57BL/6J were used for a preliminary round of mapping. At the time of mapping, few markers were known for FVB strain. Initially, many markers had to be tested to see if there was a visible difference between the FVB and C57 strains. A list of all microsatellite markers used for mapping, along with their PCR conditions, oligonucleotide sequences and electrophoresis protocol are shown in Appendix 1. When a chromosome was identified as being linked to the mutation, additional markers and mice were analysed on that chromosome to reduce the linked interval.

PCR conditions for microsatellite markers differed only in annealing temperature.

Each reaction contained approximately 100 ng of genomic DNA, 1 U of BIOTAQ™

DNA polymerase (Bioline), 200 µM each of dATP, dCTP, dGTP and dTTP, and 1x

™ BIOTAQ PCR Reaction Buffer. MgCl2 was supplemented to 1.5 mM or higher and

BSA to 0.4 µg/µL. After an initial denaturation at 94°C for 2 min, 35 cycles were performed of: denaturation (94°C, 30 s), annealing (variable temperature; 46-63°C, 30 s) and elongation (72°C, 30 s). A final elongation at 72°C for 5 min was included.

The PCR products were separated by gel electrophoresis and the data analysed by interval haplotype analysis (Neuhaus and Beier, 1998). Depending on the degree of resolution required to separate the FVB and C57 PCR products, samples were run on gels composed of either 3-4% (w/v) Certified Low Range Ultra agarose in 0.5x TBE containing ethidium bromide at a concentration of 0.5 µg/mL or 15% precast polyacrylamide gels (Bio-Rad). Samples were loaded with Ficoll stop mix at approximately 15% (v/v). Agarose gels were run in 0.5x TBE and polyacrylamide gels were run in 1x TBE at 8 V/cm until the dye front had migrated an appropriate distance. Gels were photographed under UV transillumination using a Bio-Rad Gel

Imaging System (Bio-Rad).

Single nucleotide polymorphisms

If no microsatellite markers could be found to reduce a linked interval, single nucleotide polymorphisms (SNPs) were used. The SNPs were amplified by PCR from genomic DNA as described above for microsatellite markers. A list of all SNPs used in mapping, along with their PCR conditions, oligonucleotide sequences and electrophoresis protocol are shown in Appendix 1. The majority of SNPs created or abolished a restriction endonuclease site, in which case a restriction endonuclease digest was performed to genotype the SNP. Some SNPs however were directly sequenced from PCR products to determine the genotype.

2.2.15 Progeny testing

Several mutant mice from heterozygous intercrosses of both MommeD6 and

MommeD9 were progeny tested to check that they were heterozygous and not homozygous for the mutation. This involved crossing to wildtype Line3 mice and

analysing the phenotype of the resulting offspring. If both wildtype and mutant phenotypes were observed, the parent being progeny tested was obviously heterozygous for the mutation. In all cases at least one wildtype offspring was observed in the first litter.

Similar progeny testing was performed, when possible, on F2 and F3 mice from mapping crosses that had recombined across the linked interval (i.e. that defined the interval). This involved crossing to wildtype C57 mice and analysing the phenotype of the resulting offspring. If both wildtype and mutant phenotypes were observed, the parent being progeny tested was obviously carrying the mutation and thus was a true mutant.

2.2.16 Speed congenics

During the creation of a C57 congenic strain containing a GFP transgene from the

FVB Line3 strain speed congenics was used at the F4 and F5 generations to select the

“best” mice with which to proceed to the next generation. 63 microsatellite markers spaced evenly throughout the genome that differed between FVB and C57 were used to genotype the mice at these two generations, to determine which two mice in each case had the highest proportion of C57-derived genome. The markers were amplified by PCR from genomic DNA as described previously. A list of all microsatellite markers used for speed congenics, along with their PCR conditions, oligonucleotide sequences and electrophoresis protocol are shown in Appendix 1.

2.2.17 Mapping the GFP transgene

Microsatellite markers and one SNP on chromosome 1 that differed between FVB and

C57 were used to map the location of the GFP transgene. The markers were amplified by PCR from genomic DNA as described previously. A list of the markers used for mapping the GFP transgene, along with their PCR conditions, oligonucleotide sequences and electrophoresis protocol are shown in Appendix 1.

2.2.18 Mapping the modifiers of GFP transgene expression

39 microsatellite markers evenly spaced throughout the genome that differed between

FVB and C57 were used to map modifiers of GFP transgene expression that differed between the two strains. The markers were amplified by PCR from genomic DNA as described previously. A list of the markers used for mapping the modifiers, along with their PCR conditions, oligonucleotide sequences and electrophoresis protocol are shown in Appendix 1. When a chromosome was identified as being linked to a modifier, additional markers were analysed on that chromosome to reduce the linked interval.

2.2.19 Statistics

Chi-squared test

A chi-squared test for independence returns the probability of the chi-squared statistic for the data shown (as observed numbers) against expected numbers assuming no difference in the data. A p-value of 0.05 implies that there is only a 5% probability that the data sets are statistically different by chance when they are in fact the same.

Therefore, for a p-value of 0.05 or less one can consider that the data sets being compared are statistically significantly different.

Standard student T-test

The standard student t-test assesses whether the means of two groups are statistically different from each other. A p-value of 0.05 implies that there is only a 5% probability that the two groups have statistically different means by chance when they are in fact the same. Therefore, if the p-value is 0.05 or less one can consider that the two groups have been drawn from two populations with statistically significantly different means. When comparing two groups with unequal numbers of data points, an F-test (see below) is first used to test whether the variances of the two groups are statistically different or not.

F-test

The F-test assesses whether the variances observed in two sets of data are statistically different from each other. A p-value of 0.05 implies that there is only a 5% probability that the two groups have statistically different variances by chance when they are in fact the same. Therefore, if the p-value is 0.05 or less one can consider that the two groups have been drawn from two populations with statistically significantly different variances, and this is taken into account when calculating the T-test statistic.

Chapter 3 - MommeD6

Abstract

MommeD6 is a semi-dominant, homozygous lethal mutation, and we classify it as a suppressor of variegation since it increases the expression of a GFP transgene. Unlike other Mommes characterised to date the mutation largely affects the amount of GFP per cell as opposed to the percentage of cells expressing the transgene. It may represent a class of genes involved in regulating gene expression at the post- transcriptional level. The mutation lies in a 2.5 Mbp interval on chromosome 14.

3.1 Introduction

MommeD6 was produced and identified in the dominant ENU mutagenesis screen carried out by Dr M. Blewitt. The MommeD6 line was maintained in the FVB strain as an inbred colony by M. Blewitt for the first four generations and then by myself.

We both collected the data described in this thesis that shows that MommeD6 is semi- dominant and homozygous lethal, and most of it was collected by me. Some of this material has previously been published (Blewitt et al., 2005). I am a co-author on that publication. I have continued to investigate the phenotype associated with both homozygosity and heterozygosity for the MommeD6 mutation, and I have used linkage analysis in an attempt to identify the underlying gene.

3.2 Results

3.2.1 Flow cytometry profile

The data presented in Figure 3.1 and Table 3.1 show the percentage of cells expressing the GFP transgene and the mean fluorescence of those cells in peripheral blood of offspring from crosses between heterozygous mutants (MommeD6-/+) and wildtype Line3 mice. The data presented in Table 3.1 were collected over four years by both Dr M. Blewitt and myself, although I collected the majority. MommeD6 is a suppressor of variegation. MommeD6 heterozygous mutants display a small increase in the percentage of peripheral blood cells that are expressing the transgene, and a large increase in the mean fluorescence of those expressing cells compared with that of wildtype Line3 mice (Figure 3.1 and Table 3.1). Because of this, the mean fluorescence was used to designate a mouse as either a mutant or non-mutant. In this

respect, this mutation is unusual; most Mommes produced in the dominant screen display a more obvious change in the percentage of expressing cells and the fluorescence level of an expressing cell changes less dramatically (see Blewitt et al.,

2005 and Chapter 4 - MommeD9).

100

60 Counts 40 M1

1 100 101 102 103 104 GFP Fluorescence

Figure 3.1 Histogram of GFP fluorescence analysed by flow cytometry for the MommeD6 mutation. The flow cytometry profiles of peripheral blood of five MommeD6+/+ (green line) and five MommeD6+/- (red line) mice at weaning from a representative litter are overlayed. Due to slight day to day fluctuations with the flow cytometer, data collected on different days should not be combined. The data shown are representative of that seen in 90 litters. The x-axis is an arbitrary fluorescence scale and the y-axis indicates the number of cells counted. A gate (M1) is used to determine which cells are fluorescing, and is set to exclude 99.9% of autofluorescing cells. MommeD6-/+ mice have more cells fluorescing and a significantly higher mean fluorescence than MommeD6+/+ mice.

Male wildtype offspring (MommeD6+/+) display a slightly lower percentage of expressing cells and a slightly lower mean fluorescence than female wildtype offspring (p<0.001 and p=0.01 respectively) (Table 3.1). A similar trend is seen in the wildtype Line3 colony (p=0.1 and p=0.04 for percentage of expressing cells and mean fluorescence respectively) (Table 3.1). No sex-specific effects were seen in mutant

(MommeD6-/+) offspring (Table 3.1). Sex-specific effects have been reported at other epigenetically sensitive loci such as the Avy locus, where the proportion of yellow mice is slightly higher in female than male offspring (Morgan, 2001). So, metastable

epialleles appear to be more active in females than males. Interestingly, in the

MommeD6 heterozygous mutants this difference is lost.

Table 3.1 Percentage of cells expressing the transgene and the mean fluorescence of expressing cells for MommeD6-/+, MommeD6+/+ and wildtype Line3 mice. A sample of peripheral blood was analysed at weaning. The mean ± one standard deviation of the percentage of cells expressing the transgene and mean fluorescence of those expressing cells are shown. Following crosses involving MommeD6-/+ and wildtype Line3 mice, MommeD6-/+ mice had a significantly higher percentage of expressing cells (p<<0.001) and mean fluorescence (p<<0.001) than their MommeD6+/+ littermates. Sex-specific effects were observed in wildtype Line3 mice, with males having a lower percentage of expressing cells (p=0.1) and mean fluorescence (p=0.04) than females. (All p-values are for a T-test)

3.2.2 MommeD6 is semi-dominant and homozygous lethal

An intercross between two heterozygous MommeD6 mutants produced only two classes of mice with respect to GFP expression (Figure 3.2 and data not shown), and the average litter was smaller (5.9 mice/litter) than that of wildtype Line3 (8.6 mice/litter) (Figure 3.3). In combination, these data suggest that MommeD6 homozygous mutants (MommeD6-/-) die before weaning. We can therefore assume

that the two classes represent the MommeD6+/+ and MommeD6-/+ offspring. The percentage of MommeD6-/+ and MommeD6+/+ offspring following a heterozygous intercross was 38% and 62% respectively, which is close to, but not exactly, the 1:2 ratio expected if the mutation was semi-dominant and homozygous lethal (p=0.08) since MommeD6-/- mice should theoretically contribute one-quarter of the total offspring (Table 3.2). Four female and three male mutant offspring derived from

MommeD6-/+ intercrosses were progeny tested to check that they were heterozygous and not homozygous for the mutation. This involved crossing the mutant offspring with wildtype Line3 mice (see Chapter 2 - Materials and methods). If any offspring from these crosses were wildtype for the MommeD6 mutation, then the mouse being progeny tested cannot be homozygous for the mutation. In all cases the mice produced both wildtype and mutant offspring and must therefore have been heterozygous, i.e.

MommeD6-/+, mutants (data not shown).

30 Counts 20 M1

1 100 101 102 103 104 GFP Fluorescence

Figure 3.2 Histogram of GFP fluorescence analysed by flow cytometry of offspring from MommeD6 heterozygous intercrosses. The flow cytometry profiles of peripheral blood of two wildtype (green line) and three mutant (red line) mice at weaning from a representative litter are overlayed on. Due to slight day to day fluctuations with the flow cytometer, data collected on different days should not be combined. The data shown are representative of that seen in 26 litters. The x-axis is an arbitrary fluorescence scale and the y-axis indicates the number of cells counted. A gate (M1) is used to determine which cells are fluorescing, which is set to exclude 99.9% of autofluorescing cells. Only two classes were observed.

*** 14 12 10 8.6 8 7.9 8.1

6 5.9 Litter size Litter 4 2 0 Wildtype Line3 MommeD6 -/+ sire x MommeD6 -/+ dam x MommeD6 -/+ Wildtype Line3 Wildtype Line3 intercross

(n=67) (n=51) (n=50) (n=43)

Figure 3.3 Litter size of MommeD6-/+ crosses compared to that of wildtype Line3. The boxes represent the mean ± one standard deviation with the mean in the middle of the box. The whiskers show the highest and lowest observed values. MommeD6-/+ x wildtype Line3 pairs did not have significantly different litter sizes compared to wildtype Line3 x wildtype Line3 pairs. The litter size of MommeD6-/+ intercrosses was significantly different (p<<0.001), and argues for the death of MommeD6-/- mice. (n = number of litters observed, *** indicates p<<0.001) (All p-values are for a T-test)

Table 3.2 Percentage of wildtype and mutant offspring from MommeD6 heterozygous intercrosses. Offspring were phenotyped by flow cytometry. The numbers of offspring observed were significantly different from those expected for a dominant, homozygous viable mutation, but were consistent with those expected for a semi-dominant, homozygous lethal mutation. The expected numbers for a dominant mutation are based on Mendelian ratios. The expected numbers for a semi-dominant, homozygous lethal mutation are based on the ratio if the homozygous mutants are absent. (All p-values are for a Chi-squared test)

Expected numbers Expected numbers for a semi-dominant, Phenotype Observed numbers for a dominant homozygous lethal mutation mutation Wildtype 97 63 (25%) 84 (33%) Mutant 155 189 (75%) 168 (67%) p-value <<0.001 0.08

The observed loss of ~2.7 mice/litter was slightly larger than would be expected if only MommeD6-/- mice were dying, although the extra loss is only ~0.6 mice/litter. In crosses between MommeD6-/+ and wildtype Line3 mice, a trend towards a litter size reduction of ~0.6 mice/litter was also observed following both paternal and maternal transmission of the mutation. It was not statistically significant in either case (7.9 and

8.1 mice/litter, respectively) (Figure 3.3). This suggests that there is a small, stochastic loss of MommeD6-/+ mice, and it may account for the slightly higher than expected ratio of wildtype to heterozygous mutants seen following the heterozygous intercross (Table 3.2). I decided to follow this up by analysing in more detail the offspring of crosses between heterozygous mutants and wildtype Line3 mice.

3.2.3 MommeD6 shows transmission ratio distortion (TRD) following paternal and maternal transmission

In MommeD6-/+ to wildtype Line3 crosses, significantly less mutants were detected at weaning than expected following both paternal and maternal transmission (p<0.001 and p<0.05 respectively) (Figure 3.4). This is consistent with the abnormal ratio of wildtype to mutant offspring seen following heterozygous intercrosses (Table 3.2).

This skewing away from the expected 1:1 ratio of non-mutants : mutants is referred to as transmission ratio distortion (TRD).

Following maternal transmission, both males and females displayed a statistically significant loss of MommeD6-/+ offspring (p=0.05 and p=0.02 respectively).

Following paternal transmission, the TRD was more enhanced in female offspring

(p<0.001). These results suggest a loss of ~10% of mutants, which is consistent with

the slightly smaller litter size in crosses between heterozygous mutants and wildtype

Line3 mice noted previously.

(a) Maternal transmission of MommeD6 300 * 250 232

200 171 150 * * 111 121 MommeD6 +/+

100 84 87 MommeD6 -/+ Number ofmice Number 50 0 Total Males Females

(n=403) (n=195) (n=208)

(b) Paternal transmission of MommeD6 300 ** 248 250 200 179 ** 150 119 129 100 MommeD6 +/+ 79 100 MommeD6 -/+ Number ofmice Number 50 0 Total Males Females

(n=427) (n=219) (n=208)

Figure 3.4 Transmission ratio distortion following crosses between MommeD6-/+ and wildtype Line3 mice. Following both (a) maternal and (b) paternal transmission of the MommeD6 mutation there was a significant loss of MommeD6-/+ offspring. Following maternal transmission there was no sex-specific effect, with the loss occurring in both male and female offspring. Following paternal transmission there was a sex-specific effect, with greater loss in the female offspring. There was no statistically significant difference in the overall number of male and female offspring following paternal or maternal transmission. (* indicates p<0.05 and ** indicates p<0.001) (All p-values are for a Chi-squared test)

3.2.4 MommeD6 homozygous mutants die in utero

In order to determine when the MommeD6-/- mutants were dying, pregnant females from MommeD6-/+ intercrosses were killed and embryos studied at various time points. At 14.5 dpc around one-third of the embryos were resorbed, i.e. they were invaded by macrophages (Table 3.3a). These resorbed embryos were likely to include all the MommeD6-/- mutants as well as the occasional MommeD6-/+ mutant. Until the causative point mutation has been identified, we cannot verify this by genotyping.

Since MommeD6-/- mutants would only be expected to comprise one-quarter of the total litter, and we expect ~10% of MommeD6-/+ heterozygous mutants to die, the number of resorbed embryos is close to that expected. At 10.5 dpc none of the embryos were resorbed, but one-third of them were developmentally delayed by two to three days (Table 3.3b). Figure 3.5 shows representative examples of a normal and developmentally delayed embryo at 9.5 dpc. The developmentally delayed embryo is approximately one quarter the size of a normal embryo. These embryos are likely to represent the same group of embryos that are resorbed at 14.5 dpc.

Table 3.3 Percentage of abnormal embryos from MommeD6-/+ intercrosses at 14.5 and 10.5 dpc. (a) At 14.5 dpc 38% of embryos from MommeD6-/+ intercrosses were resorbed, compared with 6% from wildtype Line3 crosses. The resorbed embryos presumably included the MommeD6-/- embryos. (b) At 10.5 dpc 37% of embryos from MommeD6-/+ intercrosses looked developmentally delayed, compared with 6% from wildtype Line3 crosses. (a) 14.5 dpc

MommeD6 -/+ intercrosses Wildtype Line3 crosses (n = 5) (n = 5) Normal 25 (62.5%) 44 (93.6%) Resorbed 15 (37.5%) 3 (6.4%) Total 40 47 Average number/litter 8 9.4

(b) 10.5 dpc MommeD6 -/+ intercrosses Wildtype Line3 crosses (n = 7) (n = 4) Normal 38 (63.3%) 34 (94.4%) Abnormal 22 (36.7%) 2 (5.6%) Total 60 36 Average number/litter 8.6 9

(a) (b)

2 mm 1 mm

Figure 3.5 A normal and an abnormal 9.5 dpc embryo from a MommeD6-/+ intercross. The normal 9.5 dpc embryo (a) is much larger than the abnormal embryo (b).

3.2.5 MommeD6-/+ mice weigh less than their wildtype littermates

Both male and female MommeD6-/+ mice weigh significantly less than their

MommeD6+/+ littermates at three weeks of age (p<0.001 and p<0.05 for males and female respectively) (Figure 3.6). So although most MommeD6-/+ mice are viable, they do show an abnormal phenotype. Further phenotyping could reveal many other subtle differences.

16 ** * 14 12 10 9.9 8.8 9.2 8 8.6 6

Body weight (g) Bodyweight 4 2 0 Male Male Female Female MommeD6 +/+ MommeD6 -/+ MommeD6 +/+ MommeD6 -/+

(n=106) (n=88) (n=131) (n=73)

Figure 3.6 Weight of MommeD6-/+ mice at weaning. The boxes represent the mean ± one standard deviation with the mean in the middle of the box, and the whiskers show the highest and lowest observed values. Both male and female MommeD6-/+ mice weighed significantly less than their counterpart MommeD6+/+ littermates at weaning (three weeks of age). (* indicates p<0.05 and ** indicates p<0.001) (All p-values are for a T-test)

3.2.6 The effect of the MommeD6 mutation on expression at the agouti viable yellow locus

In order to see whether the MommeD6 mutation had an effect on an epigenetically sensitive allele other than the GFP transgene, the MommeD6 mutation was crossed into a C57 strain containing the agouti viable yellow (Avy) allele. The Avy allele is known to be sensitive to changes in the concentration of proteins involved in the establishment and maintenance of epigenetic state, such as DNA methyltransferase 1 and DNA methyltransferase 3L (Chong et al., 2007; Gaudet et al., 2004). It has also been shown in this laboratory that expression at the Avy allele is affected by heterozygosity for some other Momme mutants (Blewitt et al., 2005; Chong et al.,

2007). This experiment is an attempt to determine whether the MommeD6 mutation affects an independent locus, i.e. one with a different promoter and coding sequence to the GFP transgene.

-/+ vy MommeD6 mice were crossed with pseudoagouti A mice, and the F1 hybrid offspring were phenotyped for coat colour at weaning. At the same time blood was collected and used to phenotype for the MommeD6 mutation by flow cytometry. So, the coat colour phenotype was determined blind with respect to the flow cytometry phenotype. Reciprocal matings were carried out because penetrance at metastable epialleles is known to be affected by parent-of-origin in the C57 background; maternal transmission of Avy results in more yellow mice than paternal transmission

(Morgan et al., 1999).

Following paternal transmission of MommeD6 and maternal transmission of Avy (a

MommeD6-/+ sire crossed with a pseudoagouti dam), there was no statistically

significant difference in coat colour penetrance between MommeD6-/+ and

MommeD6+/+ offspring, and there was no effect when these offspring were split by sex (Figure 3.7).

Figure 3.7 Paternal inheritance of MommeD6 from pseudoagouti mice carrying the Avy allele. Avy/a C57 pseudoagouti dams were mated with FVB MommeD6-/+ sires, and the offspring were split by genotype as determined by flow cytometry. Offspring not carrying the Avy allele have been omitted. Data for the MommeD6 crosses were produced from eight different breeding pairs using six different pseudoagouti dams. There was no significant difference in coat colour penetrance between MommeD6-/+ and MommeD6+/+ littermates in either male or female offspring. (All p-values are for a Chi-squared test)

Following maternal transmission of MommeD6 (a MommeD6-/+ dam and a pseudoagouti sire), there appeared to be a difference in coat colour penetrance between MommeD6-/+ and MommeD6+/+ offspring, although it was not statistically significant (Figure 3.8). There was a general shift away from mottled coat colour in the MommeD6-/+ offspring, with more yellow and more pseudoagouti offspring. When

split by sex the shift away from mottled coat colour was limited to the female offspring (p=0.01). It is not clear how to interpret this result, and the experiment would need to be repeated if one wished to pursue this further. The most conservative view would be that MommeD6 heterozygosity has little effect on expression at the Avy locus.

Figure 3.8 Maternal inheritance of MommeD6 from pseudoagouti mice carrying the Avy allele. Avy/a pseudoagouti sires were mated with MommeD6-/+ dams, and the offspring were split by genotype as determined by flow cytometry. Offspring not carrying the Avy allele have been omitted. Data for the MommeD6 crosses were produced from 10 different breeding pairs using six different pseudoagouti sires. There was a significant shift away from mottled coat colour in female MommeD6-/+ compared to MommeD6+/+ littermates. (All p-values are for a Chi-squared test)

3.2.7 MommeD6-/+ mice have increased levels of GFP mRNA

MommeD6 heterozygous mutants have almost double the level of GFP fluorescence in an expressing cell than wildtype mice (increasing from a mean of ~270 to ~490), but little change in the percentage of expressing cells (increasing from a mean of

~54% to ~63%) (Table 3.1). So, despite the fact that the number of cells that are expressing the GFP transgene in MommeD6 mutants has remained essentially the same, the amount of GFP protein product produced from those cells has almost doubled. This implies either an increase in the level of mRNA (due to either an increase in the rate of transcription of the transgene or an increase in the stability of the GFP mRNA), or no increase in the level of mRNA but an increase in translation or stability of the GFP protein product. To address this Northern analysis was used to determine the levels of GFP mRNA in MommeD6 mutant tissues.

PolyA+ mRNA obtained from the spleens of three week old male MommeD6-/+ mutants (n=3) and wildtype Line3 mice (n=3), and from six week old male

MommeD6-/+ mutants (n=2) and wildtype Line3 mice (n=2), was separated on a 1.2% agarose gel, transferred to a nylon membrane, hybridised to a radiolabelled GFP probe and GFP mRNA levels were compared (Figure 3.9a). The membranes were then stripped and rehybridised with a radiolabelled GAPDH probe as a loading control.

Although there was no statistically significant difference in GFP:GAPDH mRNA ratios between the MommeD6-/+ mutants and the wildtype Line3 mice, the mutants did have higher levels of GFP mRNA on average, with a fold increase of ~1.25 for the three week old mice and a fold increase of ~1.5 for the six week old mice (Figure

3.9b). It is difficult to know whether this increase in mRNA levels would be sufficient to explain the almost two-fold increase in GFP fluorescence.

(a) wt Line3 MommeD6-/+ wt Line3 MommeD6-/+ 1 2 3 4 5 6 7 8 9 10

GFP GFP

GAPDH GAPDH

(b) 6 4 3.28 5.09 4.79 2.74 5 4.68 3 4.34 2.40 3.79 3 4 2.90 2 1.60 3 2 2 1

1 1 Ratio of GFP:GAPDH mRNA RatioofGFP:GAPDH Ratio of GFP:GAPDH mRNA RatioofGFP:GAPDH 0 0 1 2 3 4 5 6 7 8 9 10

Wildtype Line3 MommeD6-/+ Wildtype Line3 MommeD6-/+

Figure 3.9 Northern analysis of GFP mRNA in wildtype Line3 and MommeD6-/+ mice hemizygous for the GFP transgene. (a) PolyA+ mRNA obtained from the spleens of three individual wildtype Line3 (1-3) and three individual MommeD6-/+ (4-6) three week old males was separated on a 1.2% agarose gel, transferred to a nylon membrane and hybridised to a GFP probe. A GAPDH probe was used as a loading control. The experiment was then repeated using PolyA+ mRNA from the spleens of two wildtype Line3 (7 and 8) and two MommeD6-/+ (9 and 10) six week old males. (b) The ratio of GFP mRNA levels to GAPDH mRNA levels was calculated for each sample, so that the different mice could be directly compared. The individual ratios are given for each sample. The MommeD6-/+ mice generally had higher levels of GFP mRNA, with a trend towards statistical significance (p=0.15 and 0.04, respectively for samples 4-6 and samples 9 and 10). Because the experiments were done on different days with different probes and exposure times the ratios across the two experiments cannot be directly compared. (All p-values are for a T-test)

3.2.8 Linkage analysis

Linkage analysis of the MommeD6 mutation was performed by backcrossing to C57 mice, and the detailed breeding scheme is shown in Figure 3.10. The difference in mean fluorescence between MommeD6+/+ and MommeD6-/+ offspring was still seen in the C57 background, so mice could be phenotyped into the two classes relatively easily (Table 3.4). An initial mapping screen had been performed on 67 F2 offspring

by Sarah Hemley (a research assistant) using 61 microsatellite markers, and she failed to identify a linked region (data not shown). In light of this, when I resumed the mapping I backcrossed a further generation and used 43 F3 mutant mice in a preliminary genome-wide screen using 75 microsatellite markers (Appendix 1), and then used both the F2 and F3 offspring to further narrow down the linked interval.

Founders x

MommeD6-/+ MommeD6+/+ GFP/GFP -/- FVB C57

F1 x

MommeD6+/+ MommeD6-/+ MommeD6+/+ GFP/- GFP/- -/- FVB/C57 FVB/C57 C57

F2 x MommeD6+/+ MommeD6+/+ MommeD6-/+ MommeD6-/+ MommeD6+/+ -/- GFP/- -/- GFP/- -/- FVB+C57 FVB+C57 FVB+C57 FVB+C57 C57 mixture mixture mixture mixture

MommeD6+/+ MommeD6+/+ MommeD6-/+ MommeD6-/+ -/- GFP/- -/- GFP/- FVB+C57 FVB+C57 FVB+C57 FVB+C57 mixture mixture mixture mixture

Figure 3.10 Backcross breeding scheme for mapping the MommeD6 mutation. -/+ MommeD6 mice were crossed to C57 mice. F1 mice heterozygous for the MommeD6 mutation, as determined by flow cytometry, were then backcrossed to C57 mice. Half of the resulting F2 mice did not carry the GFP transgene and so could not be phenotyped. F2 mice heterozygous for the MommeD6 mutation were used for mapping (circled), and were backcrossed again to C57 mice. Once again, half of the resulting F3 mice did not carry the GFP transgene and could not be phenotyped. F3 mice heterozygous for the MommeD6 mutation were used for mapping (circled).

Table 3.4 Percentage of cells expressing the transgene and mean fluorescence of -/+ +/+ expressing cells for MommeD6 and MommeD6 F2 and F3 mice used for mapping. A sample of peripheral blood was analysed at weaning. The mean ± one standard deviation of the percentage of cells expressing the transgene and mean fluorescence of those expressing -/+ cells are shown. F2 and F3 MommeD6 mice had a higher mean fluorescence than MommeD6+/+ mice in the mixed background and so could be reliably phenotyped by flow cytometry.

Percentage of Mean fluorescence Sex Genotype n cells expressing of the expressing the transgene cells

MommeD6 +/+ 169 31.51 ± 4.42 229.49 ± 38.67 Males MommeD6 -/+ 112 37.97 ± 4.28 404.51 ± 68.18

MommeD6 +/+ 126 30.71 ± 4.40 232.12 ± 36.25 Females MommeD6 -/+ 134 38.58 ± 5.07 411.53 ± 62.05

MommeD6 +/+ 295 31.16 ± 4.42 230.61 ± 37.61 Combined MommeD6 -/+ 246 38.30 ± 4.72 408.34 ± 64.88

Using a combination of microsatellite markers and single nucleotide polymorphisms

(SNPs) on 255 MommeD6-/+ mice, the mutation was eventually linked to a 2.5 Mbp region on chromosome 14 between the markers rs13482101 (24299096 bp) and rs6396829 (26827847 bp) (Figure 3.11). The two mice that define the interval at rs6396829 were progeny tested and they were shown to transmit the mutation to their offspring, which indicated that they were true mutants. The single mouse that defines the interval at rs13482101 was not progeny tested. However, one of the mice defining the interval one marker out from the smallest linked interval at rs13482099 (23609247 bp) was progeny tested and was found to be a true mutant. This more conservative interval, as defined by the markers rs13482099 and rs6396829, is 3.2 Mbp in size.

(a) A B C D E Mbp D14Mit11 8.7

D14Mit50 17.3

D14Mit221 19.8

D14Mit14 25.7

D14Mit60 39.8

D14Mit18 40.5

D14Mit68 64.1

D14Mit195 79.9

D14Mit264 92.8

D14Mit97 111.5

Number of Mice 10 6 1 9 6 1 8 1 1 13 2 2 1 6 4 2 1 2 3 17 12 137 7 3

(b) Mbp D14Mit221 22.3

rs13482099 23.6 C57/C57 Genotype

rs13482101 24.3 FVB/C57 Genotype

rs13482102 24.4

rs6402490 26.2

rs13482109 26.5

rs6396829 26.8

D14Mit14 28.5

Number of mice 2 2(2) 1 5(1) 4

Figure 3.11 Mapping data for MommeD6. The microsatellite markers and SNPs that were used are displayed alongside their approximate base pair location. Marker results are depicted as shaded boxes, with a black box being a C57/C57 genotype and a white box being an FVB/C57 genotype. The mutation is associated with the FVB genome, so an FVB/C57 genotype indicates that the mutation could be present at that location. (a) Crude mapping results for 255 MommeD6-/+ mice on chromosome 14. The mutation was found to be located between the microsatellite markers D14Mit221 and D14Mit14, as defined by 14 unique recombinant mice (red boxes). Mice are grouped (A-E) according to the number of markers used. Fewer markers needed to be used as the interval size was narrowed. (b) Fine mapping results of those 14 recombinants between D14Mit221 and D14Mit14. The linked interval was refined to between the SNPs rs13482101 and rs6396829, and was defined by three unique recombinant mice (red boxes). Superscript numbers indicate the number of mice with that genotype that were progeny tested and found to be true mutants.

The 2.5 Mbp interval contains 43 genes (Ensembl database version 46, NCBI build m36) (Table 3.5a). Six candidates were identified in the linked interval. Cphx,

1110051B16Rik and Hesx1 are homeobox genes, which are important transcription factors usually involved in early development (Stein et al., 1996). SNORA71 and

SNORA67 encode untranslated, small nucleolar RNAs (snoRNAs), which have been shown to play a role in the processing of mRNAs (Bachellerie et al., 2002), which is consistent with the idea that MommeD6 may be having a post-transcriptional rather than a transcriptional effect. Appl1 has links to the nucleosomal remodelling and histone deacetylase (NuRD) complex (Feng and Zhang, 2003; Zhang et al., 1999). All six candidate genes were sequenced at all exons, including exon/intron junctions and splice sites (Figure 3.12 and Appendix 1). No mutations were identified in any of these genes in the mutant animals. If we are to take the more conservative interval as defined by the markers rs13482099 and rs6396829, this 3.2 Mbp region contains an extra two genes compared with the smaller 2.5 Mbp interval, neither of which are obvious candidates (Table 3.5b).

Table 3.5 Genes in the interval linked to the MommeD6 mutation. (a) The smallest linked interval is ~2.5 Mbp in size, and is located between the SNPs rs13482101 and rs6396829. Genes that were sequenced at all exons and exon/intron boundaries are highlighted. A genomic region that appears to be triplicated is boxed. (b) A more conservative linked interval, ~3.2 Mbp in size, is located between the SNPs rs13482099 and rs6396829. Two extra genes are located in this interval.

Gene start (a) Ensembl gene ID External gene ID Description (bp) 24420451 ENSMUSG00000072685 4931406H21Rik RIKEN cDNA 4931406H21 gene 24498392 ENSMUSG00000072684 ENSMUSG00000072684 predicted gene, ENSMUSG00000072684 24527338 ENSMUSG00000021868 Ppif peptidylprolyl isomerase F (cyclophilin F) 24530158 ENSMUSG00000051186 24539690 ENSMUSG00000057293 1700054O19Rik RIKEN cDNA 1700054O19 gene 24544833 ENSMUSG00000055538 2310047A01Rik 24661180 ENSMUSG00000077081 24675354 ENSMUSG00000021866 Anxa11 annexin A11 24689961 ENSMUSG00000052323 ENSMUSG00000052323 predicted gene, ENSMUSG00000052323 24722072 ENSMUSG00000072681 Plac9 placenta specific 9 24757182 ENSMUSG00000072680 D14Ertd449e Uncharacterized protein C10orf57 homolog 24775728 ENSMUSG00000072678 Cphx cytoplasmic polyadenylated homeobox 24816189 ENSMUSG00000048502 1110051B16Rik RIKEN cDNA 1110051B16 gene 24861842 ENSMUSG00000043248 Plac9 placenta specific 9 24897724 ENSMUSG00000072676 D14Ertd449e Uncharacterized protein C10orf57 homolog 24956302 ENSMUSG00000072675 1110051B16Rik RIKEN cDNA 1110051B16 gene 25001456 ENSMUSG00000072674 Plac9 placenta specific 9 25036551 ENSMUSG00000021867 D14Ertd449e Uncharacterized protein C10orf57 homolog 25055124 ENSMUSG00000049982 Cphx cytoplasmic polyadenylated homeobox 25095951 ENSMUSG00000072672 1110051B16Rik RIKEN cDNA 1110051B16 gene 25190731 ENSMUSG00000021874 4933413J09Rik RIKEN cDNA 4933413J09 gene 25246352 ENSMUSG00000021870 Slmap sarcolemma associated protein 25330839 ENSMUSG00000065864 SNORA71 Small nucleolar RNA SNORA71 25412762 ENSMUSG00000040818 A630054L15Rik RIKEN cDNA A630054L15 gene 25461591 ENSMUSG00000053165 ENSMUSG00000053165 predicted gene, ENSMUSG00000053165 25471381 ENSMUSG00000021877 Arf4 ADP-ribosylation factor 4 25497299 ENSMUSG00000043702 E430028B21Rik RIKEN cDNA E430028B21 gene 25502333 ENSMUSG00000053922 E430028B21Rik RIKEN cDNA E430028B21 gene 25526596 ENSMUSG00000021879 4921531P07Rik RIKEN cDNA 4921531P07 gene 25597915 ENSMUSG00000064221 Dnahc12 dynein, axonemal, heavy chain 12 25625669 ENSMUSG00000072668 Dnahc12 dynein, axonemal, heavy chain 12 25631504 ENSMUSG00000072667 25721486 ENSMUSG00000021898 Asb14 ankyrin repeat and SOCS box-containing protein 14 adaptor protein, phosphotyrosine interaction, PH domain 25745872 ENSMUSG00000040760 Appl1 and leucine zipper containing 1 25827246 ENSMUSG00000040726 Hesx1 homeo box gene expressed in ES cells 25865966 ENSMUSG00000040717 Il17rd interleukin 17 receptor D 25933580 ENSMUSG00000068643 26064923 ENSMUSG00000021895 Arhgef3 Rho guanine nucleotide exchange factor (GEF) 3 Adult male testis cDNA, RIKEN full-length enriched library, clone:4933409E02 product:similar to 26255731 ENSMUSG00000040651 D14Abb1e RETINOBLASTOMA-ASSOCIATED PROTEIN RAP140 (Fragment) 26309285 ENSMUSG00000046753 Ccdc66 coiled-coil domain containing 66 26354635 ENSMUSG00000064836 SNORA67 Small nucleolar RNA SNORA67 26378072 ENSMUSG00000045240 26449326 ENSMUSG00000040640 Erc2 ELKS/RAB6-interacting/CAST family member 2

Gene start (b) Ensembl gene ID External gene ID Description (bp) 23746362 ENSMUSG00000072686 ENSMUSG00000072686 predicted gene, ENSMUSG00000072686 24292476 ENSMUSG00000007817 Zmiz1 zinc finger, MIZ-type containing 1

Cphx 13.88 Kbp

1110051B16Rik 6.39 Kbp

Hesx1 1.97 Kbp

SNORA71 131 bp

SNORA67 135 bp

Appl1 51.56 Kbp

Figure 3.12 Diagramatic maps of genes sequenced in the MommeD6 linked interval. The boxes represent the exons, with black shading indicating that the transcript is translated and no shading indicating an untranslated region. The approximate lengths of each transcript are shown above the maps. The short lines underneath the maps represent the regions sequenced by individual sets of primer pairs. A minimum of 50 bp was sequenced beyond the exon/intron junctions. Maps are not drawn to scale.

3.3 Discussion

MommeD6 is a semi-dominant, homozygous lethal mutation, with occasional premature death of heterozygous mutants. Homozygous mutants display abnormal embryonic development, dying between 10.5 and 14.5 dpc. The MommeD6 mutation shows TRD following crosses with wildtype Line3 mice, with fewer mutants than wildtypes at weaning. Embryo analysis of heterozygous intercrosses suggests

heterozygous mutants are dying in utero at the same time as homozygous mutants.

This can be tested more definitively when the mutation for MommeD6 has been identified.

With respect to the TRD, females are more affected by the MommeD6 mutation than males following paternal transmission. The effect appears to be occurring prior to mid-gestation, which is when the embryo starts to undergo sex-specific developmental changes. This suggests a link with the inheritance of an X chromosome and X-inactivation. There may be a failure of meiotic sex chromosome inactivation (MSCI) in the male gametes (Turner, 2007), resulting in a failure of imprinted X-inactivation in the extra-embryonic tissues and a subsequent failure of the placenta to develop. Disruption of imprinting has previously been linked to grandparental origin dependent TRD, with the TRD specific to disrupted imprinted regions critical for embryo survival (Croteau et al., 2002; Naumova et al., 2001).

Further investigation of this hypothesis will require the analysis of inactivation of the

X chromosome at early developmental time points; such as in the sperm, fertilised zygote and early embryo. Again, these experiments require knowledge of the point mutation in order to genotype for the MommeD6 mutation.

The MommeD6 mutation results in a unique expression profile for a Momme mutant; it increases mean fluorescence without greatly affecting the percentage of expressing cells. The fact that MommeD6 has little effect on penetrance at the Avy allele supports the idea that the mutation is not having a large impact on stochastic gene silencing.

Heterozygosity for all other MommeDs tested so far, has been found to influence expression at the Avy allele (Blewitt et al., 2005; Chong et al., 2007). Moreover, the

increase in GFP mRNA levels in MommeD6-/+ mice is subtle at best, which argues for the mutation having at least some post-translational effect. Indeed, one could imagine a scenario where the primary event is the stabilisation of the GFP protein.

Erythrocytes in the mouse have a life cycle of ~40 days (Van Putten, 1958). If we assume that the GFP protein is stable for a shorter period of time than the life cycle of erythrocytes, then a mutation that increases the stability of the GFP protein would result in a higher percentage of erythrocytes with GFP protein at any given moment in time. In other words, the increase in the fluorescence level of an expressing cell could be indirectly resulting in a slight increase in the percentage of expressing cells.

Unfortunately the sequencing of candidate genes within the linked interval has so far proved fruitless. The selection of candidate genes was based on their known functional domains and biological/biochemical activities, which limited selection to at least partially characterised genes. There are many genes in the interval that do not have immediately recognisable domains, and there are several that have no known functions. Obviously we cannot exclude these genes, but at the same time it is difficult to select the next candidate gene.

One problem with the sequencing is that it was done using heterozygous mutants because homozygous mutants die so early in development. This meant that any point mutation would appear as a double peak in the chromatogram readout, which makes it easier to miss a mutation. This is a particularly important issue for the group of genes which are triplicated or duplicated. Cphx is apparently duplicated and

1110051B16Rik is apparently triplicated. At the time of sequencing these genes, the

Ensembl database did not show a triplication of the region. This was added to the

database later. This would obviously confound sequencing of these genes. However, if there was a mutation in one of these genes then one might not expect a lethal phenotype in homozygous mutants because the remaining paralogue(s) should still be functional. Only in the rare instance of either a gain of function mutation or a dominant negative mutation would such a situation occur.

One approach which should be pursued is to carry out Northern analysis on candidate genes to check for differences in transcript levels or sizes. Either way it is critical that the point mutation be found, so that further investigation can be carried out.

Chapter 4 - MommeD9

Abstract

MommeD9 is a semi-dominant, homozygous lethal mutation and an enhancer of variegation, decreasing the expression of a GFP transgene. The effect of this mutation is to decrease the percentage of cells expressing the transgene, and in this regard it behaves like a classic enhancer of variegation. Heterozygosity for the mutation results in obesity, immune dysfunction and female infertility. The mutation lies in a 17.4

Mbp interval on chromosome 7.

4.1 Introduction

MommeD9 was identified in a dominant ENU screen carried out by Dr N.

Vickaryous. The mutant line was immediately passed to me, and all work on this mutant was done by me. I have carried out extensive breeding studies, and I have studied various phenotypes associated with homozygosity and heterozygosity for the

MommeD9 mutation using a variety of techniques. I have initiated linkage analysis to identify the underlying mutation.

4.2 Results

4.2.1 Flow cytometry profile

The data presented in Figure 4.1 and Table 4.1 shows the percentage of cells expressing the GFP transgene and the mean fluorescence of those cells in peripheral blood of offspring from crosses between heterozygous mutants (MommeD9-/+) and wildtype Line3 mice. MommeD9 is an enhancer of variegation. MommeD9 heterozygous mutants display a significantly lower percentage of peripheral blood cells that are expressing the transgene, as well as a significant decrease in the mean fluorescence of those expressing cells compared with that of wildtype Line3 mice

(Figure 4.1 and Table 4.1). There appears to be a small sex effect in MommeD9 offspring, with males having a lower percentage of expressing cells and mean fluorescence. However, as mentioned in Chapter 3, there is a similar trend in the wildtype Line3 colony (Table 3.1).

100

60 Counts 40 M1

1 100 101 102 103 104 GFP Fluorescence

Figure 4.1 Histogram of GFP fluorescence analysed by flow cytometry for the MommeD9 mutation. The flow cytometry profiles of peripheral blood of four MommeD9+/+ (green line) and four MommeD9-/+ (red line) mice at weaning from a representative litter are overlayed. Due to slight day to day fluctuations with the flow cytometer, data collected on different days should not be combined. The data shown are representative of that seen in 50 litters. The x-axis is an arbitrary fluorescence scale and the y-axis indicates the number of cells counted. A gate (M1) is used to determine which cells are fluorescing, and is set to exclude 99.9% of autofluorescing cells. MommeD9-/+ mice have significantly less cells fluorescing and a lower mean fluorescence than MommeD9+/+ mice.

Table 4.1 Percentage of cells expressing the transgene and the mean fluorescence of expressing cells for MommeD9-/+ and MommeD9+/+ mice. A sample of peripheral blood was analysed at weaning. The mean ± one standard deviation of the percentage of cells expressing the transgene and mean fluorescence of those expressing cells are shown. MommeD9-/+ mice had a significantly lower percentage of expressing cells (p<<0.001) and mean fluorescence (p<<0.001) than their MommeD9+/+ littermates. (All p-values are for a T-test)

Percentage of Mean fluorescence Sex Genotype n cells expressing of the expressing the transgene cells

MommeD9 +/+ 86 52.96 ± 4.76 268.58 ± 51.58 Males MommeD9 -/+ 69 35.90 ± 5.15 185.99 ± 34.55

MommeD9 +/+ 83 56.41 ± 5.17 286.35 ± 43.77 Females MommeD9 -/+ 64 36.93 ± 4.65 197.78 ± 31.25

Combined MommeD9 +/+ 169 55.16 ± 5.10 277.31 ± 48.59 sexes MommeD9 -/+ 133 36.39 ± 4.92 191.66 ± 33.41

4.2.2 MommeD9 is semi-dominant and homozygous lethal

The offspring of heterozygous intercrosses of MommeD9-/+ mice revealed only two classes with respect to GFP expression (Figure 4.2 and data not shown) suggesting that homozygous offspring do not survive to weaning. The ratio of non-mutant to mutant offspring is more consistent with a semi-dominant, homozygous lethal mutation than with a dominant mutation (Table 4.2). There was also a significant litter size reduction (5.1 mice/litter) compared to wildtype Line3 mice (8.6 mice/litter)

(Figure 4.3). In combination these results suggest that homozygous mutants

(MommeD9-/-) are not viable, and that MommeD9 is a semi-dominant, homozygous lethal mutation. Consistent with this hypothesis, progeny testing of four female and four male mutant offspring from MommeD9-/+ intercrosses found them to be heterozygous for the mutation, i.e. in all cases offspring were produced that expressed the transgene at wildtype levels (data not shown).

100

60 Counts 40 M1

1 100 101 102 103 104 GFP Fluorescence

Figure 4.2 Histogram of GFP fluorescence analysed by flow cytometry of offspring from MommeD9 heterozygous intercrosses. The flow cytometry profiles of peripheral blood of three wildtype (green line) and two mutant (red line) mice at weaning from a representative litter are overlayed. Due to slight day to day fluctuations with the flow cytometer, data collected on different days should not be combined. The data shown are representative of that seen in 11 litters. The x-axis is an arbitrary fluorescence scale and the y-axis indicates the number of cells counted. A gate (M1) is used to determine which cells are fluorescing, which is set to exclude 99.9% of autofluorescing cells. Only two classes were observed.

Table 4.2 Percentage of wildtype and mutant offspring from MommeD9 heterozygous intercrosses. Offspring were phenotyped by flow cytometry. The numbers of offspring observed were significantly different from those expected for a dominant, homozygous viable mutation, but were consistent with those expected for a semi-dominant, homozygous lethal mutation. The expected numbers for a dominant mutation are based on Mendelian ratios. The expected numbers for a semi-dominant, homozygous lethal mutation are based on the ratio if the homozygous mutants are absent. (All p-values are for a Chi-squared test)

Expected numbers Expected numbers for a semi-dominant, Phenotype Observed numbers for a dominant homozygous lethal mutation mutation Wildtype 25 14 (25%) 19 (33%) Mutant 31 42 (75%) 37 (67%) p-value <0.001 0.07

** * * 14

8 8.6 7.2 6.8 6

Litter size Litter 5.1 4

0 Wildtype Line3 MommeD9-/+ sire x MommeD9-/+ dam x MommeD9-/+ Wildtype Line3 Wildtype Line3 intercross

(n=67) (n=34) (n=17) (n=11)

Figure 4.3 Litter size of MommeD9-/+ crosses compared to that of wildtype Line3. The boxes represent the mean ± one standard deviation with the mean in the middle of the box, and the whiskers show the highest and lowest observed values. MommeD9-/+ x Line3 pairs had significantly smaller litter sizes compared to wildtype Line3, and MommeD9-/+ intercrosses showed an even larger difference. This argues for the death of MommeD9-/- mice and the stochastic death of the some MommeD9-/+ mice. (n = number of litters observed, * indicates p<0.05 and ** indicates p<0.001) (All p-values are for a T-test)

The percentage of non-mutant to mutant offspring following heterozygous intercrosses was 45% and 55% respectively. Although this ratio was not statistically significantly different from the expected 1:2 ratio of a semi-dominant, homozygous lethal mutation, there was a shift towards fewer mutant offspring than expected

(p=0.07) (Table 4.2). This is consistent with the fact that the litter size (5.1 mice/litter) was lower than would be expected if only MommeD9-/- mice were dying. A litter size of ~6.5 mice/litter would have been expected if only MommeD9-/- mice died (Figure

4.3). This suggests that there is stochastic loss of MommeD9-/+ mutants, and I have followed this up by analysing in more detail the crosses between heterozygous mutants and wildtype Line3 mice.

4.2.3 MommeD9 shows transmission ratio distortion (TRD)

In crosses between MommeD9-/+ and wildtype Line3 mice, a significant litter size reduction was observed at weaning following both paternal (7.2 mice/litter; p<0.05) and maternal (6.8 mice/litter; p<0.05) transmission of the mutation (Figure 4.3). This suggested that some heterozygous individuals were dying post-implantation and prior to weaning.

Following maternal transmission there was a significant loss of MommeD9-/+ mice when both sexes were grouped (p<0.05), and this appeared to be occurring in both males and females, although it was not statistically significant in either case (Figure

4.4a). Following paternal transmission of the MommeD9 mutation there was no statistically significant TRD in either male or female offspring, but there was a trend towards a loss of MommeD9-/+ mice in both cases (Figure 4.4b). The extent of the

TRD may explain the litter size reduction observed following maternal transmission,

but the extent of the TRD following paternal transmission is insufficient to explain the apparent litter size reduction in this case. This could mean that there is stochastic loss of both mutant and wildtype offspring following paternal transmission.

(a) Maternal transmission of MommeD9 140 120 100 * 80 70 MommeD9 +/+ 60 46 37 MommeD9 -/+ 40 33 Number ofmice Number 26 20 20 0 Total Males Females

(n=116) (n=63) (n=53)

(b) Paternal transmission of MommeD9

140 133 120 113 100 80 71 57 62 56 MommeD9 +/+ 60 MommeD9 -/+

40 Number ofmice Number 20 0 Total Males Females

(n=246) (n=128) (n=118)

Figure 4.4 Transmission ratio distortion following maternal but not paternal transmission of MommeD9. (a) Following maternal transmission of the MommeD9 mutation there was a significant loss of MommeD9-/+ offspring. There was no sex-specific effect, with the loss occurring in both male and female offspring, although it was only significant when the sexes were combined. (b) Following paternal transmission there was a trend toward loss of MommeD9-/+ mice. There was no significant difference in the overall number of male and female offspring following paternal or maternal transmission. (* indicates p<0.05 and ** indicates p<0.001) (All p-values are for a Chi-squared test)

4.2.4 MommeD9 homozygous mutants die in utero

Embryos from MommeD9-/+ intercrosses were studied at 14.5 and 10.5 dpc. At 14.5 dpc approximately 41% of the embryos were resorbed, implying that they had died and were macrophage invaded (Table 4.3a). Presumably the MommeD9-/- mutants made up the majority of these embryos. However, since MommeD9-/- embryos would be expected to comprise only one-quarter of the total litter, some death of MommeD9-

/+ mutants could explain the remaining embryos. At 10.5 dpc no resorbed embryos were observed, and approximately 36% appeared to be abnormal or developmentally delayed by 1-2 days (Table 4.3b). Figure 4.5 shows representative examples of normal and abnormal embryos at 10.5 dpc. The observed abnormal embryos vary in size and developmental stage. These embryos are likely to represent the same group of embryos that are resorbed at 14.5 dpc.

Table 4.3 Percentage of abnormal embryos from MommeD9-/+ intercrosses at 14.5 and 10.5 dpc. (a) At 14.5 dpc 41% of embryos from MommeD9-/+ intercrosses were resorbed, compared with 6% from wildtype Line3 crosses. The resorbed embryos presumably comprised the dead MommeD6-/- and MommeD9-/+ embryos. (b) At 10.5 dpc 36% of embryos from MommeD6-/+ intercrosses looked significantly smaller and probably developmentally delayed, compared with 6% from wildtype Line3 crosses. (a) 14.5 dpc

MommeD9 -/+ intercrosses Wildtype Line3 crosses (n = 3) (n = 5) Normal 19 (59.4%) 44 (93.6%) Resorbed 13 (40.6%) 3 (6.4%) Total 32 47 Average number/litter 10.7 9.4

(b) 10.5 dpc

MommeD9 -/+ intercrosses Wildtype Line3 crosses (n = 3) (n = 4) Normal 16 (64%) 34 (94.4%) Abnormal 9 (36%) 2 (5.6%) Total 25 36 Average number/litter 8.3 9

(a) (b)

3 mm 3 mm

Figure 4.5 Comparisons of normal and abnormal 10.5 dpc embryos from MommeD9-/+ intercrosses. (a) The normal 10.5 dpc embryo (left) is much larger than its abnormal littermate (right). (b) A normal embryo (left) and a normal but smaller littermate (right).

4.2.5 MommeD9 heterozygous mutants show premature death

MommeD9-/+ adult mice were occasionally found to die prematurely for no obvious reason, even when they appeared perfectly healthy a few days before. This occurred more often in females (n=11) than males (n=6). The average age at death was similar between males and females, 18.9 ± 8 and 17.8 ± 7 weeks, respectively. Basic necropsies were performed on several of the deceased mice, but nothing grossly abnormal was found. However, in some cases the mice appeared to have more adipose tissue than expected.

4.2.6 MommeD9-/+ mutants become obese

MommeD9-/+ mice weighed slightly less than their MommeD9+/+ littermates at three weeks of age (weaning) (Figure 4.6). Interestingly by 9 weeks of age the MommeD9-/+ offspring were heavier than their MommeD9+/+ littermates, and this difference was more noticeable at 21 weeks of age (Figure 4.7). This weight gain was seen in both male and female offspring. At 21 weeks of age female MommeD9-/+ mice displayed the largest weight gain, although their weight was surprisingly variable as indicated by the large standard deviation. This analysis was carried out on relatively few individuals and should be repeated with a larger sample size.

Because MommeD9-/+ mice were becoming obese, it was hypothesised that they might be diabetic. One crude test of diabetes is to look for glucose in urine. The urine of female MommeD9-/+ mice at 46 weeks (n=2), 19 weeks (n=3), and 11 weeks (n=2), and wildtype Line3 mice at similar ages (43 weeks, n=3; 19 weeks, n=1; 12 weeks, n=2) was tested for the presence of glucose. No detectable levels of glucose were

found in any samples (data not shown). However, it would have been useful to have diabetic control mice with which to compare.

18 * 16 14 12 10.4 10 9.5 10.0 9.4 8

6 Body weight (g) Bodyweight 4 2 0 Male Male Female Female MommeD9+/+ MommeD9-/+ MommeD9+/+ MommeD9-/+

(n=62) (n=45) (n=68) (n=46)

Figure 4.6 Weight of MommeD9-/+ mice at weaning. Both male and female MommeD9-/+ mice weighed less than their counterpart MommeD9+/+ littermates. The boxes represent the mean ± one standard deviation with the mean in the middle of the box, and the whiskers show the highest and lowest observed values. (* indicates p<0.05) (All p-values are for a T-test)

(a) Males 45 * 40 35 30 25 20 * MommeD9+/+ 15

Body weightBody (g) MommedD9-/+ 10 5 0 0 2 4 6 8 10 12 14 16 18 20 22 Age (weeks)

(b) Females 50 * 45 40 35 30 25 20 MommeD9+/+ 15

Body weight Body(g) MommedD9-/+ 10 5 0 0 2 4 6 8 10 12 14 16 18 20 22 Age (weeks)

Figure 4.7 Weight of MommeD9-/+ mice as a function of age. Although (a) male (n=45) and (b) female (n=46) MommeD9-/+ mutants weighed less than their wildtype littermates (n=62 and n=68; males and females respectively) at three weeks of age, both male (n=3) and female (n=3) MommeD9-/+ mice weighed more than their wildtype littermates (n=6 and n=3; males and females respectively) by 9 weeks of age. This difference in weight becomes even more pronounced by 21 weeks of age (MommeD9-/+ n=4 and n=6; males and females respectively) (MommeD9+/+ n=2 and n=4; males and females respectively). The error bars are ± one standard deviation from the mean. (* indicates p<0.05) (All p-values are for a T-test)

4.2.7 Haematological analysis

To investigate the premature death further, full blood counts were performed on

MommeD9-/+ and control wildtype Line3 mice. Blood was collected at 9 weeks and 21 weeks of age. At 9 weeks both male and female MommeD9-/+ mice displayed lower numbers of leukocytes, but at 21 weeks they had increased numbers (Figure 4.8a).

This increase in leukocytes was limited to lymphocytes (although the increase was only significant in male offspring) (data not shown), however since lymphocytes are the major type of leukocyte this is not surprising. Interestingly the erythrocyte count and levels of haemoglobin were consistently lower in MommeD9-/+ mice compared with controls at both 9 and 21 weeks (Figure 4.8b and c). This suggests that

MommeD9-/+ mice are mildly anaemic, though none of the results were statistically significant.

Since the peripheral blood analysis suggested haematological abnormalities in the heterozygous mutants, the spleen, another organ involved in haematopoiesis was also studied. Spleens from MommeD9-/+ and wildtype Line3 mice were examined at 9 weeks and 21 weeks of age. Preliminary analysis showed that there was a trend in both males and females towards heavier spleens in MommeD9-/+ mice (Table 4.4).

This was statistically significant in females at 21 weeks. The ratios of spleen weight to body weight revealed that female mutants at 21 weeks of age had much higher body weights in relation to the weight of their spleen compared to wildtype Line3 mice (Table 4.4).

(a) Leukocytes 20 *

L 15 m

10 cells/

3 5 10 0 Male Female Male Female

9 weeks 21 weeks

(b) Erythrocytes (c) Haemoglobin 14 20 12

L 15

m 10

8 10 g/dL

cells/ 6 6 4 5 10 2 0 0 Male Female Male Female Male Female Male Female

9 weeks 21 weeks 9 weeks 21 weeks

Wildtype Line3 -/+ MommeD9

Figure 4.8 Haematological analysis of MommeD9-/+ mice. Full blood counts were performed on MommeD9-/+ and wildtype Line3 mice. (a) Between 9 and 21 weeks the number of leukocytes increased in MommeD9-/+ mutants, compared to wildtype Line3 mice where the number decreased. (b and c) MommeD9-/+ mutants had consistently lower numbers of erythrocytes and lower levels of haemoglobin than wildtype Line3 mice. Numbers of mice: 9 weeks, wildtype males (n=4) and females (n=3), MommeD9-/+ males (n=3) and females (n=1); 21 weeks, wildtype males (n=2) and females (n=4), MommeD9-/+ males (n=4) and females (n=3). The error bars are ± one standard deviation from the mean. (* indicates p<0.05) (All p- values are for a T-test)

Table 4.4 Spleen weights and body weights at 9 and 21 weeks for MommeD9-/+ and wildtype Line3 mice. Mean values ± one standard deviation are shown. MommeD9-/+ mutants had heavier spleens than wildtype Line3 mice. When analysed as ratios with body weight, it was females at 21 weeks of age that showed the most significant difference indicating that their body weight had increased more than their spleen weight. (All p-values are for a T-test) (Note: Only one female MommeD9-/+ mouse was analysed at 9 weeks of age, so 9 week old females were not included in analysis. Also, only two male wildtype Line3 mice were analysed at 21 weeks of age, so 21 week old males were not included in analysis.)

Spleen Weight : Age Sex Genotype n Spleen Weight (g) p-value Body Weight (g) p-value p-value Body Weight

Wildtype Line3 6 0.087 ± 0.009 26.44 ± 3.06 0.0028 9 weeks Male 0.71 0.12 0.05 MommeD9 -/+ 3 0.089 ± 0.005 29.76 ± 0.92 0.0022

Wildtype Line3 4 0.106 ± 0.006 28.19 ± 1.12 0.0038 21 weeks Female 0.04 <<0.001 0.001 MommeD9 -/+ 3 0.127 ± 0.014 45.37 ± 1.27 0.0028

4.2.8 Female MommeD9-/+ mutants have reduced fertility

It was noticed that female MommeD9-/+ mutants consistently bred poorly, independent of the strain of mouse with which they were being mated. These fertility problems were not seen with MommeD9-/+ males. In inbred crosses involving a MommeD9-/+ dam and a wildtype Line3 sire, the average number of litters produced over the lifetime of the breeding pair (1.8 litters) was significantly less than observed for the reciprocal cross (5.3 litters; p<0.05) and wildtype to wildtype crosses (5 litters; p<0.05) (Figure 4.9). Each of these breeding pairs was set up for a minimum of 6 months, with the average time spent together being 32 weeks for MommeD9-/+ dams crossed with wildtype Line3 mice, 35 weeks for MommeD9-/+ sires crossed with wildtype Line3 mice, and 33 weeks for wildtype Line3 intercrosses.

MommeD9-/+ females were set up in breeding pairs between six and 14 weeks of age, and there was no indication that their age when set up influenced the number of litters produced. However, none of these MommeD9-/+ females produced a litter beyond 21 weeks of age, so there probably is an age-related effect in regards to the reduced fertility (Figure 4.10).

* 9 * 8 7 6 5 5.0 5.3 4 3

Number ofLitters Number 2 1.8 1 0 Wildtype Line3 MommeD9-/+ sire x MommeD9-/+ dam x Wildtype Line3 Wildtype Line3

(n=7) (n=6) (n=5)

Figure 4.9 Mean number of litters born to MommeD9-/+ breeding pairs. The boxes represent the mean ± one standard deviation with the mean in the middle of the box, and the whiskers show the highest and lowest observed values. The „n‟ is the number of breeding pairs. MommeD9-/+ dams crossed with wildtype Line3 mice produced fewer litters than either the reciprocal crosses or wildtype Line3 intercrosses. (* indicates p<0.05) (All p-values are for a T-test)

2 Litter number Litter 1

0 0 5 10 15 20 25 30 35 40 45 50 Age of dam (weeks)

♀ 1 ♀ 2 ♀ 3 ♀ 4 ♀ 5 ♀ 6

Figure 4.10 Ages of six MommeD9-/+ dams at the time of birth of each litter. Each coloured line (with its associated unique coloured symbol) represents an individual female, with a step up of the line indicating the birth of a litter. Each dam was set up with a wildtype Line3 sire at between six and 14 weeks of age, as indicated by the first occurrence of each coloured symbol. No MommeD9-/+ dams gave birth beyond 21 weeks of age.

4.2.9 Linkage analysis

Linkage analysis of the MommeD9 mutation was carried out by backcrossing to C57

-/+ mice, and the detailed breeding scheme is shown in Figure 4.11. F2 MommeD9 mutants could be distinguished from MommeD9+/+ mice by flow cytometry, as they retained a significantly lower percentage of expressing cells and mean fluorescence in the mixed background (Table 4.5). Using 67 F2 mice the mutation was narrowed down to a ~17.4 Mbp region on chromosome 7 between the SNP rs38424645

(9623600 bp) and the microsatellite marker D7Mit294 (26998202 bp) (Figure 4.12).

The linked interval is defined by four recombinants at the end constrained by

D7Mit294, and one recombinant at the end constrained by rs38424645.

Unfortunately, none of these recombinants were progeny tested to confirm if they were true mutants.

The linked interval contained 380 genes (Ensembl database version 46, NCBI build m36) (Appendix 2). One gene did stand out as a good candidate for the MommeD9 mutation; chromatin modifying protein 2A (chmp2a). Sequencing at all exons, including exon/intron junctions and splice sites revealed no point mutations in chmp2a in the mutant animals (Figure 4.13). No other genes in the interval were considered strong enough candidates for sequencing. More F2 mice need to be produced in order to narrow the linked interval.

Founders x

MommeD9-/+ MommeD9+/+ GFP/GFP -/- FVB C57

F1 x

MommeD9+/+ MommeD9-/+ MommeD9+/+ GFP/- GFP/- -/- FVB/C57 FVB/C57 C57

MommeD9+/+ MommeD9+/+ MommeD9-/+ MommeD9-/+ -/- GFP/- -/- GFP/- FVB+C57 FVB+C57 FVB+C57 FVB+C57 mixture mixture mixture mixture

Figure 4.11 Backcross breeding scheme for mapping the MommeD9 mutation. -/+ MommeD9 mice were crossed to C57 mice. F1 mice that were heterozygous for the MommeD9 mutation, as determined by flow cytometry, were then backcrossed to C57 mice. Half of the resulting F2 mice did not carry the GFP transgene and so could not be phenotyped. F2 mice heterozygous for the MommeD9 mutation were used for mapping (circled).

Table 4.5 Percentage of cells expressing the transgene and the mean fluorescence of -/+ +/+ expressing cells for MommeD9 and MommeD9 F2 mice used for mapping. A sample of peripheral blood was analysed at weaning. The mean ± one standard deviation of the percentage of cells expressing the transgene and mean fluorescence of those expressing cells -/+ are shown. F2 MommeD9 mice had a lower percentage of expressing cells and mean fluorescence than MommeD9+/+ mice and so could be reliably phenotyped by flow cytometry.

Percentage of Mean fluorescence Sex Genotype n cells expressing of the expressing the transgene cells

MommeD9 +/+ 41 34.57 ± 3.95 278.20 ± 52.48 Males MommeD9 -/+ 38 16.86 ± 3.12 177.35 ± 25.52

MommeD9 +/+ 33 34.65 ± 5.18 279.52 ± 58.26 Females MommeD9 -/+ 28 16.65 ± 3.25 196.76 ± 39.85

MommeD9 +/+ 74 34.61 ± 4.51 278.79 ± 54.75 Combined MommeD9 -/+ 66 16.77 ± 3.15 185.58 ± 33.52

(a) Mbp D7Mit178 0.5 cM

D7Mit57 18.9

D7Mit294 27.0

D7Mit247 36.9

D7Mit69 48.9

D7Mit220 104.3

D7Mit223 144.4

Number of mice 11 1 1 7 1 1 1 6 1 2 2 1 2 23 2 1

(b) Mbp D7Mit178 0.5 cM C57/C57 Genotype rs13479101 3.2 FVB/C57 Genotype rs38424645 9.6

rs38306571 15.1

D7Mit57 18.9

D7Mit294 27.0

D7Mit247 36.9

Number of mice 2 2 1 1

Figure 4.12 Mapping data for MommeD9. The microsatellite markers and SNPs that were used to map the MommeD9 mutation are displayed alongside their approximate base pair location. Marker results are depicted as shaded boxes, with a black box being a C57/C57 genotype and a white box being an FVB/C57 genotype. The mutation is associated with the FVB genome, so an FVB/C57 genotype indicated that the mutation could be present at that location. (a) Crude mapping results for 63 MommeD9-/+ mice on chromosome 7. The mutation was linked to an interval bounded by the microsatellite markers D7Mit178 and D7Mit294, as defined by 6 unique recombinant mice (red boxes). (b) Fine mapping results of those 6 recombinants between D7Mit178 and D7Mit247. The linked interval was refined to between the SNP rs38424645 and the microsatellite marker D7Mit294, and was defined by 5 unique recombinant mice (red boxes).

Chmp2a 2.73 Kbp

Figure 4.13 Diagramatic map of the gene Chmp2a. The boxes represent the exons, with black shading indicating that the transcript is translated and no shading indicating an untranslated region. The approximate length of the transcript is shown above the map. The short lines underneath the map represent the regions sequenced by individual sets of primer pairs. A minimum of 50 bp was sequenced beyond the exon/intron junctions. The map is not drawn to scale.

4.3 Discussion

MommeD9 is a semi-dominant, homozygous lethal mutation. Homozygous mutants appear to be developmentally delayed at ~8-9 dpc, and are dying between 10.5 and

14.5 dpc. There is also stochastic death of some heterozygous mutants prior to weaning following maternal transmission of the mutation, and possibly death of heterozygous mutants and wildtype offspring prior to weaning following paternal transmission. The heterozygous mutants probably die at the same time as the homozygous mutants.

Heterozygosity for the MommeD9 mutation could be affecting the mouse‟s ability to fight infections, and this could be responsible for the premature deaths of adults.

MommeD9 heterozygous mutants had abnormal haematological profiles, including an increased level of leukocytes at 21 weeks of age. The MommeD9 mutants were mildly anaemic. However while the effect was consistent it was also subtle, with only slightly lower numbers of erythrocytes and lower concentrations of haemoglobin in mutants compared with wildtype mice. As such we must be careful to draw any conclusions from the result.

Although heterozygous mutants weighed less than their wildtype littermates at three weeks of age, they appeared to gain weight more readily after weaning, eventually weighing more than wildtype mice. The female mutants were more affected than the male mutants. Obesity and immune status are known to be linked (Marti et al., 2001).

Clinical data suggests that obese individuals show an increased incidence and severity of certain infectious illnesses (Gottschlich et al., 1993; Weber et al., 1986). Work with mouse models of obesity supports these findings (Chandra, 1980; Ikejima et al.,

2005), particularly highlighting the role of leptin in immune function (Loffreda et al.,

1998; Matarese, 2000). Therefore the haematological abnormalities discussed above could be a consequence of the increased body weight.

Female fertility is also affected by the MommeD9 mutation. Female mutants had significantly fewer litters, with their last litter being born no later than at 21 weeks of age. This fertility loss could be related the weight gain, as other obese mice are known to have female-specific infertility (Chehab et al., 1996; Mitchell et al., 2005). This has been shown to be associated with levels of leptin and other adiponectins. Investigation of this infertility phenotype would benefit from following more females over the course of their breeding life.

The MommeD9 mutation is of interest due to its links to obesity and female infertility.

How a mutation in a gene that is affecting the epigenetic state at a transgene could be influencing these traits is unknown. Other Momme mutants being investigated in our laboratory have shown related phenotypes. One Momme shows splenomegaly and an abnormal haematology profile with high levels of leukocytes (N. Whitelaw, personal communication). Another shows severe female infertility; females do not produce

litters beyond eight weeks of age (Vickaryous, 2005). This implies that the genes being identified in our screen have wide-ranging pleiotropic effects.

More mice are needed to produce recombinants to narrow the interval. However, while there are some markers within the linked interval that differ between the FVB and C57 genomes, designing unique primers to these markers has been almost impossible. BLASTN searches consistently return multiple matches for primer sequences within the linked interval, suggesting that there have been multiple duplication events. Indeed this region contains many clusters of gene families likely to have originated from duplication events.

It would be interesting to observe the effect that the MommeD9 mutation has on a locus other than the GFP transgene that is sensitive to epigenetic state. The Avy allele would be a good candidate. Being a classic enhancer of variegation, one would predict that the presence of the MommeD9 mutation would result in a decreased probability of expression from the Avy allele. Subsequently, fewer yellow offspring and more pseudoagouti offspring should be produced.

MommeD9 may identify a novel biochemical pathway linking gene silencing and metabolism, and the mutant line may provide a useful model in which to study this at the level of the whole organism.

Chapter 5 - Creation of a congenic Line3 strain in C57 from

the Line3 strain in FVB

Abstract

To aid future ENU mutagenesis screens, a congenic Line3 strain in the C57 background has been created from the FVB Line3 strain. The new line, named

Line3C, displays a different GFP expression profile to the FVB Line3 strain, with a similar percentage of erythrocytes expressing the transgene but a higher overall mean fluorescence, indicating that there are strain-specific modifiers of epigenetic gene silencing that differ between the C57 and FVB strains. Linkage analysis has revealed three chromosomal locations that are likely to be involved.

5.1 Introduction

The mutagenesis screens carried out in our laboratory used homozygous Line3 mice that carry a GFP transgene. This transgenic line was created and maintained in an

FVB background. I decided to backcross the GFP transgene into the C57 background, i.e. produce a congenic strain. A congenic mouse strain is a strain that contains an allele, gene, or genomic region of interest in the same location as in the original strain.

The strategy requires a donor strain (in this case, FVB), which carries the region of interest (in this case, the GFP transgene), and a recipient strain (in this case, C57), into which you wish to introduce the region of interest. The creation of a congenic

Line3 strain in the C57 background would be useful for a number of reasons outlined below.

Linkage analysis on the individual Momme mutants requires the identification of microsatellite markers and SNPs in the FVB strain. These have not been as extensively characterised in FVB as they have been in some other mouse strains, such as C57. This has forced us to either test markers identified in other strains in the hope that the FVB strain is different to the C57 strain, or to find new markers. Once we have identified a gene in which a mutation has occurred, we often need to perform complementation crosses with a previously produced mouse mutant of that gene to verify that it is the one involved in the phenotype (Blewitt et al., Submitted; Chong et al., 2007). Most mouse knockouts are now maintained in the C57 strain. The interpretation of such a cross would be simpler if our mutants were in the C57 background.

A C57 transgenic line would also aid linkage analysis for mutants already produced in the FVB Line3 screens. To date, we have been crossing the mutants with C57 mice up to the F2 or F3 generation. The problem with this approach is that only half of the resultant F2 and F3 offspring carry the transgene, i.e. only half of the offspring are informative. If we were to cross the mutants with a C57 strain carrying the same transgene as the mutants, then we would maintain it in the homozygous state in the F2 and F3 generations, and all the offspring could be used for linkage analysis.

5.2 Results

5.2.1 Backcross strategy

An initial cross between the Line3 strain and the C57 strain will create an F1 mouse that is 50% FVB and 50% C57, and all mice will be hemizygous for the transgene.

Subsequent backcrossing of these F1 mice to a pure C57 mouse will produce F2 mice that are, on average, 25% FVB and 75% C57. At this stage half the offspring will be non-transgenic. Offspring carrying the GFP transgene can be selected by flow cytometry of the peripheral blood. By repeatedly backcrossing the Line3 strain to

C57, combined with selection for the region of interest (in this case, the GFP transgene by GFP fluorescence), a mouse strain can be produced that is genetically identical to the recipient (C57) strain except for the region of interest, which comes from the donor (FVB) strain. It is generally accepted practice to backcross to the F10 generation, since in theory the amount of the donor strain‟s genome at this stage will be 0.1%, i.e. the congenic strain will be 99.9% genetically identical to the recipient strain.

A speed congenic step was incorporated at the F4 and F5 generations in an attempt to accelerate the production of the congenic strain. Since each mouse born in a particular generation will have undergone recombination events to different extents, we can screen for the mice with the most recipient genome using microsatellite markers that cover the entire genome. In theory one could obtain a congenic strain with 99.9% donor genome in six or seven generations using this approach (Wakeland et al.,

1997).

A diagram of the backcross strategy used is shown in Figure 5.1 and Figure 5.2, and the theoretical quantitative contribution of each strain‟s DNA is shown in Table 5.1.

All F4 offspring (n=14) were screened with 63 microsatellite markers, i.e. approximately one marker every 20 cM. The mice were then ranked according to the percentage of markers that were C57-derived, and the two mice that had the highest percentage of C57-derived markers were used for the production of the F5 generation.

This was repeated on the F5 generation individuals. This time not all markers were needed. If the parent F4 mouse used to produce the F5 offspring was known to be homozygous for the C57-derived marker (therefore C57/C57) at any specific microsatellites, then those markers did not need to be reanalysed in the F5 offspring.

The F5 offspring (n=13) were screened with either 3 (n=7) or 4 (n=6) microsatellite markers depending on their F4 parents microsatellite marker genotype. The two mice with the highest percentage of C57-derived markers were selected. One was C57/C57 at all 63 markers, and the other was C57/C57 at 62 of the 63 markers. These two mice were used for the production of the F6 generation.

Once F10 offspring were produced, they were intercrossed to create a strain that is homozygous for the GFP transgene. The final congenic strain was called Line3C.

Founders x

Xf/Yf Xc/Xc GFP/GFP -/- FVB C57

F1 x

Xc/Yf Xc/Xc GFP/- -/- FVB/C57 C57

F2,3,4,5,6,7,8,9 x

c c c c X /Y X /X The F4 and F5 -/- GFP/- generations C57 FVB+C57 mixture involved a speed congenic approach (see Figure 5.2).

F3,4,5,6,7,8,9,10

GFP/- FVB+C57 mixture

F10 x GFP/- GFP/- C57 C57

Final congenic strain

GFP/GFP C57

Figure 5.1 Backcross breeding scheme for producing the congenic C57 strain containing the GFP transgene. A male Line3 mouse homozygous for the GFP transgene was crossed to a female C57 mouse. A male F1 mouse was then backcrossed to a female C57 mouse. By the F2 generation the sex chromosomes will be fixed as C57-derived. Only half of the F2 offspring will carry the GFP transgene, and these mice were crossed to C57 mice to produce the F3 generation. This step is repeated until the F10 generation at which point the mice are 99.9% genetically identical to wildtype C57 mice. A speed congenic step was included at the F4 and F5 generations (see Figure 5.2). The F10 offspring were crossed to each other to produce a strain that is homozygous for the GFP transgene.

100

F4 offspring with varying Select for mice with the amounts of C57 genome highest number of markers (average=93.75%) that are C57/C57

Screen with microsatellite markers

Use for producing

F5 generation

Repeat for F offspring 5

Figure 5.2 A speed congenic approach used for the F4 and F5 backcross generations. F4 offspring (n=14) were screened with 63 microsatellite markers, and the two mice that had the highest percentage of C57/C57 markers were selected for the production of the F5 generation. This process was then repeated with the F5 offspring (n=13) to produce the F6 generation.

Table 5.1 Theoretical accumulation of C57 genome and loss of FVB genome during the production of a congenic strain. In each generation you should lose half of the donor (FVB) genome of the previous generation. By the F10 generation there should only be 0.1% of donor genome remaining in the congenic strain. Note that this theory does not take into account the use of speed congenics at the F4 and F5 generations (*), so the C57 contribution is probably an underestimate from the F5 generation onwards.

101

5.2.2 Mapping the GFP transgene

Previous linkage analysis had shown that the GFP transgene in the Line3 strain resided on chromosome 1, but the precise location was not known (Blewitt, 2004).

We were keen to identify the exact location because the position of the transgene in relation to its surrounding chromosomal location could be influencing its expression and hence the type of modifiers identified in the ENU screen.

Mice produced in the backcross of Line3 into C57 from the F2 (n=16), F3 (n=34), F4

(n=14), F5 (n=13) and F6 (n=4) generations were used to address this issue. DNA from the mice was analysed with six, seven or eight chromosome 1 microsatellite markers and one single nucleotide polymorphism (SNP) (Figure 5.3). The transgene was found to be linked to a ~21.8 Mbp region between the SNP rs30790099

(45057246 bp) and the microsatellite marker D1Mit21 (66879391 bp). One mouse from the F2 generation defines the interval at rs30790099 and seven mice at later generations define the interval at D1Mit21. Using genome walking, another member of the laboratory, Nadia Whitelaw, later found that the transgene had integrated at

47635186 bp. My linkage data is consistent with this result, and I did not need to pursue this further.

102

F2 F3 F4 F5 F6 Mbp D1Mit3 19.8 ?

rs30790099 45.1

D1Mit21 66.9

D1Mit440 90.6

D1Mit136 103.8

D1Mit308 112.4

D1Mit102 149.0

D1Mit111 170.8

Number of mice 10 4 1 1 8 15 1 1 2 2 2 1 2 4 4 2 3 1 2 1 1 6 3 4

C57/C57 Genotype FVB/C57 Genotype

Figure 5.3 Mapping data for the GFP transgene. F2, F3, F4, F5 and F6 mice from the GFP transgene backcross were used. A white box indicates an FVB/C57 genotype, a black box indicates a C57/C57 genotype, and no box means that the genotype is unknown. The transgene must be associated with an

103 FVB/C57 genotype since it is in FVB genome. The transgene was mapped to a 21.8 Mbp region between the SNP rs30790099 and the microsatellite marker D1Mit21. Recombinant mice that define the linked interval are indicated by the red boxes.

5.2.3 GFP transgene expression in the C57 background

Line3 and Line3C mice hemizygous for the GFP transgene expressed the transgene in a similar percentage of cells (34.47% ± 2.31 and 33.47% ± 4.26 respectively; p=0.19)

(Table 5.2). A similar situation was observed when Line3 and Line3C mice were homozygous for the transgene (58.07% ± 4.25 and 55.78% ± 3.83 respectively; p=0.10) (Table 5.2). However, the mean fluorescence of expressing cells was significantly higher (~1.5-fold) in the Line3C than in the Line3 mice; when hemizygous (345.08 ± 24.20 and 228.19 ± 12.28 respectively; p<<0.001) and homozygous (472.08 ± 31.88 and 303.33 ± 29.87 respectively; p<<0.001) (Table 5.2).

Table 5.2 Percentage of cells expressing the transgene and the mean fluorescence of expressing cells in Line3 and Line3C mice. A sample of peripheral blood was analysed at weaning. The mean ± one standard deviation of the percentage of cells expressing the transgene and mean fluorescence of those expressing cells are shown. Line3 and Line3C mice had a similar percentage of expressing cells when hemizygous and homozygous for the transgene. The mean fluorescence was significantly higher in Line3C mice when the GFP transgene was in a hemizygous state (p<<0.001) and a homozygous (p<<0.001) state. The mean fluorescence in the Line3 mice was approximately 65% of that in Line3C mice. (All p- values are for a T-test)

Percentage of Mean fluorescence Strain n cells expressing of the expressing the transgene cells

Hemizygous for Line3 39 34.47 ± 2.31 228.19 ± 12.28 GFP Transgene Line3C 42 33.47 ± 4.26 345.08 ± 24.20

Homozygous for Line3 17 58.07 ± 4.25 303.33 ± 29.87 GFP Transgene Line3C 19 55.78 ± 3.83 472.08 ± 31.88

5.2.4 GFP mRNA levels are higher in the C57 background

Northern analysis of GFP PolyA+ mRNA from spleens of mice revealed a tendency for the GFP mRNA levels to be higher in Line3C than Line3 mice. The level of GFP

104

mRNA from spleens of Line3C mice was approximately two-fold that of the Line3 mice (p=0.04). This suggests that the difference in GFP mean fluorescence is the result of either increased transcription of the transgene or increased stability of the transgene transcript in the Line3C strain.

(a) Line3 Line3C 1 2 3 4 5 6 7 8

GFP

GAPDH

(b) 1.60 1.42 1.40 1.20 1.00 0.80 0.73 0.80 0.65 0.60 0.53 0.46 0.44 0.43 0.40

0.20 Ratio of GFP:GAPDH ofRatioGFP:GAPDH mRNA 0.00 1 2 3 4 5 6 7 8

Line3 Line3C

Figure 5.4 Northern analysis of GFP mRNA in Line3 and Line3C mice hemizygous for the transgene. (a) PolyA+ mRNA obtained from the spleens of four individual Line3 (1-4) and four individual Line3C (5-8) three week old males was separated on a 1.2% agarose gel, transferred to a nylon membrane and hybridised with a GFP probe. A GAPDH probe was used as a loading control. (b) The ratio of GFP mRNA levels to GAPDH mRNA levels was calculated for each sample, so that the different mice could be directly compared. The individual ratios are given above each sample. Line3C mice had consistently higher levels of GFP mRNA (p=0.04). (The p-value is for a T-test)

105

5.2.5 Mapping modifiers of GFP transgene expression in the two strains

To map the apparent modifiers affecting expression of the GFP transgene, genome- wide linkage analysis was carried out. This was performed by crossing Line3C mice homozygous for the GFP transgene to wildtype FVB mice for two generations (Figure

5.5).

The average mean fluorescence of F1 offspring was 292.86 ± 14.93, which is halfway between the average mean fluorescence of Line3 and Line3C mice hemizygous for the GFP transgene (Figure 5.6). This suggests considerable contribution from semi- dominant enhancers in C57 and semi-dominant suppressors in FVB. The F2 mice were found to have a wide range of expression states, and an average mean fluorescence halfway between the hemizygous Line3 mice and the F1 offspring. F2 offspring were divided into three groups with high (>278), moderate (240-278) or low

(<240) GFP expression levels. These categories were chosen to correspond to the hemizygous Line3 and F1 data. The high category limit is the F1 (mean - one standard deviation), while the low category limit is the hemizygous Line3 (mean + one standard deviation) (Figure 5.6).

106

Founders x

GFP/GFP -/- C57 FVB

F1 x

GFP/- -/- FVB/C57 FVB

-/- GFP/- FVB+C57 FVB+C57 mixture mixture

Categorised by mean fluorescence Low Moderate High

Used for mapping

Figure 5.5 Backcross breeding scheme for mapping the modifiers of GFP transgene expression. A C57 Line3C mouse was crossed with an FVB Line3 mouse to produce F1 offspring that were hemizygous for the GFP transgene. These F1 offspring were then backcrossed to FVB Line3 mice. Half of the resulting F2 mice did not carry the transgene and could therefore not be used. The F2 mice that were hemizygous for the transgene were split into three categories based on their mean fluorescence at weaning. The low and high expressors were used for mapping.

107

(a) 9 8 7 6 5 4 3

Number Number Miceof 2 1

200 230 240 250 260 290 300 310 340 350 360 390 400 210 220 270 280 320 330 370 380

------

195 225 235 245 255 285 295 305 335 345 355 385 395 205 215 265 275 315 325 365 375 Mean Fluorescence

9 Low Moderate High 8 7 6 5 4 3

Number Number Miceof 2 1

200 230 240 250 260 290 300 310 340 350 360 390 400 210 220 270 280 320 330 370 380

------

195 225 235 245 255 285 295 305 335 345 355 385 395 205 215 265 275 315 325 365 375 Mean Fluorescence

Line3 Line3C F1 Hybrids F2 Hybrids

(b) 430.00

380.00

330.00

High 280.00 Moderate

Mean Fluorescence Mean 230.00 Low

180.00 Line3 Line3C F1 Hybrids F2 Hybrids

n=39 n=42 n=47 n=37

Figure 5.6 Comparison of the mean fluorescence of cells expressing the GFP transgene in Line3, Line3C, F1 hybrids and F2 hybrids hemizygous for the transgene. (a) Histograms showing the range of mean fluorescence observed for Line3, Line3C, F1 hybrids and F2 hybrids. Moving averages are overlayed on the histograms. F1 hybrids fall between the Line3 and Line3C mice. The F2 hybrids have a wider range of mean fluorescence, and their mean falls between the Line3 mice and F1 hybrids. The F2 offspring were split into three categories based on their mean fluorescence; high (>278), moderate (240-278) and low (<240). (b) Box whisker plot of the same data as in (a). The box represents the mean ± one standard deviation, and the whiskers show the highest and lowest observed values.

108

The high expressors were indistinguishable from F1 offspring in terms of mean fluorescence, and so we can assume they retain most of the C57 enhancer modifiers in the heterozygous state. The low expressors were indistinguishable from hemizygous

Line3 mice in terms of mean fluorescence, and so we assume they have lost most of the C57 enhancer modifiers and therefore to have gained homozygosity for the FVB suppressor modifiers. Only the high and low expressors were used in linkage analysis.

In order to map the modifiers that are enhancing expression of the transgene in the

C57 background we want to locate chromosomal regions where all the high expressors have an FVB/C57 genotype and all the low expressors have an FVB/FVB genotype. However, we are dealing with the mapping of multiple interacting loci simultaneously, i.e. some high expressors may not have retained all of the C57 enhancers and would be FVB/FVB at those loci. Furthermore, the probability of recombination occurring in the F1 gametes is not equivalent across the entire genome, i.e. distance from the telomere can influence recombination frequency. Therefore chromosomal regions were chosen where the percentage of mice with a FVB/C57 genotype is statistically significantly greater in high expressors than in low expressors.

37 F2 mice were used for linkage analysis; 19 high expressors and 18 low expressors.

Using 39 microsatellite markers that covered all 19 autosomes at a density of at least two markers per chromosome, four genomic regions were identified as having a statistically significantly greater percentage of an FVB/C57 genotype in the high expressors than in the low expressors, and were therefore linked to potential C57 enhancers of transgene expression (Figure 5.7). These regions are found on

109

chromosomes 1, 7, 10 and 15, and are linked to the microsatellite markers D1Mit111,

D7Mit69, D10Mit267 and D15Mit16 respectively.

Although D1Mit111 is located on chromosome 1, along with the transgene, the marker is a sufficient cM distance from the transgene to assume that it is unlinked.

The transgene is known to be located at ~47.6 Mbp, which is very close to the microsatellite marker D1Mit175 (located at 26.2 cM), therefore we can assume that the transgene is also at ~26.2 cM. Since D1Mit111 is located at 92.3 cM, the transgene is ~66.1 cM away from D1Mit111, which is in excess of the 50 cM distance that defines linked loci.

110

Figure 5.7 Mapping data for modifiers of GFP transgene expression. Microsatellite markers are shown with their chromosome location and approximate cM location (Ensembl database version 46, NCBI build m36). The mean fluorescence of GFP transgene expression of each individual mouse is shown. The genotypes of high GFP expressing F2 mice (n=19) were compared to those for low GFP expressing F2 mice (n=18), with the percentage of markers that are FVB/C57 shown. If high expressors had statistically significantly more FVB/C57 markers than low expressors, those regions were linked to C57 enhancers of expression (red boxes). (* indicates p<0.05) (All p-values are for a Chi-squared test)

111

Marker D1Mit3 D1Mit440 D1Mit111 D2Mit92 D2Mit48 D3Mit63 D3Mit17 D4Mit17 D4Mit251 D5Mit233 D5Mit101 D6Mit188 D6Mit373 D7Mit69 D7Mit223 cM 11 54 92.3 41.4 87 22 71.8 31.4 66 29 81 32.5 74.3 24.5 72.4 Mean Fl 325.98 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 321.42 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB 313.75 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 309.83 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 309.42 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 308.39 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 305.92 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 299.56 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 289.16 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/FVB 288.71 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB 288.62 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 287.84 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/FVB

High ExpressorsHigh 285.18 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 284.84 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB 281.91 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/C57 280.84 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 278.83 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB 278.29 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/FVB 278.16 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/FVB % FVB/C57 84 89 68 47 37 42 42 32 37 11 42 63 37 47 58

238.79 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 236.74 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 232.66 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 231.97 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/C57 231.1 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB 230.99 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB 229.53 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 229.01 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB 226.27 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 224.73 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB 222.25 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB

220.06 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB Low ExpressorsLow 218.58 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB 214.44 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB 213.2 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 211.85 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB 209.29 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 198.42 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 % FVB/C57 94 67 33 56 72 56 44 44 50 72 56 44 44 22 50

112 * *

Marker D8Mit190 D8Mit215 D9Mit248 D9Mit1000 D10Mit3 D10Mit267 D11Mit20 D11Mit333 D12Mit2 D12Mit28 D13Mit16 D13Mit260 D14Mit18 D14Mit97 cM 21 59 31 61 21 67.5 20 66 19 52 10 65 16.5 58 Mean Fl 325.98 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB 321.42 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB 313.75 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 309.83 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 309.42 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB 308.39 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB 305.92 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 299.56 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB 289.16 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB 288.71 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 288.62 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB 287.84 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57

HighExpressors 285.18 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 284.84 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 281.91 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 280.84 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB 278.83 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 278.29 FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB 278.16 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB % FVB/C57 58 53 53 74 58 68 68 68 58 63 63 37 47 47

238.79 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 236.74 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB 232.66 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/C57 231.97 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 231.1 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB 230.99 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB 229.53 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 229.01 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB 226.27 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB 224.73 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB 222.25 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB

220.06 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB Low Expressors Low 218.58 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 214.44 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB 213.2 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB 211.85 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 209.29 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB 198.42 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB % FVB/C57 44 44 56 61 56 39 56 61 72 56 72 50 56 33

113 *

Marker D15Mit53 D15Mit16 D16Mit4 D16Mit86 D17Mit133 D17Mit123 D18Mit177 D18Mit48 D19Mit41 D19Mit11 cM 12.3 61.7 27.3 66 10.4 56.7 20 50 16 41 Mean Fl 325.98 FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB 321.42 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB 313.75 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB 309.83 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB 309.42 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 308.39 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 305.92 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB 299.56 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 289.16 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB 288.71 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB 288.62 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB 287.84 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57

HighExpressors 285.18 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 284.84 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 281.91 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 280.84 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB 278.83 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 278.29 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 278.16 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB % FVB/C57 63 68 53 53 58 47 53 47 47 47

238.79 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB 236.74 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 232.66 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 231.97 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 231.1 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/C57 230.99 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB 229.53 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 229.01 FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 226.27 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 224.73 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 222.25 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57

220.06 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 Low Expressors Low 218.58 FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 214.44 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB 213.2 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 211.85 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 209.29 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/FVB 198.42 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB % FVB/C57 50 33 50 56 56 44 44 44 61 72

114 *

To increase confidence in the mapping, the modifiers located on chromosomes 7, 10 and 15 were mapped with several more microsatellite markers (Figure 5.8). The chromosome 1 enhancer could not be further analysed because the presence of the transgene would have interfered with the linkage. The initial linkage was confirmed for all three modifiers, with surrounding microsatellite markers consistent with the previous mapping. This also gave more precise mapping locations of the modifiers.

The modifier on chromosome 7 is probably located between the markers D7Mit57 and D7Mit247, although it is likely to be closer to D7Mit247. The modifier on chromosome 10 is probably located between the markers D10Mit230 and D10Mit267.

The modifier on chromosome 15 is probably located between the markers D15Mit92 and D15Mit70. The modifiers on chromosomes 7 and 15 show the most promise.

115

Figure 5.8 Fine mapping of modifiers linked to chromosomes 7, 10 and 15. Microsatellite markers are shown with their chromosome location and approximate cM location (Ensembl database version 46, NCBI build m36). The mean fluorescence of GFP transgene expression of each individual mouse is shown. The genotypes of high GFP expressing F2 mice (n=19) were compared to those for low GFP expressing F2 mice (n=18), with the percentage of markers that are FVB/C57 shown. The microsatellite markers identified in the preliminary linkage analysis as being linked to an enhancer (Figure 5.7) are highlighted. In all cases the initial linkage was confirmed by surrounding markers and has further narrowed down the locations of the modifiers to more precise chromosomal locations (red boxes). (* indicates p<0.05; ** indicates p<0.001; *** indicates p<<0.001) (All p-values are for a Chi-squared test)

116

Chromosome 7 10 15 Marker D7Mit57 D7Mit247 D7Mit69 D7Mit350 D7Mit220 D7Mit223 D10Mit213 D10Mit3 D10Mit61 D10Mit230 D10Mit267 D15Mit53 D15Mit92 D15Mit70D15Mit16 cM 4 16 24.5 41 52.4 72.4 11 21 32 49 67.5 12.3 35.3 47.7 61.7 Mean Fl 325.98 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 321.42 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 313.75 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 309.83 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB 309.42 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/FVB 308.39 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 305.92 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 299.56 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 289.16 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 288.71 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 288.62 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 287.84 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB

High Expressors High 285.18 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVBFVB/FVB 284.84 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVBFVB/FVB 281.91 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 280.84 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 278.83 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 278.29 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVBFVB/FVB 278.16 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 % FVB/C57 58 58 47 37 42 58 58 58 74 68 68 63 84 84 68

238.79 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 236.74 FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVBFVB/FVB 232.66 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB 231.97 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 231.1 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVBFVB/FVB 230.99 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVBFVB/FVB 229.53 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVBFVB/FVB 229.01 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/C57 226.27 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 224.73 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 222.25 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVBFVB/FVB

220.06 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVBFVB/FVB Low Expressors Low 218.58 FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 214.44 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/FVB FVB/FVBFVB/FVB 213.2 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVBFVB/FVB 211.85 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB 209.29 FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVB FVB/FVB FVB/FVBFVB/FVB 198.42 FVB/FVB FVB/FVB FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/C57 FVB/FVB FVB/FVBFVB/FVB % FVB/C57 22 17 22 33 33 50 44 56 56 44 39 50 33 33 33

117 ** *** * * * *** *** *

5.3 Discussion

I have created a congenic C57 strain containing a GFP transgene, Line3C. These

Line3C mice will prove useful in both current and future ENU mutagenesis screens for modifiers of epigenetic reprogramming. In particular, having the GFP transgene in both mouse strains allows for more efficient linkage analysis, because every mouse is useful. This is true regardless of the strain in which the mutation was produced. From an animal ethics viewpoint, we must always attempt to minimise the number of mice used in any experiment.

In addition, we now have the potential to produce and maintain mutant lines in a C57 background. Because many known mutants are maintained in the C57 background, this makes the mutant lines better suited to complementation experiments.

The finding that there are modifiers that influence the expression of the GFP transgene that differ between the FVB and C57 strains is not surprising. We have known for some time that different mouse strains can influence the epigenetic state of particular loci in different ways, particularly in the case of transgenes (Opsahl et al.,

2002; Sapienza et al., 1989; Schweizer et al., 1998).

We have shown here that there are semi-dominant enhancers within the C57 strain that do not affect the number of cells expressing the transgene, but that do cause those cells that are expressing the transgene to produce more GFP protein than they otherwise would have in the FVB strain. Whether these modifiers would have the same effect on all transgenes, or if the GFP transgene is specifically targeted is

118

unknown. To pursue this further, we could observe the expression of other transgenes when they are in the FVB and C57 strains.

To proceed further with the mapping of these modifiers, more F2 mice and microsatellite markers need to be analysed to narrow the linked interval. The higher levels of GFP mRNA in Line3C mice suggest that the difference in GFP fluorescence occurs at the level of transcription and/or transcript stability. The increase in the level of GFP fluorescence (1.5x in a hemizygous cell) is similar to that found at the level of

GFP mRNA.

This could be pursued further. However, unlike the identification of a point mutation from the ENU mutagenesis screen (where the point mutation will be unique in the linked interval), the identification of relevant sequence differences in this case will be difficult because within a linked interval there will be many strain-specific sequence differences (eg. SNPs). This highlights the power of the ENU mutagenesis screen.

119

Chapter 6 - General Discussion

120

The sensitised ENU mutagenesis screen developed in our laboratory is proving to be useful in identifying modifiers of epigenetic gene silencing in the mouse. We have already found the underlying genes involved in several Momme mutants; Smchd1

(structural maintenance of chromosomes flexible hinge domain containing 1)

(MommeD1) (Blewitt et al., Submitted), the DNA methyltransferase Dnmt1

(MommeD2) and the chromatin remodeler Snf2h (sucrose non-fermenting 2 homolog)

(MommeD4) (Chong et al., 2007), WSTF (Williams syndrome transcription factor)

(MommeD10) and Uble1b (ubiquitin-like (sentrin) activating enzyme 1b) (MommeR2)

(Ashe, 2007), and Foxo3a (forkhead box O3a) (MommeR1) (Vickaryous, 2005).

The two Momme mutants discussed in this thesis, MommeD6 and MommeD9, show interesting differences. Not only are they examples of a suppressor and an enhancer of variegation respectively, but they show a significant difference with respect to their effect on the GFP transgene. MommeD9 behaves like the majority of Momme mutants identified in our screen, with the mutation affecting the percentage of erythrocytes expressing the transgene, which is what one would expect for a classic modifier of variegation. On the other hand, MommeD6 shows little effect on variegation, but a significant effect on expression levels. The reason behind this result can only be fully understood once the underlying genes have been identified.

Both the MommeD6 and MommeD9 mutations show similar phenotypes with respect to the time of death of homozygous mutants, with both mutants dying mid-gestation.

MommeD2 (the Dnmt1 mutant) also shows homozygous death at mid-gestation (Ashe,

2007; Chong et al., 2007), as does MommeD1 (the Smchd1 mutant) (Blewitt et al.,

Submitted; Blewitt et al., 2005). This is despite the fact that all four Momme mutants

121

have very different GFP transgene expression profiles and are linked to different chromosomal locations. This suggests that mid-gestation is a critical time for epigenetic gene silencing, and that future genes identified by this screen will also be vital for correct differentiation and development.

Comparisons between epigenetic modifiers in mammals and those in other organisms show that there are common mechanisms controlling epigenetic phenomena. For example, modifiers of histone methylation have been identified in mammals,

Drosophila and Arabidopsis (Naumann et al., 2005; Peters et al., 2001; Schotta et al.,

2002). Furthermore, mutants of epigenetic modifiers in the mouse, such as the

Momme mutants identified in our laboratory, appear to show functional deficiencies similar to related mutants in other organisms, such as the Su(var) and E(var) mutants identified in the Drosophila PEV screens. This emphasises that the work discussed in this thesis could not only have an impact on our understanding of epigenetic mechanisms in the mouse, but also those in other organisms.

It is interesting to note that Waddington, the biologist who first coined the term

“epigenetic” saw epigenetics as the explanation for the fact that cells of the same genotype can differentiate into cells with different phenotypes. Although this screen was designed to identify genes involved in transgene silencing, it turns out that these same genes are also involved in differentiation and development. This underscores the value of studying transgene silencing in mammals.

122

References

Allfrey, V. G. (1966). Structural modifications of histones and their possible role in the regulation of ribonucleic acid synthesis. Proc Can Cancer Conf 6, 313-335.

Ashe, A. (2007) Modifiers of epigenetic gene silencing, PhD Thesis, University of Sydney, Sydney.

Baarends, W. M., Wassenaar, E., van der Laan, R., Hoogerbrugge, J., Sleddens- Linkels, E., Hoeijmakers, J. H., de Boer, P., and Grootegoed, J. A. (2005). Silencing of unpaired chromatin and histone H2A ubiquitination in mammalian meiosis. Mol Cell Biol 25, 1041-1053.

Bachellerie, J. P., Cavaille, J., and Huttenhofer, A. (2002). The expanding snoRNA world. Biochimie 84, 775-790.

Baksa, K., Morawietz, H., Dombradi, V., Axton, M., Taubert, H., Szabo, G., Torok, I., Udvardy, A., Gyurkovics, H., Szoor, B., and et al. (1993). Mutations in the protein phosphatase 1 gene at 87B can differentially affect suppression of position-effect variegation and mitosis in Drosophila melanogaster. Genetics 135, 117-125.

Bannister, A. J., Zegerman, P., Partridge, J. F., Miska, E. A., Thomas, J. O., Allshire, R. C., and Kouzarides, T. (2001). Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410, 120-124.

Belyaev, D. K., Ruvinsky, A. O., and Borodin, P. M. (1981). Inheritance of alternative states of the fused gene in mice. J Hered 72, 107-112.

Blewitt, M., Gendrel, A. V., Pang, Z., Sparrow, D. B., Craig, J. M., Apedaile, A., Hilton, D. J., Dunwoodie, S. L., Brockdorff, N., Kay, G. F., and Whitelaw, E. (Submitted). SmcHD1, a protein containing a structural maintenance of chromosomes hinge domain, is involved in methylation of the inactive X chromosome. Nat Genet.

Blewitt, M. E. (2004) Epigenetic reprogramming in the mouse, PhD Thesis, University of Sydney, Sydney.

Blewitt, M. E., Vickaryous, N. K., Hemley, S. J., Ashe, A., Bruxner, T. J., Preis, J. I., Arkell, R., and Whitelaw, E. (2005). An N-ethyl-N-nitrosourea screen for genes involved in variegation in the mouse. Proc Natl Acad Sci U S A 102, 7629-7634.

Blewitt, M. E., Vickaryous, N. K., Paldi, A., Koseki, H., and Whitelaw, E. (2006). Dynamic reprogramming of DNA methylation at an epigenetically sensitive allele in mice. PLoS Genet 2, e49.

123

Cambareri, E. B., Foss, H. M., Rountree, M. R., Selker, E. U., and Kinsey, J. A. (1996). Epigenetic control of a transposon-inactivated gene in Neurospora is dependent on DNA methylation. Genetics 143, 137-146.

Carlson, L. L., Page, A. W., and Bestor, T. H. (1992). Properties and localization of DNA methyltransferase in preimplantation mouse embryos: implications for genomic imprinting. Genes Dev 6, 2536-2541.

Cavaille, J., Seitz, H., Paulsen, M., Ferguson-Smith, A. C., and Bachellerie, J. P. (2002). Identification of tandemly-repeated C/D snoRNA genes at the imprinted human 14q32 domain reminiscent of those at the Prader-Willi/Angelman syndrome region. Hum Mol Genet 11, 1527-1538.

Chandra, R. K. (1980). Cell-mediated immunity in genetically obese C57BL/6J ob/ob) mice. Am J Clin Nutr 33, 13-16.

Chehab, F. F., Lim, M. E., and Lu, R. (1996). Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin. Nat Genet 12, 318-320.

Chong, S., Vickaryous, N., Ashe, A., Zamudio, N., Youngson, N., Hemley, S., Stopka, T., Skoultchi, A., Matthews, J., Scott, H. S., et al. (2007). Modifiers of epigenetic reprogramming show paternal effects in the mouse. Nat Genet 39, 614-622.

Cleard, F., Matsarskaia, M., and Spierer, P. (1995). The modifier of position-effect variegation Suvar(3)7 of Drosophila: there are two alternative transcripts and seven scattered zinc fingers, each preceded by a tryptophan box. Nucleic Acids Res 23, 796- 802.

Croteau, S., Andrade, M. F., Huang, F., Greenwood, C. M., Morgan, K., and Naumova, A. K. (2002). Inheritance patterns of maternal alleles in imprinted regions of the mouse genome at different stages of development. Mamm Genome 13, 24-29.

Czermin, B., Melfi, R., McCabe, D., Seitz, V., Imhof, A., and Pirrotta, V. (2002). Drosophila enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites. Cell 111, 185-196.

De Rubertis, F., Kadosh, D., Henchoz, S., Pauli, D., Reuter, G., Struhl, K., and Spierer, P. (1996). The histone deacetylase RPD3 counteracts genomic silencing in Drosophila and yeast. Nature 384, 589-591.

Dean, W., Santos, F., and Reik, W. (2003). Epigenetic reprogramming in early mammalian development and following somatic nuclear transfer. Semin Cell Dev Biol 14, 93-100.

Delaval, K., and Feil, R. (2004). Epigenetic regulation of mammalian genomic imprinting. Curr Opin Genet Dev 14, 188-195.

124

Dorn, R., Krauss, V., Reuter, G., and Saumweber, H. (1993). The enhancer of position-effect variegation of Drosophila, E(var)3-93D, codes for a chromatin protein containing a conserved domain common to several transcriptional regulators. Proc Natl Acad Sci U S A 90, 11376-11380.

Druker, R., Bruxner, T. J., Lehrbach, N. J., and Whitelaw, E. (2004). Complex patterns of transcription at the insertion site of a retrotransposon in the mouse. Nucleic Acids Res 32, 5800-5808.

Duncan, B. K., and Miller, J. H. (1980). Mutagenic deamination of cytosine residues in DNA. Nature 287, 560-561.

Eissenberg, J. C., James, T. C., Foster-Hartnett, D. M., Hartnett, T., Ngan, V., and Elgin, S. C. (1990). Mutation in a heterochromatin-specific chromosomal protein is associated with suppression of position-effect variegation in Drosophila melanogaster. Proc Natl Acad Sci U S A 87, 9923-9927.

Farkas, G., Gausz, J., Galloni, M., Reuter, G., Gyurkovics, H., and Karch, F. (1994). The Trithorax-like gene encodes the Drosophila GAGA factor. Nature 371, 806-808.

Feng, Q., and Zhang, Y. (2003). The NuRD complex: linking histone modification to nucleosome remodeling. Curr Top Microbiol Immunol 274, 269-290.

Garzino, V., Pereira, A., Laurenti, P., Graba, Y., Levis, R. W., Le Parco, Y., and Pradel, J. (1992). Cell lineage-specific expression of modulo, a dose-dependent modifier of variegation in Drosophila. Embo J 11, 4471-4479.

Gaudet, F., Rideout, W. M., 3rd, Meissner, A., Dausman, J., Leonhardt, H., and Jaenisch, R. (2004). Dnmt1 expression in pre- and postimplantation embryogenesis and the maintenance of IAP silencing. Mol Cell Biol 24, 1640-1648.

Gerasimova, T. I., Gdula, D. A., Gerasimov, D. V., Simonova, O., and Corces, V. G. (1995). A Drosophila protein that imparts directionality on a chromatin insulator is an enhancer of position-effect variegation. Cell 82, 587-597.

Gottschlich, M. M., Mayes, T., Khoury, J. C., and Warden, G. D. (1993). Significance of obesity on nutritional, immunologic, hormonal, and clinical outcome parameters in burns. J Am Diet Assoc 93, 1261-1268.

Grewal, S. I., and Klar, A. J. (1996). Chromosomal inheritance of epigenetic states in fission yeast during mitosis and meiosis. Cell 86, 95-101.

Grienenberger, A., Miotto, B., Sagnier, T., Cavalli, G., Schramke, V., Geli, V., Mariol, M. C., Berenger, H., Graba, Y., and Pradel, J. (2002). The MYST domain acetyltransferase Chameau functions in epigenetic mechanisms of transcriptional repression. Curr Biol 12, 762-766.

125

Gruenbaum, Y., Stein, R., Cedar, H., and Razin, A. (1981). Methylation of CpG sequences in eukaryotic DNA. FEBS Lett 124, 67-71.

Hajkova, P., Erhardt, S., Lane, N., Haaf, T., El-Maarri, O., Reik, W., Walter, J., and Surani, M. A. (2002). Epigenetic reprogramming in mouse primordial germ cells. Mech Dev 117, 15-23.

Hannon, G. J. (2002). RNA interference. Nature 418, 244-251.

Hatada, S., Grant, D. J., and Maeda, N. (2003). An intronic endogenous retrovirus- like sequence attenuates human haptoglobin-related gene expression in an orientation- dependent manner. Gene 319, 55-63.

Henderson, D. S., Banga, S. S., Grigliatti, T. A., and Boyd, J. B. (1994). Mutagen sensitivity and suppression of position-effect variegation result from mutations in mus209, the Drosophila gene encoding PCNA. Embo J 13, 1450-1459.

Hitotsumachi, S., Carpenter, D. A., and Russell, W. L. (1985). Dose-repetition increases the mutagenic effectiveness of N-ethyl-N-nitrosourea in mouse spermatogonia. Proc Natl Acad Sci U S A 82, 6619-6621.

Howell, C. Y., Bestor, T. H., Ding, F., Latham, K. E., Mertineit, C., Trasler, J. M., and Chaillet, J. R. (2001). Genomic imprinting disrupted by a maternal effect mutation in the Dnmt1 gene. Cell 104, 829-838.

Huang, Y., Greene, E., Murray Stewart, T., Goodwin, A. C., Baylin, S. B., Woster, P. M., and Casero, R. A., Jr. (2007). Inhibition of lysine-specific demethylase 1 by polyamine analogues results in reexpression of aberrantly silenced genes. Proc Natl Acad Sci U S A 104, 8023-8028.

Ikejima, S., Sasaki, S., Sashinami, H., Mori, F., Ogawa, Y., Nakamura, T., Abe, Y., Wakabayashi, K., Suda, T., and Nakane, A. (2005). Impairment of host resistance to Listeria monocytogenes infection in liver of db/db and ob/ob mice. Diabetes 54, 182- 189.

Jeltsch, A. (2002). Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA methyltransferases. Chembiochem 3, 274-293.

Jin, Y., Wang, Y., Walker, D. L., Dong, H., Conley, C., Johansen, J., and Johansen, K. M. (1999). JIL-1: a novel chromosomal tandem kinase implicated in transcriptional regulation in Drosophila. Mol Cell 4, 129-135.

Jin, Y., Wang, Y., Johansen, J., and Johansen, K. M. (2000). JIL-1, a chromosomal kinase implicated in regulation of chromatin structure, associates with the male specific lethal (MSL) dosage compensation complex. J Cell Biol 149, 1005-1010.

126

Joanis, V., and Lloyd, V. K. (2002). Genomic imprinting in Drosophila is maintained by the products of Suppressor of variegation and trithorax group, but not Polycomb group, genes. Mol Genet Genomics 268, 103-112.

Johansen, K. M., and Johansen, J. (2006). Regulation of chromatin structure by histone H3S10 phosphorylation. Chromosome Res 14, 393-404.

Jones, R. S., and Gelbart, W. M. (1993). The Drosophila Polycomb-group gene Enhancer of zeste contains a region with sequence similarity to trithorax. Mol Cell Biol 13, 6357-6366.

Jordan, I. K., Rogozin, I. B., Glazko, G. V., and Koonin, E. V. (2003). Origin of a substantial fraction of human regulatory sequences from transposable elements. Trends Genet 19, 68-72.

Kim, S. H., Kang, Y. K., Koo, D. B., Kang, M. J., Moon, S. J., Lee, K. K., and Han, Y. M. (2004). Differential DNA methylation reprogramming of various repetitive sequences in mouse preimplantation embryos. Biochem Biophys Res Commun 324, 58-63.

Kono, T., Obata, Y., Wu, Q., Niwa, K., Ono, Y., Yamamoto, Y., Park, E. S., Seo, J. S., and Ogawa, H. (2004). Birth of parthenogenetic mice that can develop to adulthood. Nature 428, 860-864.

Lachner, M., O'Carroll, D., Rea, S., Mechtler, K., and Jenuwein, T. (2001). Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410, 116-120.

Laible, G., Wolf, A., Dorn, R., Reuter, G., Nislow, C., Lebersorger, A., Popkin, D., Pillus, L., and Jenuwein, T. (1997). Mammalian homologues of the Polycomb-group gene Enhancer of zeste mediate gene silencing in Drosophila heterochromatin and at S. cerevisiae telomeres. Embo J 16, 3219-3232.

LaJeunesse, D., and Shearn, A. (1996). E(z): a polycomb group gene or a trithorax group gene? Development 122, 2189-2197.

Lane, N., Dean, W., Erhardt, S., Hajkova, P., Surani, A., Walter, J., and Reik, W. (2003). Resistance of IAPs to methylation reprogramming may provide a mechanism for epigenetic inheritance in the mouse. Genesis 35, 88-93.

Larsson, J., Zhang, J., and Rasmuson-Lestander, A. (1996). Mutations in the Drosophila melanogaster gene encoding S-adenosylmethionine synthetase [corrected] suppress position-effect variegation. Genetics 143, 887-896.

Lavie, L., Kitova, M., Maldener, E., Meese, E., and Mayer, J. (2005). CpG methylation directly regulates transcriptional activity of the human endogenous retrovirus family HERV-K(HML-2). J Virol 79, 876-883.

127

Leonhardt, H., Page, A. W., Weier, H. U., and Bestor, T. H. (1992). A targeting sequence directs DNA methyltransferase to sites of DNA replication in mammalian nuclei. Cell 71, 865-873.

Lerach, S., Zhang, W., Bao, X., Deng, H., Girton, J., Johansen, J., and Johansen, K. M. (2006). Loss-of-function alleles of the JIL-1 kinase are strong suppressors of position effect variegation of the wm4 allele in Drosophila. Genetics 173, 2403-2406.

Li, E., Bestor, T. H., and Jaenisch, R. (1992). Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915-926.

Li, E. (2002). Chromatin modification and epigenetic reprogramming in mammalian development. Nat Rev Genet 3, 662-673.

Lloyd, V. (2000). Parental imprinting in Drosophila. Genetica 109, 35-44.

Loffreda, S., Yang, S. Q., Lin, H. Z., Karp, C. L., Brengman, M. L., Wang, D. J., Klein, A. S., Bulkley, G. B., Bao, C., Noble, P. W., et al. (1998). Leptin regulates proinflammatory immune responses. Faseb J 12, 57-65.

Luger, K., and Richmond, T. J. (1998). The histone tails of the nucleosome. Curr Opin Genet Dev 8, 140-146.

Marhold, J., Kramer, K., Kremmer, E., and Lyko, F. (2004). The Drosophila MBD2/3 protein mediates interactions between the MI-2 chromatin complex and CpT/A- methylated DNA. Development 131, 6033-6039.

Marmorstein, R., and Roth, S. Y. (2001). Histone acetyltransferases: function, structure, and catalysis. Curr Opin Genet Dev 11, 155-161.

Marti, A., Marcos, A., and Martinez, J. A. (2001). Obesity and immune function relationships. Obes Rev 2, 131-140.

Martin, D. I., and Whitelaw, E. (1996). The vagaries of variegating transgenes. Bioessays 18, 919-923.

Matarese, G. (2000). Leptin and the immune system: how nutritional status influences the immune response. Eur Cytokine Netw 11, 7-14.

Mayer, W., Niveleau, A., Walter, J., Fundele, R., and Haaf, T. (2000). Demethylation of the zygotic paternal genome. Nature 403, 501-502.

Michalak, P. (2006). RNA world - the dark matter of evolutionary genomics. J Evol Biol 19, 1768-1774.

128

Miotto, B., Sagnier, T., Berenger, H., Bohmann, D., Pradel, J., and Graba, Y. (2006). Chameau HAT and DRpd3 HDAC function as antagonistic cofactors of JNK/AP-1- dependent transcription during Drosophila metamorphosis. Genes Dev 20, 101-112.

Mitchell, M., Armstrong, D. T., Robker, R. L., and Norman, R. J. (2005). Adipokines: implications for female fertility and obesity. Reproduction 130, 583-597.

Moore, T., and Haig, D. (1991). Genomic imprinting in mammalian development: a parental tug-of-war. Trends Genet 7, 45-49.

Morgan, H. D., Sutherland, H. G., Martin, D. I., and Whitelaw, E. (1999). Epigenetic inheritance at the agouti locus in the mouse. Nat Genet 23, 314-318.

Morgan, H. D. (2001) Epigenetic inheritance at the murine agouti locus, PhD Thesis, University of Sydney, Sydney.

Morgan, H. D., Santos, F., Green, K., Dean, W., and Reik, W. (2005). Epigenetic reprogramming in mammals. Hum Mol Genet 14 Spec No 1, R47-58.

Namekawa, S. H., Park, P. J., Zhang, L. F., Shima, J. E., McCarrey, J. R., Griswold, M. D., and Lee, J. T. (2006). Postmeiotic sex chromatin in the male germline of mice. Curr Biol 16, 660-667.

Naumann, K., Fischer, A., Hofmann, I., Krauss, V., Phalke, S., Irmler, K., Hause, G., Aurich, A. C., Dorn, R., Jenuwein, T., and Reuter, G. (2005). Pivotal role of AtSUVH2 in heterochromatic histone methylation and gene silencing in Arabidopsis. Embo J 24, 1418-1429.

Naumova, A. K., Greenwood, C. M., and Morgan, K. (2001). Imprinting and deviation from Mendelian transmission ratios. Genome 44, 311-320.

Neuhaus, I. M., and Beier, D. R. (1998). Efficient localization of mutations by interval haplotype analysis. Mamm Genome 9, 150-154.

Newman, B. L., Lundblad, J. R., Chen, Y., and Smolik, S. M. (2002). A Drosophila homologue of Sir2 modifies position-effect variegation but does not affect life span. Genetics 162, 1675-1685.

Okamoto, I., Otte, A. P., Allis, C. D., Reinberg, D., and Heard, E. (2004). Epigenetic dynamics of imprinted X inactivation during early mouse development. Science 303, 644-649.

Okano, M., Bell, D. W., Haber, D. A., and Li, E. (1999). DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247-257.

129

Olek, A., and Walter, J. (1997). The pre-implantation ontogeny of the H19 methylation imprint. Nat Genet 17, 275-276.

Opsahl, M. L., McClenaghan, M., Springbett, A., Reid, S., Lathe, R., Colman, A., and Whitelaw, C. B. (2002). Multiple effects of genetic background on variegated transgene expression in mice. Genetics 160, 1107-1112.

Oswald, J., Engemann, S., Lane, N., Mayer, W., Olek, A., Fundele, R., Dean, W., Reik, W., and Walter, J. (2000). Active demethylation of the paternal genome in the mouse zygote. Curr Biol 10, 475-478.

Parkhurst, S. M., Harrison, D. A., Remington, M. P., Spana, C., Kelley, R. L., Coyne, R. S., and Corces, V. G. (1988). The Drosophila su(Hw) gene, which controls the phenotypic effect of the gypsy transposable element, encodes a putative DNA-binding protein. Genes Dev 2, 1205-1215.

Perry, W. L., Copeland, N. G., and Jenkins, N. A. (1994). The molecular basis for dominant yellow agouti coat color mutations. Bioessays 16, 705-707.

Persson, K. (1976). A minute mutant with suppressor effect on the eye-colour gene zeste in Drosophila melanogaster. Hereditas 82, 57-62.

Peters, A. H., O'Carroll, D., Scherthan, H., Mechtler, K., Sauer, S., Schofer, C., Weipoltshammer, K., Pagani, M., Lachner, M., Kohlmaier, A., et al. (2001). Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability. Cell 107, 323-337.

Pillai, R. S., Bhattacharyya, S. N., Artus, C. G., Zoller, T., Cougot, N., Basyuk, E., Bertrand, E., and Filipowicz, W. (2005). Inhibition of translational initiation by Let-7 MicroRNA in human cells. Science 309, 1573-1576.

Pillai, R. S., Bhattacharyya, S. N., and Filipowicz, W. (2007). Repression of protein synthesis by miRNAs: how many mechanisms? Trends Cell Biol 17, 118-126.

Plath, K., Fang, J., Mlynarczyk-Evans, S. K., Cao, R., Worringer, K. A., Wang, H., de la Cruz, C. C., Otte, A. P., Panning, B., and Zhang, Y. (2003). Role of histone H3 lysine 27 methylation in X inactivation. Science 300, 131-135.

Preis, J. I., Downes, M., Oates, N. A., Rasko, J. E., and Whitelaw, E. (2003). Sensitive flow cytometric analysis reveals a novel type of parent-of-origin effect in the mouse genome. Curr Biol 13, 955-959.

Qi, D., Jin, H., Lilja, T., and Mannervik, M. (2006). Drosophila Reptin and other TIP60 complex components promote generation of silent chromatin. Genetics 174, 241-251.

130

Rakyan, V. K., Blewitt, M. E., Druker, R., Preis, J. I., and Whitelaw, E. (2002). Metastable epialleles in mammals. Trends Genet 18, 348-351.

Rakyan, V. K., Chong, S., Champ, M. E., Cuthbert, P. C., Morgan, H. D., Luu, K. V., and Whitelaw, E. (2003). Transgenerational inheritance of epigenetic states at the murine Axin(Fu) allele occurs after maternal and paternal transmission. Proc Natl Acad Sci U S A 100, 2538-2543.

Rasko, J. E., Battini, J. L., Gottschalk, R. J., Mazo, I., and Miller, A. D. (1999). The RD114/simian type D retrovirus receptor is a neutral amino acid transporter. Proc Natl Acad Sci U S A 96, 2129-2134.

Reed, S. C. (1937). The Inheritance and Expression of Fused, a New Mutation in the House Mouse. Genetics 22, 1-13.

Reik, W., and Walter, J. (2001). Genomic imprinting: parental influence on the genome. Nat Rev Genet 2, 21-32.

Rosenberg, M. I., and Parkhurst, S. M. (2002). Drosophila Sir2 is required for heterochromatic silencing and by euchromatic Hairy/E(Spl) bHLH repressors in segmentation and sex determination. Cell 109, 447-458.

Santos, F., Hendrich, B., Reik, W., and Dean, W. (2002). Dynamic reprogramming of DNA methylation in the early mouse embryo. Dev Biol 241, 172-182.

Sapienza, C., Paquette, J., Tran, T. H., and Peterson, A. (1989). Epigenetic and genetic factors affect transgene methylation imprinting. Development 107, 165-168.

Schotta, G., Ebert, A., Krauss, V., Fischer, A., Hoffmann, J., Rea, S., Jenuwein, T., Dorn, R., and Reuter, G. (2002). Central role of Drosophila SU(VAR)3-9 in histone H3-K9 methylation and heterochromatic gene silencing. Embo J 21, 1121-1131.

Schotta, G., Ebert, A., Dorn, R., and Reuter, G. (2003). Position-effect variegation and the genetic dissection of chromatin regulation in Drosophila. Semin Cell Dev Biol 14, 67-75.

Schweizer, J., Valenza-Schaerly, P., Goret, F., and Pourcel, C. (1998). Control of expression and methylation of a hepatitis B virus transgene by strain-specific modifiers. DNA Cell Biol 17, 427-435.

Seum, C., Spierer, A., Pauli, D., Szidonya, J., Reuter, G., and Spierer, P. (1996). Position-effect variegation in Drosophila depends on dose of the gene encoding the E2F transcriptional activator and cell cycle regulator. Development 122, 1949-1956.

Shiio, Y., and Eisenman, R. N. (2003). Histone sumoylation is associated with transcriptional repression. Proc Natl Acad Sci U S A 100, 13225-13230.

131

Shin, S., and Janknecht, R. (2007). Diversity within the JMJD2 histone demethylase family. Biochem Biophys Res Commun 353, 973-977.

Sinclair, D. A., Clegg, N. J., Antonchuk, J., Milne, T. A., Stankunas, K., Ruse, C., Grigliatti, T. A., Kassis, J. A., and Brock, H. W. (1998). Enhancer of Polycomb is a suppressor of position-effect variegation in Drosophila melanogaster. Genetics 148, 211-220.

Solari, A. J. (1974). The behavior of the XY pair in mammals. Int Rev Cytol 38, 273- 317.

Stein, S., Fritsch, R., Lemaire, L., and Kessel, M. (1996). Checklist: vertebrate homeobox genes. Mech Dev 55, 91-108.

Strodicke, M., Karberg, S., and Korge, G. (2000). Domina (Dom), a new Drosophila member of the FKH/WH gene family, affects morphogenesis and is a suppressor of position-effect variegation. Mech Dev 96, 67-78.

Tachibana, M., Sugimoto, K., Fukushima, T., and Shinkai, Y. (2001). Set domain- containing protein, G9a, is a novel lysine-preferring mammalian histone methyltransferase with hyperactivity and specific selectivity to lysines 9 and 27 of histone H3. J Biol Chem 276, 25309-25317.

Takagi, N., and Sasaki, M. (1975). Preferential inactivation of the paternally derived X chromosome in the extraembryonic membranes of the mouse. Nature 256, 640-642.

Takeda, S., and Paszkowski, J. (2006). DNA methylation and epigenetic inheritance during plant gametogenesis. Chromosoma 115, 27-35.

Thomas, T., Loveland, K. L., and Voss, A. K. (2007). The genes coding for the MYST family histone acetyltransferases, Tip60 and Mof, are expressed at high levels during sperm development. Gene Expr Patterns 7, 657-665.

Tremblay, K. D., Duran, K. L., and Bartolomei, M. S. (1997). A 5' 2-kilobase-pair region of the imprinted mouse H19 gene exhibits exclusive paternal methylation throughout development. Mol Cell Biol 17, 4322-4329.

Tschiersch, B., Hofmann, A., Krauss, V., Dorn, R., Korge, G., and Reuter, G. (1994). The protein encoded by the Drosophila position-effect variegation suppressor gene Su(var)3-9 combines domains of antagonistic regulators of homeotic gene complexes. Embo J 13, 3822-3831.

Turner, J. M. (2007). Meiotic sex chromosome inactivation. Development 134, 1823- 1831.

132

Ukai, H., Ishii-Oba, H., Ukai-Tadenuma, M., Ogiu, T., and Tsuji, H. (2003). Formation of an active form of the interleukin-2/15 receptor beta-chain by insertion of the intracisternal A particle in a radiation-induced mouse thymic lymphoma and its role in tumorigenesis. Mol Carcinog 37, 110-119.

Valley, C. M., Pertz, L. M., Balakumaran, B. S., and Willard, H. F. (2006). Chromosome-wide, allele-specific analysis of the histone code on the human X chromosome. Hum Mol Genet 15, 2335-2347.

Van Putten, L. M. (1958). The life span of red cells in the rat and the mouse as determined by labeling with DFP32 in vivo. Blood 13, 789-794.

Vickaryous, N. K. (2005) An ENU mutagenesis screen to identify modifiers of gene silencing in the mouse, PhD Thesis, University of Sydney, Sydney.

Wakeland, E., Morel, L., Achey, K., Yui, M., and Longmate, J. (1997). Speed congenics: a classic technique in the fast lane (relatively speaking). Immunol Today 18, 472-477.

Walsh, C. P., Chaillet, J. R., and Bestor, T. H. (1998). Transcription of IAP endogenous retroviruses is constrained by cytosine methylation. Nat Genet 20, 116- 117.

Wang, J., Mager, J., Chen, Y., Schneider, E., Cross, J. C., Nagy, A., and Magnuson, T. (2001a). Imprinted X inactivation maintained by a mouse Polycomb group gene. Nat Genet 28, 371-375.

Wang, Y., Zhang, W., Jin, Y., Johansen, J., and Johansen, K. M. (2001b). The JIL-1 tandem kinase mediates histone H3 phosphorylation and is required for maintenance of chromatin structure in Drosophila. Cell 105, 433-443.

Weber, D. J., Rutala, W. A., Samsa, G. P., Bradshaw, S. E., and Lemon, S. M. (1986). Impaired immunogenicity of hepatitis B vaccine in obese persons. N Engl J Med 314, 1393.

Weber, J. S., Salinger, A., and Justice, M. J. (2000). Optimal N-ethyl-N-nitrosourea (ENU) doses for inbred mouse strains. Genesis 26, 230-233.

Weber, M., Davies, J. J., Wittig, D., Oakeley, E. J., Haase, M., Lam, W. L., and Schubeler, D. (2005). Chromosome-wide and promoter-specific analyses identify sites of differential DNA methylation in normal and transformed human cells. Nat Genet 37, 853-862.

West, J. D., Frels, W. I., Chapman, V. M., and Papaioannou, V. E. (1977). Preferential expression of the maternally derived X chromosome in the mouse yolk sac. Cell 12, 873-882.

133

Yamamoto, Y., Girard, F., Bello, B., Affolter, M., and Gehring, W. J. (1997). The cramped gene of Drosophila is a member of the Polycomb-group, and interacts with mus209, the gene encoding Proliferating Cell Nuclear Antigen. Development 124, 3385-3394.

Yoder, J. A., Walsh, C. P., and Bestor, T. H. (1997). Cytosine methylation and the ecology of intragenomic parasites. Trends Genet 13, 335-340.

Zhang, Y., Ng, H. H., Erdjument-Bromage, H., Tempst, P., Bird, A., and Reinberg, D. (1999). Analysis of the NuRD subunits reveals a histone deacetylase core complex and a connection with DNA methylation. Genes Dev 13, 1924-1935.

134

Appendix

Appendix 1

Microsatellite markers used in the preliminary round of mapping MommeD6

Average A. Tm MgCl Chr Locus bp 2 Resolution Forward primer Reverse primer product (oC) (mM) size (bp) 2 D2Mit1 3803361 58 2 15% polyacrylamide CTTTTTCGTATGTGGTGGGG AACATTGGGCCTCTATGCAC 123 2 D2Mit296 31146564 56 1.5 4%(w/v) agarose CAACTGTAAATCCAGTCGTAGGG CTCTGCTGAGGTTACTGTGGG 147 2 D2Mit92 71380492 56 1.5 3%(w/v) agarose TGTATGCACAGGTATTTCCCC TGAGGAAAGGGGATAAAATTTG 149 2 D2Mit395 119216354 56 2 3%(w/v) agarose AGGTCAGCCTGGACTATATGG AGCATCCATGGGATAATGGT 125 2 D2Mit48 155816121 49.5 1.5 3%(w/v) agarose GCTCTGCAGAAGATGCTGC GCTGAGACGCAGAGTCGC 130 2 D2Mit74 180774022 60 1.5 3%(w/v) agarose CCAAGCTTGCAGTTTGTTAGC AGGTGTTATTGAGCCCTGTATAGC 153 3 D3Mit164 7504187 56 1.5 3%(w/v) agarose GCTCCTGGGAAAGGAAGAAT GATACTTGGGGTTGTGCATACA 135 3 D3Mit63 41138949 57 1.5 3%(w/v) agarose CTATGGACTTGTGACATAGGAGTG ACCACAAGATGGAGTAAGAGTTCA 162 3 D3Mit49 89318587 56 1.5 3%(w/v) agarose CTTTTCTCGCCCCACTTTC TCCTTTTAGTTTTTGATCCTCTGG 132 3 D3Mit17 143584611 51 1.5 3%(w/v) agarose CATGGCTCCATGGTTCTTG CCACGGAGAACAACTGAAGA 198 3 D3Mit88 153878328 56 1.5 3%(w/v) agarose GGCTGCTACTCTCACACGC GTCCTTGGTGGCTGAACG 167 4 D4Mit193 32496577 58 1.5 3%(w/v) agarose TATTTTAATTTTAGCCCATCAGGG AAAGACATACAATTGATCCACAGG 136 4 D4Mit17 62851655 54.5 1.5 3%(w/v) agarose TGGCCAACCTCTGTGCTTCC ACAGTTGTCCTCTGACATCC 147 4 D4Mit146 109298867 56 1.5 3%(w/v) agarose AAAAATGACAGCATTATGTTGGG CTCCCTCAGTCTTGCTTTGG 125 4 D4Mit251 136199272 49 1.5 3%(w/v) agarose AAAAATCGTTCTTTGACTTCTACATG TTTAAAAGGGTTTCTTTATCCTGTG 114 4 D4Mit42 150413794 49 1.5 4%(w/v) agarose CATGTTTGCCACCCTGAAAC CCTCACTTAGGCAGGTGACTC 100 5 D5Mit48 8803678 59 1.5 15% polyacrylamide GACTATCATCCAAGCCAAGACC AAAAGACACTTTCCCTGACATAGC 199 5 D5Mit148 32252471 61 1.5 3%(w/v) agarose GCTGCAAAGAAGAGAGAGGG CCTCTGGCCAGCATGATATA 149 5 D5Mit205 94208131 60 1.5 3%(w/v) agarose GAGACGGCAGTTGGAGAGTC GGCTAAGTGATAATCATTGCAGG 132 5 D5Mit158 115224168 58 1.5 15% polyacrylamide AAAGACGCTGAGGAGTCACTG CAGGAGACCTTGTAATAAAGGAAA 313 6 D6Mit138 4453823 59 1.5 3%(w/v) agarose GCTCTTATTAATGAAGAAGAAGGAGG CAAAGAAAGCATTTCAAGACTGC 135 6 D6Mit188 75377092 59 1.5 3%(w/v) agarose CTTTAGTCATTATTAGGATTGCCTATG TGGGATAGCATTGGAAACGT 128 6 D6Mit105 107761387 59 1.5 3%(w/v) agarose CTGTCTCCACTACTTCTATTCCTGG CAAAAGCCTTATATATTACACCTCACC 237 6 D6Mit373 147009626 58 1.8 3%(w/v) agarose TTCTGGGGTGAGAGGCAG AGAACATTGACAAAAAGTGATTGTG 106 7 D7Mit178 0.5 cM 46 2.5 3%(w/v) agarose ACCTCTGATTTCAGAACCCTTG TAGAGAGCCACTAGCATATCATAACC 200 7 D7Mit69 48933118 55 1.5 3%(w/v) agarose CCCACCAGAGATCACCAAGT CACAATGAAGGCTGAAAGCA 233 7 D7Mit220 104268546 56 1.5 3%(w/v) agarose AAGCATGCAAGCACACTCAC ATGCACACAGGCAGTCACTC 135 7 D7Mit223 144419262 54.5 1.5 3%(w/v) agarose ATGCACATGAGTGTGTGTATGC TCCTGTGTCTGACGCTCATC 106 8 D8Mit124 14723137 61 1.5 3%(w/v) agarose CAACTGTGTATCATAAACTGGGAA GAAGAATCACTCAGCAGTGTATGG 129 8 D8Mit190 37456381 56 1.5 3%(w/v) agarose CTTTGTTGCTGTTTCATTCTGG AGTCATATACAAGGTCAACCTGAGC 133 8 D8Mit248 95574476 56 1.5 3%(w/v) agarose ATCCCTCAAGCAGTACCCCT AGCAGAGGACCACACCTTACA 148 8 D8Mit215 118746715 53 1.8 3%(w/v) agarose AATACACAAGGTTGGCCTCA ATGTGTGGATATTCATGTGCTC 178 8 D8Mit121 126781454 59 1.5 15% polyacrylamide CGGTCAATCCCGAGTTTG CAAGGCTGTCAGTCAGTGTAGG 256 9 D9Mit43 10041139 55 1.5 3%(w/v) agarose CAGTACAGCATTTACCACACAGC CCCCATGTTATTTCCTGGG 132 9 D9Mit285 40405563 56 1.5 3%(w/v) agarose AAATACATTGCTGATTATATCAGAGA GGACTCTAGATCTCATCAGGGA 125 9 D9Mit336 65375870 56 1.5 3%(w/v) agarose AAGTGGTTCACAGAAATGTATACAGG TTTTCTTTCTGTGGTAAAGGGG 122 D9Mit1000 / 9 114527594 53.5 1.5 3%(w/v) agarose GCCTGGGCTACATGAGACTC GGGAATTCCAATACACTAAAGGG 224 D8Mit46 9 D9Mit18 120138143 56 1.5 3%(w/v) agarose TCACTGTAGCCCAGAGCAGT CCTGTTGTCAACACCTGATG 180 10 D10Mit28 9103565 59 1.5 3%(w/v) agarose CCTCCTGTATGTGTATTTAAAGCA CTGCCCATCTGACCCTGATA 147 10 D10Mit3 28841602 55 1.5 3%(w/v) agarose GTTGATAGTCCCACCTCACTCA TGAGAAATTCCATCTGTGGC 244 10 D10Mit20 66407765 57 1.5 4%(w/v) agarose CACCCTCACACAGATATGCG GCATTGGGAAGTCCATGAGT 234 10 D10Mit230 89622039 55 1.5 3%(w/v) agarose AGATAGCCTAGGGGGTGCAT ATCAGTTTCCAATCGCTGCT 115 10 D10Mit267 119074063 58 1.5 3%(w/v) agarose ACACTTACAGTACCCTGGTGTGG GTGTGTGGGCGGATGTAAG 103 11 D11Mit19 25322121 59 1.5 3%(w/v) agarose CTAGCTGCTTCTAGAACCTTCCC TTTGATCCTGAGCACAAACG 141 11 D11Mit20 44582634 58 1.5 3%(w/v) agarose CCTGTCCAGGTTTGAGAGGA CTTGGGAGCCTCTTCGGT 114 11 D11Mit29 69610176 56 1.8 15% polyacrylamide TTGAGGCATGAGGGGATTAG TTTCCGTCATTGCTAAAGGG 147 11 D11Mit333 108529872 56 1.5 3%(w/v) agarose CATGTGGTTATTTTCTAGCCCC AGGCATCAATAACTATTTTTCAGTG 125 12 D12Mit2 42516678 52 1.5 3%(w/v) agarose ACACAGGCTAAAACATGGGC GCATCTGTATTCCACAGGCA 134 12 D12Mit143 80799115 54 1.5 3%(w/v) agarose CCCTATGCATGTACATTGTGAA CGTGGGCATTTATCTTTCCT 147 12 D12Mit28 105743145 55 1.5 4%(w/v) agarose TTGGCAGTCCAGAGGAGGT CCAGTTCTGGTGTCAGTTTTACC 142 13 D13Mit16 20300907 58.5 1.5 3%(w/v) agarose CCAGCTGAAGGCTTACTCGT AAAGTTAGAATCAGCCATTCAAGG 207 13 D13Mit139 51780403 56 1.5 3%(w/v) agarose AGAATAAGTCAAGGCTATGATGTGG TTGTTTGTTTGTTTGAAGTAGAACG 139

135

13 D13Mit144 97203236 60 1.5 3%(w/v) agarose AGGAGAATGCTAGGATTGTTTCC GAAAAGATGCATATACATGTGATGC 118 13 D13Mit78 119948098 58.5 1.5 3%(w/v) agarose ACAGCACGGGTTTATCATCC TATGCCTGCCAGGCTTCTAT 229 14 D14Mit18 46739212 59 1.5 3%(w/v) agarose AAGGTGGACCAGGAAGGAGT GACATTGAGAGACCAAAAAATGC 191 14 D14Mit68 71249390 56 1.5 15% polyacrylamide GTGGCATGCACAACCGTATA CCCTTTTGAGGTGCTTGTTT 153 14 D14Mit97 117317851 59 1.5 3%(w/v) agarose TCAGTCCAAACTCTGTTAATCTTCC CAGCTCCACATTTTTGCTCA 136 15 D15Mit53 13225180 56 1.5 3%(w/v) agarose CTCCCTTACCTTCGGCTCTT AGGGTAATTTCAATTAAACTCGTG 137 15 D15Mit92 71049723 59 1.5 3%(w/v) agarose AGTCTCTCTCCCCCTTCTCTC TGCCACAAGCACAATAGTATCC 147 15 D15Mit70 81029340 56 1.5 3%(w/v) agarose CATTGAGGGTTTGTAGGTTGG ACCCCTGCAAGTTGTCTTTG 149 15 D15Mit16.2 102816142 56 1.5 3%(w/v) agarose GACAGACAAAAGCCGAGACA CTTGGAATGCGACTGTCCTG 114 16 D16Mit131 7234363 57 1.5 3%(w/v) agarose TGGTGGTGGTGTTGATGGTA AAGACCATTTCTAATAAACAACACCC 140 16 D16Mit4 36159726 57 1.5 3%(w/v) agarose AGTTCCAGGCTACTTGGGGT GAGCCCTCATTGCAAATCAT 132 16 D16Mit139 65587777 60 1.5 3%(w/v) agarose GTATGTAAGGAATGGTCAAATTCTTG TCATTGTGATTGTGAAAGAATGC 150 16 D16Mit86 93022607 59 1.5 3%(w/v) agarose TAATGTGGCAAGCAACCAAA GCATGTTTCCATGTGTCTGG 128 17 D17Mit164 3881097 55 1.5 3%(w/v) agarose AGGCCCTAACATGTAGCAGG TATTATTGAGACTGTGGTTGTTGTTG 133 17 D17Mit24 37090217 55 1.5 3%(w/v) agarose ACCTCTCACCTCTCTCTGTG TGGAGAGACGTCCTATGATG 131 17 D17Mit217.3 65786795 55 1.5 3%(w/v) agarose GCATTTGTAGCCTTATCCCTGTA AATATGTAAGACGCCTCCTTTCC 158 17 D17Mit123 93503910 56 1.5 4%(w/v) agarose CACAAGGAGGGAGCCTGTAG CACCGTAAGAGTCTAATAATAAGGGG 133 18 D18Mit60 32632119 59 1.5 15% polyacrylamide ACCTGACACCATTTTCAGGC ATCCTTGAGCCTGTTAAAAGACA 205 18 D18Mit91 55505517 56 1.5 4%(w/v) agarose TCCACAAATGTTGGCAAAGA TTTCTGGCCATATTGGAAGC 140 18 D18Mit48 77012578 57 1.5 3%(w/v) agarose TTGCACTCACAGGGCACAT TCAGAGTTTCCAGAAGACACCA 168 19 D19Mit68 3645155 57 1.5 3%(w/v) agarose CCAATACAAATCAGACTCAATAGTCG AGGGTCTCCCCATCTTCCTA 132 19 D19Mit11 42447920 59 1.5 3%(w/v) agarose TCAAAGTCAAGGTGGGCAG ACTTTCCAGATGTTGGGCAC 146 19 D19Mit71 59653090 56 1.5 3%(w/v) agarose ATGATTCCCGCAGTTTTGTC TCTCAACTGTTATTCCTCAATAGCC 136

Microsatellite markers and SNPs used for chromosome 14 fine mapping (MommeD6)

Average A. Tm MgCl Locus bp 2 Resolution Forward primer Reverse primer product (oC) (mM) size (bp) D14Mit11 10883087 55 1.5 3%(w/v) agarose AATATTTTCATGTTTGGAGTCGTG CACTGCAGTGTCAATTTCTACTTT 150 D14Mit50 19800934 55 1.5 3%(w/v) agarose GAGGGGGAATCCTAGTGCTC AGCAAAGCCCTATCCACATG 190 D14Mit221 22279769 53 1.5 15% polyacrylamide ATTCTTACTGGAAGAAACAAACTATGC CATGGTGTACTCTCTCACTCGTG 108 2%(w/v) agarose, rs13482099 23609247 59 1.5 CCAACTCTGTCTGCTTGTGC TTGCTAACCACATGCTCTGC 413 sequence product 2%(w/v) agarose, rs13482101 24299096 63 1.5 TTTCCCACATGCTTGTCAAA GCCCAGGTTCTGGGTTTAGT 419 sequence product Cut with AlwNI, rs13482102 24422495 59 1.5 CCATTCTTCCTGCCTCTCAC GCCTGTCCAGGAGATGAGAA 699 2%(w/v) agarose Cut with BglII, rs6402490 26225733 59 1.5 ACCTACGGCAGAAGGATGAA CTCTGCGTTATCCACGGTTT 623 2%(w/v) agarose Cut with BtsI, rs13482109 26458650 59 1.5 AAAGCTCCCTGCTACCCATT CAAAGGAAATTCAGGCCAGT 409 2%(w/v) agarose Cut with NsiI, rs6396829 26827847 55 1.5 CATGCAAACAGCAAACACCT TCCTTGGATATTTGGGCATAA 569 2%(w/v) agarose D14Mit14 28495267 59 1.5 3%(w/v) agarose GCACATTCCAAAACACATGC GGGATGGTGTCAATCAATCC 236 D14Mit60 46019670 55 1.5 3%(w/v) agarose AGGCTGCCCATAAAAGGG GTTTGTGCTAATGTTCTCATCTGG 108 D14Mit18 46739212 59 1.5 3%(w/v) agarose AAGGTGGACCAGGAAGGAGT GACATTGAGAGACCAAAAAATGC 191 D14Mit68 71249390 56 1.5 15% polyacrylamide GTGGCATGCACAACCGTATA CCCTTTTGAGGTGCTTGTTT 153 D14Mit195 86820406 59 1.5 3%(w/v) agarose AGAGATAATCAATTCACACAAATTGG TTCTGTGTTCAATGTCCACACA 121 D14Mit264 98838948 55 1.5 15% polyacrylamide TTCTCTGGAGCTGGGGTTAC CATTTAAGAAACTGCTCTGGGG 138 D14Mit97 117317851 59 1.5 3%(w/v) agarose TCAGTCCAAACTCTGTTAATCTTCC CAGCTCCACATTTTTGCTCA 136

136

Primers used for the sequencing of MommeD6 candidate genes

Cphx

Exons Product size A. Tm MgCl 2 Forward primer Reverse primer covered (bp) (oC) (mM)

1 273 58 1.5 CCTAAGGTGATCCTGGTGGA AGACATCCCTGGGTAGAGCA 2 490 58 1.5 CCTTTTGTGGGTAGAAATGAGG TGTTAACACTGCCCCTGACA 3+4 681 58 1.5 TTTGGAGCCCATCTACAGAAC TGTACAACGAGCCAGTCTGC 4 677 58 1.5 TGACCAGCTTGAGACACAGG GCCACTGAGAAGGTCAAAGC 4 696 58 1.5 TTTCCTTCCCCTCACTGTCA CTGGTGACACACCCACTCTC

1110051B16Rik

Exons Product size A. Tm MgCl 2 Forward primer Reverse primer covered (bp) (oC) (mM)

1 375 58 1.5 CCATCAAGGAGTTAAACCAGTG TCCTGAGGTTTGAGGAAACAA 2 300 58 1.5 CATGGAGCAGACAAAGCAG CTGCCCAGAAGAACTTAGCC 3 249 58 1.5 GTTTGGCTGATGTTCGGAGT CCACAACATGCAGTTGCTTA 4 376 58 1.5 AAGGCTGTGTCCTGTCACCT GTAGCCTGGCTTTGTCCATC 5 650 58 1.5 TGGAAGGGTTAGCCTGTGTC TTTGTCAGGCATCCATTCAG 5 641 58 1.5 CAGAGGGCATAATCCCAGAG GGCAGGAAATTGACTTGAGC 5 672 58 1.5 GGCGTGAAATCAGTGGCTAT GCATCTAGTTACCGCTGCTG

Hesx1

Exons Product size A. Tm MgCl 2 Forward primer Reverse primer covered (bp) (oC) (mM)

1 723 56 1.5 CTGCTCTTCTATCCACCCTGT CTATTCCCTTCCGACCCTGT 2 406 56 1.5 TGGGGGAGACAATTCTTTTG ACTGGGACCCCAGAGTGTCT 3+4 697 56 1.5 AAGGCCCAAATTTAAGTGCAT GGGAAGTGTTAGGGGAGGAA

SNORA71

Exons Product size A. Tm MgCl 2 Forward primer Reverse primer covered (bp) (oC) (mM)

1 408 55 1.5 ATCCAGGATGGAGTGCAAAG AGGATGTGACCAGGGTTGAC

SNORA67

Exons Product size A. Tm MgCl 2 Forward primer Reverse primer covered (bp) (oC) (mM)

1 526 63 1.5 AGTCTCTGACCCCCACACAC ACGTACCCAGAGCCAAGAGA

Appl1

Exons Product size A. Tm MgCl 2 Forward primer Reverse primer covered (bp) (oC) (mM)

1 600 53 1.5 GCCACTGCTCTGGTAGAGAAC CGCGATAAAAACAGCACCTC 2 300 55 1.5 TGAAAAGTCAGAATTACTAGAGTTCAA TGAGCCTAAGAGGAAGTAAGGTG 3 229 55 1.5 CGAAGGTGGCCTAGATATTGTC GAATCTGGATGAAAGTTTATCTACACA 4 230 55 1.5 TGGAAGCATAAATTGTGTTACTCAG GTTTTATAGTTTGCAGTAGAAAGCTG 5 395 55 1.5 CAAAAACAAAACAAAATTTTAACCA GGTATGTTGTGGTCAGAACTGG 6 225 55 1.5 CTTTTGCTTGAGTTTTAGTTGTGA CACAAAAACCAGCCATGATGT 7 250 55 1.5 GCTATGGGGAGAATTTGAACA CACATCCAGGCAAAACATCT 8 376 55 1.5 AAAACAGGAAGGCTGTGTTCA AAAACCACAATACAAATCTACTGACAA 9 277 55 1.5 TCCTAGAAATGGTTGATGTGATTT TGACAGGAAAACTAACCCTTAGGA 10 320 55 1.5 ATTTGGGCAAATAGGATTGA CCAAAACAATCCAGCAAACA 11 500 55 1.5 CTGCAAGTCGTTTTGTTTTCA CCAACACCCTCACACAGACA 12 419 55 1.5 GGCCTTCTTTCCCTGAAGTC TTGGGAACAGATTAGCTAGGG 13 250 53 1.5 TGCTTCTAATGAAAATTCCGTCT AAATGAGTCAATTAATACAAGCCATA 14 588 65 1.5 TGCTAGGTGATAGCAGTACAAACC GGCTGCACTCTGGAACTCAC 15 400 54 1.5 TGCTTTTCATTGGAGTTTGATTT GGCAATTACAGAAAAGACAGAACA 16 239 57 1.5 GAGCTTTTCTTTGTAAACATGTGAAG AGCAGCAGAACAAATGCAAA 17+18 456 60 1.5 GAGTTAAGGCCAACCTGTGC CGTACCAGGATACATAGGACCA 19 373 54 1.5 TGTGTCTTTAAAATTCTTACCTGTGA CATCAAGCAGCTCAACTGGA 20 249 56 1.5 TCATCCATTTGGGTATTTGAA CCTACCATTGATCTTGTTTAGCA 21 296 56 1.5 AAATGTACTGAAAAGTTACAAAGCTCA TGCAATGAATCAACAAGCAGA 22 294 60 1.5 AGAGCTCCATCGGAAACTGC CCATGATCTTCAGCATGGAT

137

Microsatellite markers used in the preliminary round of mapping MommeD9

Average A. Tm MgCl Chr Locus bp 2 Resolution Forward primer Reverse primer product (oC) (mM) size (bp) 2 D2Mit1 3803361 58 2 15% polyacrylamide CTTTTTCGTATGTGGTGGGG AACATTGGGCCTCTATGCAC 123 2 D2Mit74 180774022 60 1.5 3%(w/v) agarose CCAAGCTTGCAGTTTGTTAGC AGGTGTTATTGAGCCCTGTATAGC 153 3 D3Mit164 7504187 56 1.5 3%(w/v) agarose GCTCCTGGGAAAGGAAGAAT GATACTTGGGGTTGTGCATACA 135 3 D3Mit88 153878328 56 1.5 3%(w/v) agarose GGCTGCTACTCTCACACGC GTCCTTGGTGGCTGAACG 167 4 D4Mit193 32496577 58 1.5 3%(w/v) agarose TATTTTAATTTTAGCCCATCAGGG AAAGACATACAATTGATCCACAGG 136 4 D4Mit42 150413794 49 1.5 4%(w/v) agarose CATGTTTGCCACCCTGAAAC CCTCACTTAGGCAGGTGACTC 100 5 D5Mit48 8803678 59 1.5 15% polyacrylamide GACTATCATCCAAGCCAAGACC AAAAGACACTTTCCCTGACATAGC 199 5 D5Mit158 115224168 58 1.5 15% polyacrylamide AAAGACGCTGAGGAGTCACTG CAGGAGACCTTGTAATAAAGGAAA 313 6 D6Mit138 4453823 59 1.5 3%(w/v) agarose GCTCTTATTAATGAAGAAGAAGGAGG CAAAGAAAGCATTTCAAGACTGC 135 6 D6Mit373 147009626 58 1.8 3%(w/v) agarose TTCTGGGGTGAGAGGCAG AGAACATTGACAAAAAGTGATTGTG 106 7 D7Mit178 0.5 cM 46 2.5 3%(w/v) agarose ACCTCTGATTTCAGAACCCTTG TAGAGAGCCACTAGCATATCATAACC 200 7 D7Mit223 144419262 54.5 1.5 3%(w/v) agarose ATGCACATGAGTGTGTGTATGC TCCTGTGTCTGACGCTCATC 106 8 D8Mit124 14723137 61 1.5 3%(w/v) agarose CAACTGTGTATCATAAACTGGGAA GAAGAATCACTCAGCAGTGTATGG 129 8 D8Mit121 126781454 59 1.5 15% polyacrylamide CGGTCAATCCCGAGTTTG CAAGGCTGTCAGTCAGTGTAGG 256 9 D9Mit43 10041139 55 1.5 3%(w/v) agarose CAGTACAGCATTTACCACACAGC CCCCATGTTATTTCCTGGG 132 9 D9Mit18 120138143 56 1.5 3%(w/v) agarose TCACTGTAGCCCAGAGCAGT CCTGTTGTCAACACCTGATG 180 10 D10Mit28 9103565 59 1.5 3%(w/v) agarose CCTCCTGTATGTGTATTTAAAGCA CTGCCCATCTGACCCTGATA 147 10 D10Mit267 119074063 58 1.5 3%(w/v) agarose ACACTTACAGTACCCTGGTGTGG GTGTGTGGGCGGATGTAAG 103 11 D11Mit19 25322121 59 1.5 3%(w/v) agarose CTAGCTGCTTCTAGAACCTTCCC TTTGATCCTGAGCACAAACG 141 11 D11Mit333 108529872 56 1.5 3%(w/v) agarose CATGTGGTTATTTTCTAGCCCC AGGCATCAATAACTATTTTTCAGTG 125 12 D12Mit2 42516678 52 1.5 3%(w/v) agarose ACACAGGCTAAAACATGGGC GCATCTGTATTCCACAGGCA 134 12 D12Mit28 105743145 55 1.5 4%(w/v) agarose TTGGCAGTCCAGAGGAGGT CCAGTTCTGGTGTCAGTTTTACC 142 13 D13Mit16 20300907 58.5 1.5 3%(w/v) agarose CCAGCTGAAGGCTTACTCGT AAAGTTAGAATCAGCCATTCAAGG 207 13 D13Mit78 119948098 58.5 1.5 3%(w/v) agarose ACAGCACGGGTTTATCATCC TATGCCTGCCAGGCTTCTAT 229 14 D14Mit18 46739212 59 1.5 3%(w/v) agarose AAGGTGGACCAGGAAGGAGT GACATTGAGAGACCAAAAAATGC 191 14 D14Mit97 117317851 59 1.5 3%(w/v) agarose TCAGTCCAAACTCTGTTAATCTTCC CAGCTCCACATTTTTGCTCA 136 15 D15Mit53 13225180 56 1.5 3%(w/v) agarose CTCCCTTACCTTCGGCTCTT AGGGTAATTTCAATTAAACTCGTG 137 15 D15Mit16.2 102816142 56 1.5 3%(w/v) agarose GACAGACAAAAGCCGAGACA CTTGGAATGCGACTGTCCTG 114 16 D16Mit131 7234363 57 1.5 3%(w/v) agarose TGGTGGTGGTGTTGATGGTA AAGACCATTTCTAATAAACAACACCC 140 16 D16Mit86 93022607 59 1.5 3%(w/v) agarose TAATGTGGCAAGCAACCAAA GCATGTTTCCATGTGTCTGG 128 17 D17Mit164 3881097 55 1.5 3%(w/v) agarose AGGCCCTAACATGTAGCAGG TATTATTGAGACTGTGGTTGTTGTTG 133 17 D17Mit123 93503910 56 1.5 4%(w/v) agarose CACAAGGAGGGAGCCTGTAG CACCGTAAGAGTCTAATAATAAGGGG 133 18 D18Mit60 32632119 59 1.5 15% polyacrylamide ACCTGACACCATTTTCAGGC ATCCTTGAGCCTGTTAAAAGACA 205 18 D18Mit48 77012578 57 1.5 3%(w/v) agarose TTGCACTCACAGGGCACAT TCAGAGTTTCCAGAAGACACCA 168 19 D19Mit68 3645155 57 1.5 3%(w/v) agarose CCAATACAAATCAGACTCAATAGTCG AGGGTCTCCCCATCTTCCTA 132 19 D19Mit71 59653090 56 1.5 3%(w/v) agarose ATGATTCCCGCAGTTTTGTC TCTCAACTGTTATTCCTCAATAGCC 136

Microsatellite markers and SNPs used for chromosome 7 fine mapping (MommeD9)

Average A. Tm MgCl Locus bp 2 Resolution Forward primer Reverse primer product (oC) (mM) size (bp) D7Mit178 0.5 cM 46 2.5 3%(w/v) agarose ACCTCTGATTTCAGAACCCTTG TAGAGAGCCACTAGCATATCATAACC 200 2%(w/v) agarose, rs13479101 3214872 58 1.5 TGATAACTGCCCCCTTGG AGGACCCAAGCAGTCTTTTT 250 sequence product Cut with BsrI, rs38424645 9623600 55 1.5 AAGAACTCCTTCAGTCCCCATA TTTTGTGGTTTTTACTGAAGTTGA 660 2%(w/v) agarose Cut with DdeI, rs38306571 15145305 63 1.5 GGTCTCATGTAGCCCATGCT GTGGGTGGGATGATGGATAG 776 2%(w/v) agarose D7Mit57 18856293 61 1.5 3%(w/v) agarose TTCCCTCTAGAACTCTGACCTCC AGTTCAGAGCCGAGACTAGGC 146 D7Mit294 26998202 56 1.5 3%(w/v) agarose TAGTGGGAAAGAGAGAAACAATCC TAATGTTTAATCTTGTCGTCTTAGTGG 118 D7Mit247 36851585 55 1.5 3%(w/v) agarose TCTTTTGACTTGATTTTGGCG TGGAGGAGACATATCTTTGCG 118 D7Mit69 48933118 55 1.5 3%(w/v) agarose CCCACCAGAGATCACCAAGT CACAATGAAGGCTGAAAGCA 233 D7Mit220 104268546 56 1.5 3%(w/v) agarose AAGCATGCAAGCACACTCAC ATGCACACAGGCAGTCACTC 135 D7Mit223 144419262 54.5 1.5 3%(w/v) agarose ATGCACATGAGTGTGTGTATGC TCCTGTGTCTGACGCTCATC 106

Primers used for the sequencing of the MommeD9 candidate gene Chmp2a

Exons Product size A. Tm MgCl 2 Forward primer Reverse primer covered (bp) (oC) (mM)

1 300 56 1.5 TCCTAGCACTTCCCAGCATT AGGTGAAGACCTCGATGTGG 2 383 56 1.5 TCAAGCGTACCTCCAGTTTCT TTGCTCAAAGCACTGTTTCAC 3-6 952 56 1.5 GAGAGGGGTGAGATTTTGGA ATCTTCACTCGAGCCTGGAA

138

Microsatellite markers used for speed congenics

Average A. Tm MgCl Chr Locus bp 2 Resolution Forward primer Reverse primer product (oC) (mM) size (bp) 1 D1Mit3 19797184 52 1.5 3%(w/v) agarose TTTTTGTTTTCTTTTCTTTTCCC CCCTCTTCTGGTTTCCACAT 160 1 D1Mit440 90617199 57 1.5 3%(w/v) agarose TCCACACAAGGTGTCCTCTG GCTCAGGTGACCTCCAAAAC 114 1 D1Mit102 149011738 55 1.5 3%(w/v) agarose AAATACCAGCAAAACAATAAAGGC TGAATTAAAATTGCAGAGGCG 113 1 D1Mit111 170844217 52.5 1.5 3%(w/v) agarose ATTGCCTGACTCCAGTATTCTACC TTAGGTGTGTGAAAGACATTCCC 171 2 D2Mit296 31146564 56 1.5 4%(w/v) agarose CAACTGTAAATCCAGTCGTAGGG CTCTGCTGAGGTTACTGTGGG 147 2 D2Mit92 71380492 56 1.5 3%(w/v) agarose TGTATGCACAGGTATTTCCCC TGAGGAAAGGGGATAAAATTTG 149 2 D2Mit395 119216354 56 2 3%(w/v) agarose AGGTCAGCCTGGACTATATGG AGCATCCATGGGATAATGGT 125 2 D2Mit48 155816121 49.5 1.5 3%(w/v) agarose GCTCTGCAGAAGATGCTGC GCTGAGACGCAGAGTCGC 130 3 D3Mit164 7504187 56 1.5 3%(w/v) agarose GCTCCTGGGAAAGGAAGAAT GATACTTGGGGTTGTGCATACA 135 3 D3Mit63 41138949 57 1.5 3%(w/v) agarose CTATGGACTTGTGACATAGGAGTG ACCACAAGATGGAGTAAGAGTTCA 162 3 D3Mit49 89318587 56 1.5 3%(w/v) agarose CTTTTCTCGCCCCACTTTC TCCTTTTAGTTTTTGATCCTCTGG 132 3 D3Mit17 143584611 51 1.5 3%(w/v) agarose CATGGCTCCATGGTTCTTG CCACGGAGAACAACTGAAGA 198 4 D4Mit193 32496577 58 1.5 3%(w/v) agarose TATTTTAATTTTAGCCCATCAGGG AAAGACATACAATTGATCCACAGG 136 4 D4Mit17 62851655 54.5 1.5 3%(w/v) agarose TGGCCAACCTCTGTGCTTCC ACAGTTGTCCTCTGACATCC 147 4 D4Mit146 109298867 56 1.5 3%(w/v) agarose AAAAATGACAGCATTATGTTGGG CTCCCTCAGTCTTGCTTTGG 125 4 D4Mit251 136199272 49 1.5 3%(w/v) agarose AAAAATCGTTCTTTGACTTCTACATG TTTAAAAGGGTTTCTTTATCCTGTG 114 5 D5Mit148 32252471 61 1.5 3%(w/v) agarose GCTGCAAAGAAGAGAGAGGG CCTCTGGCCAGCATGATATA 149 5 D5Mit233 52985474 57 1.5 3%(w/v) agarose TCCCCTCTGATCTCCTCAGA CCTCCTAGAATACAATTCAATGTGG 147 5 D5Mit158 115224168 58 1.5 15% polyacrylamide AAAGACGCTGAGGAGTCACTG CAGGAGACCTTGTAATAAAGGAAA 313 5 D5Mit101 141967054 58 1.5 3%(w/v) agarose GCCAGCCAGCCTTGACTC GGCTTCGTGCATGTAAAACA 132 6 D6Mit138 4453823 59 1.5 3%(w/v) agarose GCTCTTATTAATGAAGAAGAAGGAGG CAAAGAAAGCATTTCAAGACTGC 135 6 D6Mit188 75377092 59 1.5 3%(w/v) agarose CTTTAGTCATTATTAGGATTGCCTATG TGGGATAGCATTGGAAACGT 128 6 D6Mit105 107761387 59 1.5 3%(w/v) agarose CTGTCTCCACTACTTCTATTCCTGG CAAAAGCCTTATATATTACACCTCACC 237 6 D6Mit373 147009626 58 1.8 3%(w/v) agarose TTCTGGGGTGAGAGGCAG AGAACATTGACAAAAAGTGATTGTG 106 7 D7Mit178 0.5 cM 46 2.5 3%(w/v) agarose ACCTCTGATTTCAGAACCCTTG TAGAGAGCCACTAGCATATCATAACC 200 7 D7Mit69 48933118 55 1.5 3%(w/v) agarose CCCACCAGAGATCACCAAGT CACAATGAAGGCTGAAAGCA 233 7 D7Mit220 104268546 56 1.5 3%(w/v) agarose AAGCATGCAAGCACACTCAC ATGCACACAGGCAGTCACTC 135 7 D7Mit223 144419262 54.5 1.5 3%(w/v) agarose ATGCACATGAGTGTGTGTATGC TCCTGTGTCTGACGCTCATC 106 8 D8Mit190 37456381 56 1.5 3%(w/v) agarose CTTTGTTGCTGTTTCATTCTGG AGTCATATACAAGGTCAACCTGAGC 133 8 D8Mit248 95574476 56 1.5 3%(w/v) agarose ATCCCTCAAGCAGTACCCCT AGCAGAGGACCACACCTTACA 148 8 D8Mit215 118746715 53 1.8 3%(w/v) agarose AATACACAAGGTTGGCCTCA ATGTGTGGATATTCATGTGCTC 178 9 D9Mit64 103743217 56 1.5 3%(w/v) agarose TTCACCAAACCTTATCTTACTCCA TGGAAGAAACAGTGTTGGGT 190 9 D9Mit248 58160696 56 1.8 3%(w/v) agarose TCAGAGTTCAGGAGGGCTGT TCTGAGAGGCCACATGTCTG 130 D9Mit1000 / 9 114527594 53.5 1.5 3%(w/v) agarose GCCTGGGCTACATGAGACTC GGGAATTCCAATACACTAAAGGG 224 D8Mit46 10 D10Mit3 28841602 55 1.5 3%(w/v) agarose GTTGATAGTCCCACCTCACTCA TGAGAAATTCCATCTGTGGC 244 10 D10Mit20 66407765 57 1.5 4%(w/v) agarose CACCCTCACACAGATATGCG GCATTGGGAAGTCCATGAGT 234 10 D10Mit267 119074063 58 1.5 3%(w/v) agarose ACACTTACAGTACCCTGGTGTGG GTGTGTGGGCGGATGTAAG 103 11 D11Mit20 44582634 58 1.5 3%(w/v) agarose CCTGTCCAGGTTTGAGAGGA CTTGGGAGCCTCTTCGGT 114 11 D11Mit29 69610176 56 1.8 15% polyacrylamide TTGAGGCATGAGGGGATTAG TTTCCGTCATTGCTAAAGGG 147 11 D11Mit333 108529872 56 1.5 3%(w/v) agarose CATGTGGTTATTTTCTAGCCCC AGGCATCAATAACTATTTTTCAGTG 125 12 D12Mit2 42516678 52 1.5 3%(w/v) agarose ACACAGGCTAAAACATGGGC GCATCTGTATTCCACAGGCA 134 12 D12Mit143 80799115 54 1.5 3%(w/v) agarose CCCTATGCATGTACATTGTGAA CGTGGGCATTTATCTTTCCT 147 12 D12Mit28 105743145 55 1.5 4%(w/v) agarose TTGGCAGTCCAGAGGAGGT CCAGTTCTGGTGTCAGTTTTACC 142 13 D13Mit275 37330987 57 1.5 3%(w/v) agarose TTAGCAAGGGAACAGAGAGAGG CAATCAAGGTATCCCTGTCTCC 107 13 D13Mit144 97203236 60 1.5 3%(w/v) agarose AGGAGAATGCTAGGATTGTTTCC GAAAAGATGCATATACATGTGATGC 118 13 D13Mit260 113486474 56 1.5 3%(w/v) agarose TAAATTTGGATGCAGACAATGG TTAAAAATAGAAATGGCTCTGTGTG 115 13 D13Mit78 119948098 58.5 1.5 3%(w/v) agarose ACAGCACGGGTTTATCATCC TATGCCTGCCAGGCTTCTAT 229 14 D14Mit18 46739212 59 1.5 3%(w/v) agarose AAGGTGGACCAGGAAGGAGT GACATTGAGAGACCAAAAAATGC 191 14 D14Mit68 71249390 56 1.5 15% polyacrylamide GTGGCATGCACAACCGTATA CCCTTTTGAGGTGCTTGTTT 153 14 D14Mit97 117317851 59 1.5 3%(w/v) agarose TCAGTCCAAACTCTGTTAATCTTCC CAGCTCCACATTTTTGCTCA 136 15 D15Mit53 13225180 56 1.5 3%(w/v) agarose CTCCCTTACCTTCGGCTCTT AGGGTAATTTCAATTAAACTCGTG 137 15 D15Mit92 71049723 59 1.5 3%(w/v) agarose AGTCTCTCTCCCCCTTCTCTC TGCCACAAGCACAATAGTATCC 147 15 D15Mit16.2 102816142 56 1.5 3%(w/v) agarose GACAGACAAAAGCCGAGACA CTTGGAATGCGACTGTCCTG 114 16 D16Mit4 36159726 57 1.5 3%(w/v) agarose AGTTCCAGGCTACTTGGGGT GAGCCCTCATTGCAAATCAT 132 16 D16Mit139 65587777 60 1.5 3%(w/v) agarose GTATGTAAGGAATGGTCAAATTCTTG TCATTGTGATTGTGAAAGAATGC 150 16 D16Mit86 93022607 59 1.5 3%(w/v) agarose TAATGTGGCAAGCAACCAAA GCATGTTTCCATGTGTCTGG 128 17 D17Mit133 24585209 59 1.5 3%(w/v) agarose TCTGCTGTGTTCACAGGTGA GCCCCTGCTAGATCTGACAG 188 17 D17Mit20 56912782 62 1.5 3%(w/v) agarose AGAACAGGACACCGGACATC TCATAAGTAGGCACACCAATGC 165 17 D17Mit123 93503910 56 1.5 4%(w/v) agarose CACAAGGAGGGAGCCTGTAG CACCGTAAGAGTCTAATAATAAGGGG 133 18 D18Mit177 41108480 56 1.5 15% polyacrylamide CTGTAGTTTATCAGTTCACCCTGTG TGTGCTGTTAAACAAATATCTCTGG 171 18 D18Mit48 77012578 57 1.5 3%(w/v) agarose TTGCACTCACAGGGCACAT TCAGAGTTTCCAGAAGACACCA 168 19 D19Mit41 18743419 56 1.5 3%(w/v) agarose AGCCCTCCACCCAGTTTC TCTGGGGAAAAAGGATGAGA 164 19 D19Mit11 42447920 59 1.5 3%(w/v) agarose TCAAAGTCAAGGTGGGCAG ACTTTCCAGATGTTGGGCAC 146

139

Microsatellite markers and SNPs used for mapping the GFP transgene on chromosome 1

Average A. Tm MgCl Locus bp 2 Resolution Forward primer Reverse primer product (oC) (mM) size (bp) D1Mit3 19797184 52 1.5 3%(w/v) agarose TTTTTGTTTTCTTTTCTTTTCCC CCCTCTTCTGGTTTCCACAT 160 Cut with BamHI, rs30790099 45057246 59 1.5 GTCACTCAAGGCCACTTGCT TCCTTCTGTACCCCTCCCTA 788 3%(w/v) agarose D1Mit21 66879391 59 1.5 3%(w/v) agarose CGCTGGACAATCTTATAATTGCA TCGAATCCCAACAACCACAT 243 D1Mit440 90617199 57 1.5 3%(w/v) agarose TCCACACAAGGTGTCCTCTG GCTCAGGTGACCTCCAAAAC 114 D1Mit136 103825697 59 1.5 3%(w/v) agarose TAGCCCTACACACTGTAGAAATGC TGAACACAAAGTAGTAAATGCGTG 103 D1Mit308 112435744 53 1.5 3%(w/v) agarose GAGGCTATGAGTCAAATGGACC TTTATGAGGTGCTGAGATGCA 149 D1Mit102 149011738 55 1.5 3%(w/v) agarose AAATACCAGCAAAACAATAAAGGC TGAATTAAAATTGCAGAGGCG 113 D1Mit111 170844217 52.5 1.5 3%(w/v) agarose ATTGCCTGACTCCAGTATTCTACC TTAGGTGTGTGAAAGACATTCCC 171

Microsatellite markers used for mapping modifiers of GFP transgene expression

Average A. Tm MgCl Chr Locus bp 2 Resolution Forward primer Reverse primer product (oC) (mM) size (bp) 1 D1Mit3 19797184 52 1.5 3%(w/v) agarose TTTTTGTTTTCTTTTCTTTTCCC CCCTCTTCTGGTTTCCACAT 160 1 D1Mit440 90617199 57 1.5 3%(w/v) agarose TCCACACAAGGTGTCCTCTG GCTCAGGTGACCTCCAAAAC 114 1 D1Mit111 170844217 52.5 1.5 3%(w/v) agarose ATTGCCTGACTCCAGTATTCTACC TTAGGTGTGTGAAAGACATTCCC 171 2 D2Mit92 71380492 56 1.5 3%(w/v) agarose TGTATGCACAGGTATTTCCCC TGAGGAAAGGGGATAAAATTTG 149 2 D2Mit48 155816121 49.5 1.5 3%(w/v) agarose GCTCTGCAGAAGATGCTGC GCTGAGACGCAGAGTCGC 130 3 D3Mit63 41138949 57 1.5 3%(w/v) agarose CTATGGACTTGTGACATAGGAGTG ACCACAAGATGGAGTAAGAGTTCA 162 3 D3Mit17 143584611 51 1.5 3%(w/v) agarose CATGGCTCCATGGTTCTTG CCACGGAGAACAACTGAAGA 198 4 D4Mit17 62851655 54.5 1.5 3%(w/v) agarose TGGCCAACCTCTGTGCTTCC ACAGTTGTCCTCTGACATCC 147 4 D4Mit251 136199272 49 1.5 3%(w/v) agarose AAAAATCGTTCTTTGACTTCTACATG TTTAAAAGGGTTTCTTTATCCTGTG 114 5 D5Mit233 52985474 57 1.5 3%(w/v) agarose TCCCCTCTGATCTCCTCAGA CCTCCTAGAATACAATTCAATGTGG 147 5 D5Mit101 141967054 58 1.5 3%(w/v) agarose GCCAGCCAGCCTTGACTC GGCTTCGTGCATGTAAAACA 132 6 D6Mit188 75377092 59 1.5 3%(w/v) agarose CTTTAGTCATTATTAGGATTGCCTATG TGGGATAGCATTGGAAACGT 128 6 D6Mit373 147009626 58 1.8 3%(w/v) agarose TTCTGGGGTGAGAGGCAG AGAACATTGACAAAAAGTGATTGTG 106 7 D7Mit57 18856293 61 1.5 3%(w/v) agarose TTCCCTCTAGAACTCTGACCTCC AGTTCAGAGCCGAGACTAGGC 146 7 D7Mit247 36851585 55 1.5 3%(w/v) agarose TCTTTTGACTTGATTTTGGCG TGGAGGAGACATATCTTTGCG 118 7 D7Mit69 48933118 55 1.5 3%(w/v) agarose CCCACCAGAGATCACCAAGT CACAATGAAGGCTGAAAGCA 233 7 D7Mit350 83462274 55 1.5 3%(w/v) agarose TCTGCATCTCACTGTCCCAG ATCTACAAATGAGTTTCTAAGGACTGC 119 7 D7Mit220 104268546 56 1.5 3%(w/v) agarose AAGCATGCAAGCACACTCAC ATGCACACAGGCAGTCACTC 135 7 D7Mit223 144419262 54.5 1.5 3%(w/v) agarose ATGCACATGAGTGTGTGTATGC TCCTGTGTCTGACGCTCATC 106 8 D8Mit190 37456381 56 1.5 3%(w/v) agarose CTTTGTTGCTGTTTCATTCTGG AGTCATATACAAGGTCAACCTGAGC 133 8 D8Mit215 118746715 53 1.8 3%(w/v) agarose AATACACAAGGTTGGCCTCA ATGTGTGGATATTCATGTGCTC 178 9 D9Mit248 58160696 56 1.8 3%(w/v) agarose TCAGAGTTCAGGAGGGCTGT TCTGAGAGGCCACATGTCTG 130 D9Mit1000 / 9 114527594 53.5 1.5 3%(w/v) agarose GCCTGGGCTACATGAGACTC GGGAATTCCAATACACTAAAGGG 224 D8Mit46 10 D10Mit213 20096855 51.5 1.5 3%(w/v) agarose CTCCTCCTACTGATTGTCCCC GGGACAAACTTTTAAAAATTGCA 150 10 D10Mit3 28841602 55 1.5 3%(w/v) agarose GTTGATAGTCCCACCTCACTCA TGAGAAATTCCATCTGTGGC 244 10 D10Mit61 66262144 58 1.5 3%(w/v) agarose GTCATCTCAGGGCACAACCT ACACTCGTGCACACGCAT 145 10 D10Mit230 89622039 55 1.5 3%(w/v) agarose AGATAGCCTAGGGGGTGCAT ATCAGTTTCCAATCGCTGCT 115 10 D10Mit267 119074063 58 1.5 3%(w/v) agarose ACACTTACAGTACCCTGGTGTGG GTGTGTGGGCGGATGTAAG 103 11 D11Mit20 44582634 58 1.5 3%(w/v) agarose CCTGTCCAGGTTTGAGAGGA CTTGGGAGCCTCTTCGGT 114 11 D11Mit333 108529872 56 1.5 3%(w/v) agarose CATGTGGTTATTTTCTAGCCCC AGGCATCAATAACTATTTTTCAGTG 125 12 D12Mit2 42516678 52 1.5 3%(w/v) agarose ACACAGGCTAAAACATGGGC GCATCTGTATTCCACAGGCA 134 12 D12Mit28 105743145 55 1.5 4%(w/v) agarose TTGGCAGTCCAGAGGAGGT CCAGTTCTGGTGTCAGTTTTACC 142 13 D13Mit16 20300907 58.5 1.5 3%(w/v) agarose CCAGCTGAAGGCTTACTCGT AAAGTTAGAATCAGCCATTCAAGG 207 13 D13Mit260 113486474 56 1.5 3%(w/v) agarose TAAATTTGGATGCAGACAATGG TTAAAAATAGAAATGGCTCTGTGTG 115 14 D14Mit18 46739212 59 1.5 3%(w/v) agarose AAGGTGGACCAGGAAGGAGT GACATTGAGAGACCAAAAAATGC 191 14 D14Mit97 117317851 59 1.5 3%(w/v) agarose TCAGTCCAAACTCTGTTAATCTTCC CAGCTCCACATTTTTGCTCA 136 15 D15Mit53 13225180 56 1.5 3%(w/v) agarose CTCCCTTACCTTCGGCTCTT AGGGTAATTTCAATTAAACTCGTG 137 15 D15Mit92 71049723 59 1.5 3%(w/v) agarose AGTCTCTCTCCCCCTTCTCTC TGCCACAAGCACAATAGTATCC 147 15 D15Mit70 81029340 56 1.5 3%(w/v) agarose CATTGAGGGTTTGTAGGTTGG ACCCCTGCAAGTTGTCTTTG 149 15 D15Mit16.2 102816142 56 1.5 3%(w/v) agarose GACAGACAAAAGCCGAGACA CTTGGAATGCGACTGTCCTG 114 16 D16Mit4 36159726 57 1.5 3%(w/v) agarose AGTTCCAGGCTACTTGGGGT GAGCCCTCATTGCAAATCAT 132 16 D16Mit86 93022607 59 1.5 3%(w/v) agarose TAATGTGGCAAGCAACCAAA GCATGTTTCCATGTGTCTGG 128 17 D17Mit133 24585209 59 1.5 3%(w/v) agarose TCTGCTGTGTTCACAGGTGA GCCCCTGCTAGATCTGACAG 188 17 D17Mit123 93503910 56 1.5 4%(w/v) agarose CACAAGGAGGGAGCCTGTAG CACCGTAAGAGTCTAATAATAAGGGG 133 18 D18Mit177 41108480 56 1.5 15% polyacrylamide CTGTAGTTTATCAGTTCACCCTGTG TGTGCTGTTAAACAAATATCTCTGG 171 18 D18Mit48 77012578 57 1.5 3%(w/v) agarose TTGCACTCACAGGGCACAT TCAGAGTTTCCAGAAGACACCA 168 19 D19Mit41 18743419 56 1.5 3%(w/v) agarose AGCCCTCCACCCAGTTTC TCTGGGGAAAAAGGATGAGA 164 19 D19Mit11 42447920 59 1.5 3%(w/v) agarose TCAAAGTCAAGGTGGGCAG ACTTTCCAGATGTTGGGCAC 146

140

Appendix 2

Genes in the MommeD9 linked interval

Gene start Ensembl gene ID External gene ID Description (bp) 9669698 ENSMUSG00000070826 9720328 ENSMUSG00000074390 9833330 ENSMUSG00000076224 mmu-mir-297-1 mmu-mir-297-1 9863459 ENSMUSG00000070835 XR_004965.1 similar to aurora kinase C (LOC676765), mRNA 9880638 ENSMUSG00000054272 Zscan4c zinc finger and SCAN domain containing 4C 9982513 ENSMUSG00000074389 10056491 ENSMUSG00000066814 NM_001080965.1 aurora kinase C (Aurkc), transcript variant 1, mRNA 10123295 ENSMUSG00000057084 10225343 ENSMUSG00000076988 10255513 ENSMUSG00000070827 XR_002295.2 predicted gene, EG665798 (EG665798), misc RNA 10272831 ENSMUSG00000070828 Zscan4c zinc finger and SCAN domain containing 4C 10323888 ENSMUSG00000074387 10471446 ENSMUSG00000074386 10575463 ENSMUSG00000070824 V1rg1 vomeronasal 1 receptor, G1 10662121 ENSMUSG00000051687 V1rg2 vomeronasal 1 receptor, G2 10752639 ENSMUSG00000047655 V1rg5 vomeronasal 1 receptor, G5 10786207 ENSMUSG00000043308 V1rg6 vomeronasal 1 receptor, G6 10836175 ENSMUSG00000045713 V1rg4 vomeronasal 1 receptor, G4 10873014 ENSMUSG00000075773 U6 U6 spliceosomal RNA 10991786 ENSMUSG00000066807 11058328 ENSMUSG00000061602 V1rg7 vomeronasal 1 receptor, G7 11082057 ENSMUSG00000046443 V1rg10 vomeronasal 1 receptor, G10 11098829 ENSMUSG00000057161 V1rg3 vomeronasal 1 receptor, G3 11165623 ENSMUSG00000066805 V1rg9 vomeronasal 1 receptor, G9 11210666 ENSMUSG00000058132 V1rg12 vomeronasal 1 receptor, G12 11227072 ENSMUSG00000066804 V1rg8 vomeronasal 1 receptor, G8 11264762 ENSMUSG00000077138 11267672 ENSMUSG00000066803 V1rg11 vomeronasal 1 receptor, G11 11321015 ENSMUSG00000034071 Zfp551 zinc fingr protein 551 11354911 ENSMUSG00000030384 11384167 ENSMUSG00000030386 Zfp606 zinc finger protein 606 11418400 ENSMUSG00000030385 2900092C05Rik RIKEN cDNA 2900092C05 gene 11508924 ENSMUSG00000034033 11521202 ENSMUSG00000066801 11589331 ENSMUSG00000053088 11642865 ENSMUSG00000059582 11668269 ENSMUSG00000070822 Zscan18 zinc finger and SCAN domain containing 18 11705161 ENSMUSG00000057894 Zfp329 zinc finger protein 329 11735662 ENSMUSG00000058638 Zfp110 zinc finger protein 110 11759101 ENSMUSG00000077662 11781474 ENSMUSG00000060397 Zfp128 zinc finger protein 128 11797994 ENSMUSG00000054715 Zscan22 zinc finger and SCAN domain containing 22 11822497 ENSMUSG00000012848 Rps5 ribosomal protein S5 11827595 ENSMUSG00000033967 2310014L17Rik RIKEN cDNA 2310014L17 gene 11830953 ENSMUSG00000065006 U6 U6 spliceosomal RNA 11866043 ENSMUSG00000004500 Zfp324 zinc finger protein 324 11878027 ENSMUSG00000033961 Zfp446 zinc finger protein 446 11888525 ENSMUSG00000030382 Slc27a5 solute carrier family 27 (fatty acid transporter) 11906303 ENSMUSG00000049600 ZBTB45 zinc finger and BTB domain containing 45 11924331 ENSMUSG00000005566 Trim28 tripartite motif protein 28 11932198 ENSMUSG00000033916 Chmp2a chromatin modifying protein 2A ubiquitin-conjugating enzyme E2M (UBC12 homolog, 11935417 ENSMUSG00000005575 Ube2m yeast) 11943329 ENSMUSG00000030380 Mzf1 myeloid zinc finger 1 11984468 ENSMUSG00000070817 V1rj3 vomeronasal 1 receptor, J3 12002302 ENSMUSG00000070816 ENSMUSG00000070816 predicted gene, ENSMUSG00000070816 12031650 ENSMUSG00000070815 V1rk1 vomeronasal 1 receptor, K1

141

12079027 ENSMUSG00000048261 12119518 ENSMUSG00000050453 V1rj2 vomeronasal 1 receptor, J2 12159146 ENSMUSG00000070814 6330408A02Rik RIKEN cDNA 6330408A02 gene 12177462 ENSMUSG00000056394 Lig1 ligase I, DNA, ATP-dependent phospholipase A2, group IVC (cytosolic, calcium- 12229819 ENSMUSG00000033847 Pla2g4c independent) 12298311 ENSMUSG00000005649 Cabp5 calcium binding protein 5 12351020 ENSMUSG00000074378 Gm767 gene model 767, (NCBI) 12541606 ENSMUSG00000072469 12633738 ENSMUSG00000070811 RIKEN cDNA C730007P19 gene (C730007P19Rik), 12809996 ENSMUSG00000074377 NM_009286.1 mRNA 12916121 ENSMUSG00000074376 sulfotransferase family 2A, dehydroepiandrosterone 12967735 ENSMUSG00000074375 Sult2a2 (DHEA)-preferring, member 2 13093464 ENSMUSG00000070810 NP_001074794.1 predicted gene, EG629219 13204960 ENSMUSG00000070809 13206837 ENSMUSG00000050977 13310899 ENSMUSG00000030378 2810007J24Rik RIKEN cDNA 2810007J24 gene 13430832 ENSMUSG00000041596 B430211C08Rik RIKEN cDNA B430211C08 gene 13559888 ENSMUSG00000055942 EG194588 predicted gene, EG194588 13678856 ENSMUSG00000064977 U6 U6 spliceosomal RNA 13812538 ENSMUSG00000075887 U6 U6 spliceosomal RNA 14289151 ENSMUSG00000074372 Obox2 oocyte specific homeobox 2 14331842 ENSMUSG00000074371 14343178 ENSMUSG00000074370 14427624 ENSMUSG00000066772 Obox3 oocyte specific homeobox 3 14427968 ENSMUSG00000074369 Obox3 oocyte specific homeobox 3 14495078 ENSMUSG00000054310 Obox1 oocyte specific homeobox 1 14541030 ENSMUSG00000075966 U6 U6 spliceosomal RNA 14700981 ENSMUSG00000074368 Obox2 oocyte specific homeobox 2 14760634 ENSMUSG00000074367 Obox4 oocyte specific homeobox 4 14853902 ENSMUSG00000075896 U6 U6 spliceosomal RNA 14916226 ENSMUSG00000074366 Obox5 oocyte specific homeobox 5 14991997 ENSMUSG00000041583 Obox6 oocyte specific homeobox 6 15024471 ENSMUSG00000041578 Crx cone-rod homeobox containing gene 15054646 ENSMUSG00000074365 Crxos1 Crx opposite strand transcript 1 15075737 ENSMUSG00000041571 Sepw1 selenoprotein W, muscle 1 15096692 ENSMUSG00000041560 Gltscr2 glioma tumor suppressor candidate region gene 2 15105488 ENSMUSG00000074364 Ehd2 EH-domain containing 2 15129194 ENSMUSG00000070808 Gltscr1 glioma tumor suppressor candidate region gene 1 N-ethylmaleimide sensitive fusion protein attachment 15257165 ENSMUSG00000006024 Napa protein alpha [Source:MarkerSymbol;Acc:MGI:104563] 15278417 ENSMUSG00000006021 Kptn kaptin solute carrier family 8 (sodium/calcium exchanger), 15288682 ENSMUSG00000030376 Slc8a2 member 2 15333549 ENSMUSG00000041420 Mrg2 myeloid ecotropic viral integration site-related gene 2 15355819 ENSMUSG00000006019 Dhx34 DEAH (Asp-Glu-Ala-His) box polypeptide 34 15395446 ENSMUSG00000074361 Gpr77 G protein-coupled receptor 77 15405265 ENSMUSG00000049130 C5ar1 complement component 5a receptor 1 15434171 ENSMUSG00000041375 Ccdc9 coiled-coil domain containing 9 15468160 ENSMUSG00000002083 Bbc3 Bcl-2 binding component 3 15485575 ENSMUSG00000052833 Sae1 SUMO1 activating enzyme subunit 1 15559388 ENSMUSG00000059273 BC057627 cDNA sequence BC057627 15611301 ENSMUSG00000019158 Tmem160 transmembrane protein 160 15614243 ENSMUSG00000001988 Npas1 neuronal PAS domain protein 1 15630335 ENSMUSG00000065893 5S_rRNA 5S ribosomal RNA 15653561 ENSMUSG00000058230 Grlf1 glucocorticoid receptor DNA binding factor 1 15783097 ENSMUSG00000064745 U6 U6 spliceosomal RNA 15880451 ENSMUSG00000007209 Ceacam9 CEA-related cell adhesion molecule 9 15896984 ENSMUSG00000008036 Ap2s1 adaptor-related protein complex 2, sigma 1 subunit solute carrier family 1 (neutral amino acid transporter), 15939891 ENSMUSG00000001918 Slc1a5 member 5 15967789 ENSMUSG00000048920 Fkrp fukutin related protein 15974411 ENSMUSG00000030374 Strn4 striatin, calmodulin binding protein 4 16001456 ENSMUSG00000041187 Prkd2 protein kinase D2 guanine nucleotide binding protein (G protein), gamma 8 16050308 ENSMUSG00000063594 Gng8 subunit 16065012 ENSMUSG00000043017 Ptgir prostaglandin I receptor (IP)

142

16075470 ENSMUSG00000019370 Calm3 calmodulin 3 16103615 ENSMUSG00000070802 EG434128 predicted gene, EG434128 16111917 ENSMUSG00000064689 U6 U6 spliceosomal RNA 16118317 ENSMUSG00000041141 0710005I19Rik RIKEN cDNA 0710005I19 gene 16153110 ENSMUSG00000041117 16163174 ENSMUSG00000003099 Ppp5c protein phosphatase 5, catalytic subunit 16190124 ENSMUSG00000004328 Hif3a hypoxia inducible factor 3, alpha subunit 16232562 ENSMUSG00000066760 Psg16 pregnancy specific glycoprotein 16 16310047 ENSMUSG00000053228 Psg24 pregnancy-specific glycoprotein 24 16362190 ENSMUSG00000023159 Psg29 pregnancy-specific glycoprotein 29 16769288 ENSMUSG00000077108 16776692 ENSMUSG00000077083 16844369 ENSMUSG00000072468 16871774 ENSMUSG00000008789 Psg30 pregnancy-specific glycoprotein 30 16951096 ENSMUSG00000066758 16971204 ENSMUSG00000023185 Ceacam14 CEA-related cell adhesion molecule 14 17130646 ENSMUSG00000030368 Ceacam11 CEA-related cell adhesion molecule 11 17168411 ENSMUSG00000057195 Ceacam13 CEA-related cell adhesion molecule 13 17224449 ENSMUSG00000030366 Ceacam12 CEA-related cell adhesion molecule 12 17335049 ENSMUSG00000066756 Igfl3 IGF-like family member 3 17344211 ENSMUSG00000076355 17403869 ENSMUSG00000054005 Mill1 MHC I like leukocyte 1 17504366 ENSMUSG00000003505 Psg18 pregnancy specific glycoprotein 18 17576361 ENSMUSG00000076332 17581063 ENSMUSG00000030373 Psg28 pregnancy-specific glycoprotein 28 17633104 ENSMUSG00000070799 EG574429 predicted gene, EG574429 17678224 ENSMUSG00000070798 Psg25 pregnancy-specific glycoprotein 25 17707607 ENSMUSG00000065313 U6 U6 spliceosomal RNA 17715036 ENSMUSG00000070797 EG545925 predicted gene, EG545925 17764982 ENSMUSG00000074359 Psg23 pregnancy-specific glycoprotein 23 17805375 ENSMUSG00000070796 Psg21 pregnancy-specific glycoprotein 21 17842940 ENSMUSG00000063305 EG434540 predicted gene, EG434540 17844449 ENSMUSG00000054308 Psg20 pregnancy-specific glycoprotein 20 17876604 ENSMUSG00000044903 BC050099 cDNA sequence BC050099 17903625 ENSMUSG00000065368 U6 U6 spliceosomal RNA 17948316 ENSMUSG00000004542 Psg19 pregnancy specific glycoprotein 19 17972462 ENSMUSG00000004540 Psg17 pregnancy specific glycoprotein 17 17998488 ENSMUSG00000040987 Mill2 MHC I like leukocyte 2 18043202 ENSMUSG00000030413 Pglyrp1 peptidoglycan recognition protein 1 18049406 ENSMUSG00000074358 C530028I08Rik RIKEN cDNA C530028I08 gene 18083871 ENSMUSG00000030411 NP_001025048.1 neuro-oncological ventral antigen 2 18146046 ENSMUSG00000051965 Nanos2 nanos homolog 2 (Drosophila) 18149841 ENSMUSG00000048481 P42pop Myb protein P42POP Adult male thymus cDNA, RIKEN full-length enriched 18150467 ENSMUSG00000074355 Q3V3W0_MOUSE library, clone:5830487H05 product:hypothetical Arginine- rich region profile containing protein, full insert sequence. 18162575 ENSMUSG00000044030 Irf2bp1 interferon regulatory factor 2 binding protein 1 18171805 ENSMUSG00000040891 Foxa3 forkhead box A3 18182954 ENSMUSG00000023118 Sympk symplekin 18213216 ENSMUSG00000040866 Rshl1 radial spokehead-like 1 18234837 ENSMUSG00000030410 Dmwd dystrophia myotonica-containing WD repeat motif 18242463 ENSMUSG00000030409 Dmpk dystrophia myotonica-protein kinase 18253066 ENSMUSG00000040841 Six5 sine oculis-related homeobox 5 homolog (Drosophila) 18278392 ENSMUSG00000050428 Fbxo46 F-box protein 46 18298739 ENSMUSG00000030407 Qpctl glutaminyl-peptide cyclotransferase-like 18308360 ENSMUSG00000040824 Snrpd2 small nuclear ribonucleoprotein D2 18321065 ENSMUSG00000030406 Gipr gastric inhibitory polypeptide receptor 18339726 ENSMUSG00000040811 Eml2 echinoderm microtubule associated protein like 2 18339987 ENSMUSG00000065543 mmu-mir-330 mmu-mir-330 18371060 ENSMUSG00000044317 Gpr4 G protein-coupled receptor 4 18386875 ENSMUSG00000052214 Opa3 optic atrophy 3 (human) 18400652 ENSMUSG00000064580 U1 U1 spliceosomal RNA 18416239 ENSMUSG00000030403 Vasp vasodilator-stimulated phosphoprotein 18435328 ENSMUSG00000030402 C79127 expressed sequence C79127 18441189 ENSMUSG00000030401 Rtn2 reticulon 2 (Z-band associated protein) 18463363 ENSMUSG00000003545 Fosb FBJ osteosarcoma oncogene B excision repair cross-complementing rodent repair 18503622 ENSMUSG00000003549 Ercc1 deficiency, complementation group 1

143

18515371 ENSMUSG00000047649 Cd3eap CD3E antigen, epsilon polypeptide associated protein protein phosphatase 1, regulatory (inhibitor) subunit 13 18519768 ENSMUSG00000040734 Ppp1r13l like excision repair cross-complementing rodent repair 18540561 ENSMUSG00000030400 Ercc2 deficiency, complementation group 2 18545150 ENSMUSG00000076315 18552965 ENSMUSG00000040714 Klc3 kinesin light chain 3 18569616 ENSMUSG00000030399 Ckm creatine kinase, muscle 18577334 ENSMUSG00000040705 Ckm creatine kinase, muscle 18584597 ENSMUSG00000030397 Mark4 MAP/microtubule affinity-regulating kinase 4 18619320 ENSMUSG00000065388 U1 U1 spliceosomal RNA 18647578 ENSMUSG00000011263 Exoc3l2 exocyst complex component 3-like 2 biogenesis of lysosome-related organelles complex-1, 18664326 ENSMUSG00000057667 Bloc1s3 subunit 3 18667247 ENSMUSG00000002043 Trappc6a trafficking protein particle complex 6A 18682200 ENSMUSG00000060621 Nkpd1 NTPase, KAP family P-loop domain containing 1 18689323 ENSMUSG00000051403 BC024868 cDNA sequence BC024868 18723471 ENSMUSG00000044709 Gemin7 gem (nuclear organelle) associated protein 7 18735844 ENSMUSG00000011267 Zfp296 zinc finger protein 296 splicing factor, arginine/serine-rich 16 (suppressor-of-white- 18739561 ENSMUSG00000061028 Sfrs16 apricot homolog, Drosophila) 18764744 ENSMUSG00000002983 Relb avian reticuloendotheliosis viral (v-rel) oncogene related B 18791765 ENSMUSG00000002981 Clptm1 cleft lip and palate associated transmembrane protein 1 18830106 ENSMUSG00000002992 Apoc2 apolipoprotein C-II 18836616 ENSMUSG00000074336 Apoc4 apolipoprotein C-IV 18848006 ENSMUSG00000040564 Apoc1 apolipoprotein C-I 18854795 ENSMUSG00000002985 Apoe apolipoprotein E translocase of outer mitochondrial membrane 40 homolog 18859887 ENSMUSG00000002984 Tomm40 (yeast) translocase of outer mitochondrial membrane 40 homolog 18859892 ENSMUSG00000053573 Tomm40 (yeast) 18875186 ENSMUSG00000062300 Pvrl2 poliovirus receptor-related 2 18914905 ENSMUSG00000002980 Bcam basal cell adhesion molecule 18939488 ENSMUSG00000040525 Cblc Casitas B-lineage lymphoma c 18966984 ENSMUSG00000053175 Bcl3 B-cell leukemia/lymphoma 3 19012095 ENSMUSG00000014686 NP_001028591.2 CEA-related cell adhesion molecule 16 19035585 ENSMUSG00000049848 Ceacam19 CEA-related cell adhesion molecule 19 19062101 ENSMUSG00000040511 Pvr poliovirus receptor 19100170 ENSMUSG00000040498 2210010C17Rik RIKEN cDNA 2210010C17 gene similar to 60S ribosomal protein L7a (LOC671487), 19112294 ENSMUSG00000070778 XR_003443.1 mRNA 19123934 ENSMUSG00000070777 Ceacam20 CEA-related cell adhesion molecule 20 19166545 ENSMUSG00000060508 Nlrp9b NLR family, pyrin domain containing 9B 19168606 ENSMUSG00000070150 5S_rRNA 5S ribosomal RNA 19280178 ENSMUSG00000074334 19296645 ENSMUSG00000074333 V1rd21 vomeronasal 1 receptor, D21 19491694 ENSMUSG00000074332 19576464 ENSMUSG00000074331 V1rd14 vomeronasal 1 receptor, D14 19632482 ENSMUSG00000074330 ENSMUSG00000074330 predicted gene, ENSMUSG00000074330 19668184 ENSMUSG00000074329 similar to vomeronasal 1 receptor, D14 (LOC672039), 19703404 ENSMUSG00000074328 XR_003678.1 mRNA 19838500 ENSMUSG00000042466 19979574 ENSMUSG00000074327 XR_003717.1 similar to KP78a CG6715-PA (LOC672121), mRNA 20015426 ENSMUSG00000074326 V1rd13 vomeronasal 1 receptor, D13 20135630 ENSMUSG00000074325 20274583 ENSMUSG00000074324 XR_003156.1 20596500 ENSMUSG00000074323 20636462 ENSMUSG00000074322 EG667283 predicted gene, EG667283 20681692 ENSMUSG00000074321 EG381936 predicted gene, EG381936 20844760 ENSMUSG00000074320 V1rd13 vomeronasal 1 receptor, D13 20878236 ENSMUSG00000074319 EG545929 predicted gene, EG545929 21019018 ENSMUSG00000074317 21110398 ENSMUSG00000074316 EG667283 predicted gene, EG667283 21296417 ENSMUSG00000074315 XR_003717.1 similar to KP78a CG6715-PA (LOC672121), mRNA 21584711 ENSMUSG00000074314 V1rd13 vomeronasal 1 receptor, D13 21618187 ENSMUSG00000074313 EG545929 predicted gene, EG545929 21758965 ENSMUSG00000074312 21785666 ENSMUSG00000074311 EG667283 predicted gene, EG667283

144

21826082 ENSMUSG00000072467 21842536 ENSMUSG00000074310 V1rd21 vomeronasal 1 receptor, D21 22122343 ENSMUSG00000074309 V1rd14 vomeronasal 1 receptor, D14 22171870 ENSMUSG00000074308 ENSMUSG00000074330 predicted gene, ENSMUSG00000074330 22207382 ENSMUSG00000074307 XR_004366.1 22422371 ENSMUSG00000074306 EG381936 predicted gene, EG381936 22449082 ENSMUSG00000074304 EG667283 predicted gene, EG667283 22498653 ENSMUSG00000074301 22515186 ENSMUSG00000074299 V1rd21 vomeronasal 1 receptor, D21 22552231 ENSMUSG00000074298 22807900 ENSMUSG00000074297 V1rd13 vomeronasal 1 receptor, D13 22841376 ENSMUSG00000074296 EG545929 predicted gene, EG545929 22981561 ENSMUSG00000074295 EG381936 predicted gene, EG381936 23010035 ENSMUSG00000045693 Nlrp4e NLR family, pyrin domain containing 4E 23094649 ENSMUSG00000015721 Nlrp5 NLR family, pyrin domain containing 5 23249501 ENSMUSG00000074291 23285987 ENSMUSG00000074290 23341112 ENSMUSG00000062483 V1rd7 vomeronasal 1 receptor, D7 23367076 ENSMUSG00000035523 V1rd9 vomeronasal 1 receptor, D9 23461135 ENSMUSG00000074289 V1rd22 vomeronasal 1 receptor, D22 23517082 ENSMUSG00000074288 23543587 ENSMUSG00000074287 23574280 ENSMUSG00000057513 V1rd12 vomeronasal 1 receptor, D12 23602289 ENSMUSG00000062598 V1rd13 vomeronasal 1 receptor, D13 23637146 ENSMUSG00000046924 V1rd17 vomeronasal 1 receptor, D17 ATP synthase, H+ transporting, mitochondrial F0 complex, 23655552 ENSMUSG00000066724 Atp5g2 subunit c (subunit 9), isoform 2 23661174 ENSMUSG00000061127 V1rd16 vomeronasal 1 receptor, D16 23692872 ENSMUSG00000057946 V1rd20 vomeronasal 1 receptor, D20 23711871 ENSMUSG00000060690 V1rd18 vomeronasal 1 receptor, D18 23763534 ENSMUSG00000066723 V1rd15 vomeronasal 1 receptor, D15 23790684 ENSMUSG00000057101 Zfp180 zinc finger protein 180 23821074 ENSMUSG00000052675 Zfp112 zinc finger protein 112 23845788 ENSMUSG00000047603 Zfp235 zinc finger protein 235 23886492 ENSMUSG00000068962 Zfp114 zinc finger protein 114 23905637 ENSMUSG00000062862 Zfp111 zinc finger protein 111 23936808 ENSMUSG00000074283 Zfp109 zinc finger protein 109 23966404 ENSMUSG00000030486 Zfp108 zinc finger protein 108 23981996 ENSMUSG00000055305 Zfp93 zinc finger protein 93 23996068 ENSMUSG00000050605 Zfp61 zinc finger protein 61 24010556 ENSMUSG00000074282 Zfp94 zinc finger protein 94 24041963 ENSMUSG00000055826 1700008P20Rik RIKEN cDNA 1700008P20 gene 24057982 ENSMUSG00000030484 Lypd5 Ly6/Plaur domain containing 5 potassium intermediate/small conductance calcium- 24079023 ENSMUSG00000054342 Kcnn4 activated channel, subfamily N, member 4 24108388 ENSMUSG00000002210 1500002O20Rik RIKEN cDNA 1500002O20 gene 24140914 ENSMUSG00000062028 NM_199013.2 immunity-related GTPase family, cinema 1 (Irgc1), mRNA 24171260 ENSMUSG00000046223 Plaur plasminogen activator, urokinase receptor 24190859 ENSMUSG00000054793 Cadm4 cell adhesion molecule 4 24215941 ENSMUSG00000064264 Zfp428 zinc finger protein 428 24239449 ENSMUSG00000041037 Irgq immunity-related GTPase family, Q 24250418 ENSMUSG00000011632 2310033E01Rik RIKEN cDNA 2310033E01 gene X-ray repair complementing defective repair in Chinese 24255049 ENSMUSG00000051768 Xrcc1 hamster cells 1 24292598 ENSMUSG00000066721 Zfp575 zinc finger protein 575 24296303 ENSMUSG00000064254 Ethe1 ethylmalonic encephalopathy 1 24320105 ENSMUSG00000061511 24325592 ENSMUSG00000074277 24345293 ENSMUSG00000057454 Lypd3 Ly6/Plaur domain containing 3 24376767 ENSMUSG00000062773 Tex101 testis expressed gene 101 24418019 ENSMUSG00000045587 BC049730 cDNA sequence BC049730 24431492 ENSMUSG00000058717 EG210155 predicted gene, EG210155 24452743 ENSMUSG00000052212 Cd177 CD177 antigen 24486036 ENSMUSG00000054169 Ceacam10 CEA-related cell adhesion molecule 10 24527555 ENSMUSG00000011350 EG545936 predicted gene, EG545936 24570962 ENSMUSG00000050347 Tmsb10 thymosin, beta 10 24573380 ENSMUSG00000062732 Lypd4 Ly6/Plaur domain containing 4 doublesex and mab-3 related transcription factor like 24578817 ENSMUSG00000011349 Dmrtc2 family C2

145

24593896 ENSMUSG00000040952 Rps19 ribosomal protein S19 24606271 ENSMUSG00000003379 Cd79a CD79A antigen (immunoglobulin-associated alpha) 24611809 ENSMUSG00000074276 Arhgef1 Rho guanine nucleotide exchange factor (GEF) 1 24616494 ENSMUSG00000040940 Arhgef1 Rho guanine nucleotide exchange factor (GEF) 1 24637553 ENSMUSG00000042789 EG232974 24678508 ENSMUSG00000003380 Rabac1 Rab acceptor 1 (prenylated) 24686927 ENSMUSG00000040907 Atp1a3 ATPase, Na+/K+ transporting, alpha 3 polypeptide 24718613 ENSMUSG00000003378 Grik5 glutamate receptor, ionotropic, kainate 5 (gamma 2) 24786025 ENSMUSG00000045252 Zfp574 zinc finger protein 574 24801222 ENSMUSG00000008496 Pou2f2 POU domain, class 2, transcription factor 2 24861217 ENSMUSG00000074274 D930028M14Rik RIKEN cDNA D930028M14 gene 24911600 ENSMUSG00000054499 Dedd2 death effector domain-containing DNA binding protein 2 24930211 ENSMUSG00000046541 Zfp526 zinc finger protein 526 24937019 ENSMUSG00000057177 Gsk3a glycogen synthase kinase 3 α 24937783 ENSMUSG00000056719 9130221H12Rik RIKEN cDNA 9130221H12 gene 24951314 ENSMUSG00000040857 Erf Ets2 repressor factor 24979606 ENSMUSG00000005442 Cic capicua homolog (Drosophila) 24990200 ENSMUSG00000074272 Ceacam1 CEA-related cell adhesion molecule 1 platelet-activating factor acetylhydrolase, isoform 1b, 25003809 ENSMUSG00000005447 Pafah1b3 alpha1 subunit 25011639 ENSMUSG00000058741 25014868 ENSMUSG00000043843 Tmem145 transmembrane protein 145 25039579 ENSMUSG00000045039 Megf8 multiple EGF-like-domains 8 25076382 ENSMUSG00000063651 Cnfn cornifelin 25088287 ENSMUSG00000003123 Lipe lipase, hormone sensitive 25093565 ENSMUSG00000053714 Lipe lipase, hormone sensitive 25108813 ENSMUSG00000060188 BC024561 cDNA sequence BC024561 25150912 ENSMUSG00000074262 25224800 ENSMUSG00000054385 Ceacam2 CEA-related cell adhesion molecule 2 25310084 ENSMUSG00000060819 25323381 ENSMUSG00000074261 EG632778 predicted gene, EG632778 25328194 ENSMUSG00000057229 2310004L02Rik RIKEN cDNA 2310004L02 gene UDP-GlcNAc:betaGal beta-1,3-N- 25336384 ENSMUSG00000059479 B3gnt8 acetylglucosaminyltransferase 8 branched chain ketoacid dehydrogenase E1, alpha 25338707 ENSMUSG00000060376 Bckdha polypeptide 25367924 ENSMUSG00000061286 Exosc5 exosome component 5 25377902 ENSMUSG00000061702 Tmem91 transmembrane protein 91 25389918 ENSMUSG00000063439 BC028440 cDNA sequence BC028440 25395762 ENSMUSG00000002603 Tgfb1 transforming growth factor, beta 1 25419877 ENSMUSG00000002608 Ccdc97 coiled-coil domain containing 97 25430244 ENSMUSG00000040725 Hnrpul1 heterogeneous nuclear ribonucleoprotein U-like 1 25466034 ENSMUSG00000002602 Axl AXL receptor tyrosine kinase 25511235 ENSMUSG00000040703 Cyp2s1 cytochrome P450, family 2, subfamily s, polypeptide 1 25537362 ENSMUSG00000062502 Rpl37a ribosomal protein L37a 25537392 ENSMUSG00000076445 25577705 ENSMUSG00000064538 Y Y RNA 25590781 ENSMUSG00000074257 25606418 ENSMUSG00000030483 Cyp2b10 cytochrome P450, family 2, subfamily b, polypeptide 10 25659013 ENSMUSG00000053435 Cyp2b13 cytochrome P450, family 2, subfamily b, polypeptide 13 25770255 ENSMUSG00000040583 Cyp2b13 cytochrome P450, family 2, subfamily b, polypeptide 13 RN170_MOUSE Isoform 2 of Q8CBG9 - Mus musculus 25780832 ENSMUSG00000045149 Q8CBG9-2 (Mouse) 25882169 ENSMUSG00000040660 Cyp2b9 cytochrome P450, family 2, subfamily b, polypeptide 9 26015929 ENSMUSG00000074254 Cyp2a4 cytochrome P450, family 2, subfamily a, polypeptide 4 26063146 ENSMUSG00000040614 Nlrp9c NLR family, pyrin domain containing 9C 26143873 ENSMUSG00000040601 Nlrp4a NLR family, pyrin domain containing 4A 26239944 ENSMUSG00000077509 26259572 ENSMUSG00000054102 Nlrp9a NLR family, pyrin domain containing 9A 26319879 ENSMUSG00000046130 V1re12 vomeronasal 1 receptor, E12 26373993 ENSMUSG00000040650 EG243881 predicted gene, EG243881 26465947 ENSMUSG00000066704 Cyp2b19 cytochrome P450, family 2, subfamily b, polypeptide 19 26517682 ENSMUSG00000049685 Cyp2g1 cytochrome P450, family 2, subfamily g, polypeptide 1 26544101 ENSMUSG00000005547 Cyp2a5 cytochrome P450, family 2, subfamily a, polypeptide 5 26641111 ENSMUSG00000040590 26737850 ENSMUSG00000060407 Cyp2a12 cytochrome P450, family 2, subfamily a, polypeptide 12 26757261 ENSMUSG00000074253 26828735 ENSMUSG00000052974 Cyp2f2 cytochrome P450, family 2, subfamily f, polypeptide 2 26867419 ENSMUSG00000058709 Egln2 EGL nine homolog 2 (C. elegans)

146

26877193 ENSMUSG00000053291 Rab4b RAB4B, member RAS oncogene family 26888504 ENSMUSG00000058217 Mia1 melanoma inhibitory activity 1 26895767 ENSMUSG00000061479 Snrpa small nuclear ribonucleoprotein polypeptide A 26915930 ENSMUSG00000003752 Itpkc inositol 1,4,5-trisphosphate 3-kinase C 26941783 ENSMUSG00000003762 Adck4 aarF domain containing kinase 4 26967521 ENSMUSG00000063160 Numbl numb-like

147