Genetic Background: Understanding its importance in mouse-based biomedical research A Jackson Laboratory Resource Manual

This resource manual highlights the importance of using genetically well-defined mice for biomedical research.

It briefly describes the following:

• The importance of genetic background • Resources for helping researchers choose the appropriate mouse model • Proper nomenclature to communicate the genetic makeup of mouse models • The Jackson Laboratory’s Genetic Quality Control and Genetic Stability programs Cover Photos

Front cover, left: JAX® Mice strain C3H/HeJ (000659)

Front cover, middle: Technician displaying holders with straws in the liquid nitrogen storage tank in our Cryopreservation Repository.

Front cover, right: JAX® Mice strain C57BL/6J (000664) Table of Contents

Introduction...... 1

Genetic Background Definition and Examples...... 2 Genetic Background Makes a Difference...... 2 The Influence of 129 Substrain Backgrounds on Targeted Mutations...... 4 Consequences of Using Inappropriate Backgrounds...... 4 Minimizing the Confounding Effects of Genetic Background...... 5 How Substrains Arise...... 5

Resources to Help You Choose Appropriate Models The Mouse Phenome Database...... 6 The JAX® Mice Database...... 6 The JAX® Mice Catalog...... 6 JAX Technical Support...... 6

Correct Nomenclature...... 7

How We Ensure Genetic Quality & Stability...... 8

Our Genetic Quality Control Program...... 8

Our Genome Scanning Service...... 9

Our Genetic Stability Program...... 10

The Jackson Laboratory: Pioneer in Cryopreservation...... 11

Do Your Part to Lessen the Impact of Genetic Drift...... 12

References...... 13

Introduction

The utility of the laboratory mouse as a research • Mouse biology databases, such as the Mouse model of human biology is increasing every year. Genome Informatics Database (MGI, Following are some of the reasons: www.informatics.jax.org), the JAX® Mice Database (jaxmice.jax.org), the Mouse SNP Database • The laboratory mouse is biologically similar to (mousesnp.roche.com), and the Mouse Phenome humans, is susceptible to many of the same diseases, Database (www.jax.org/phenome) are continually is easy to maintain, reproduces quickly, and is very being expanded and improved. amenable to genetic manipulation and analysis. • The number of available mouse models, including • The C57BL/6J strain was selected by the Mouse congenics, consomics, recombinant inbred strains, Genome Sequencing Initiative to be the first mouse spontaneous mutants, ENU-generated mutants, strain to be sequenced. targeted mutants, and transgenics, is increasing almost exponentially. • Fifteen JAX® Mice strains, including DBA/2J and C3H/HeJ have been resequenced by the National As the amount of mouse-based biomedical research Institute of Environmental Health Sciences increases, researchers must be more mindful than Resequencing Project (NIH News 2006). Dense ever of the genetic makeup of the models they use. If SNP maps of virtually the entire mouse genome for research is to be reliable and reproducible over time and these strains are available from the Mouse Phenome place, and, most importantly, if it is to have the greatest Database (www.jax.org/phenome). This information potential for improving human health, it must be facilitates comparative genomics among mouse conducted with mice of well-defined, stable, and clearly strains, humans, and other sequenced species. communicated genetic backgrounds. In the following pages, we discuss how these criteria can be met.

The Jackson Laboratory 1 Genetic Background

Definition and Examples

You may occasionally see the following cautionary note on of the targeted mutant strains NOD.129S7(B6)-Rag1tm1Mom/J strain data sheets of some of our JAX® Mice models: (003729) and NOD.Cg-Rag1tm1Mom Prf1tm1Sdz/Sz (004848) is This strain is on a genetic background different from that NOD. However, the first strain carries a targeted mutation on which the allele was first characterized. It should be noted of the Rag1 gene, likely a few Rag1-linked alleles from that the phenotype could vary from that originally described. 129S7-derived ES cells, and possibly some B6 alleles from We will modify the strain description if necessary as published crosses in the strain’s breeding history. In contrast, the second results become available. strain is a congenic (Cg) with more than one donor strain We include this note because the variety of genetic in its breeding history. It carries targeted mutations of the backgrounds and the mutations characterized and published Rag1 and Prf1 genes and possibly some background alleles on them are continually increasing. As a result, researchers from those other strains. Similarly, the genetic background of must be more mindful than ever of the genetic backgrounds transgenic strains FVB/N-Tg(MMTVneu)202Mul/J (002376) of the mouse models they use. and FVB/N-Tg(MMTV-PyVT)634Mul/J (002374) is FVB/N. As applied to a mutant mouse strain, genetic background However, whereas the first strain carries an MMTVneu refers to its genetic makeup (all its alleles at all loci) except transgene, the second carries an MMTV-PyVT transgene. the mutated gene of interest and a very small amount of On the other hand, strains B6.129S7-Rag1tm1Mom/J (002216) other genetic material, generally from one or two other and NOD.129S7(B6)-Rag1tm1Mom/J (003729) each have the strains. As we shall see, that “other” genetic material can same targeted mutation of the Rag1 gene, but on different significantly influence a mutant strain’s phenotype. backgrounds: C57BL/6J (B6) and NOD. Correct strain nomenclature indicates what a mutant strain’s background is. For example, the genetic background

Genetic Background Makes a Difference

The technology for producing genetically engineered them unexplainable. Such modifier genes are the reason why mice has been substantially refined, resulting in an normal development and physiology often vary significantly ever-increasing number, variety, and availability of mutant among inbred strains. mouse models. Generally, alleles of interest (such as One of the first documented instances of the influence of spontaneous mutations, targeted mutations, transgenes, genetic background on gene expression was the discovery and congenic regions) are maintained on one to several that, on a B6 background, the diabetes (db) and obese (ob) backgrounds that are more vigorous, better characterized, mutations cause obesity and transient diabetes, but, on a more amenable to scientific experiments, reproduce better, C57BLKS/J (BKS) background, they cause obesity and overt display a phenotype better, or have some other advantages diabetes (Coleman and Hummel 1973; Coleman 1978). over other backgrounds. However, alleles are sometimes (Fig. 1, page 3). Since those results were published, genetic transferred to backgrounds that are not well characterized. background has been shown to influence the expression In any case, inattention to a mutant’s genetic background can of many other genes, including the following: seriously confound research results. Each strain has unique background alleles that may interact with and modify the • The multiple intestinal neoplasia mutation (Min) of expression of a mutation, transgene, or other genetic insert. the adenomatous polyposis coli (Apc) gene (ApcMin). The likelihood of such modifier genes having a confounding B6 mice heterozygous for the ApcMin mutation are very effect is especially high in an uncharacterized background susceptible to developing intestinal polyps. Offspring of or in a segregating or mixed background of unspecified these mice mated with AKR/J, MA/MyJ, or CAST mice origin. Even in a well-characterized strain, undiscovered are significantly less susceptible, indicating that the latter modifier genes may confound results, sometimes making three strains harbor strain-unique ApcMin modifier loci,

The Jackson Laboratory 2 Genetic Background

Genetic Background Makes a Difference (continued)

named modifier of Min 1 (Mom1) (Moser et al. 1992; Many phenotypic differences among substrains can Dietrich et al. 1993). The AKR allele of Mom1 has been only be explained by the presence of as yet undiscovered shown to actually contain two genes (MacPhee et al. 1995; modifiers. Following are several examples: Cormier et al. 1997, 2000). • The Prkdcscid mutation. On the NOD background, the • Differences in tissue rejection among related 129 strains Prkdcscid mutation is associated with low natural killer (Simpson et al. 1997) (NK) cell activity, no complement activity, impaired • Differences in susceptibility to proteoglycan-induced macrophage development, and impaired antigen arthritis and spondylitis among C3H substrains presenting cell functions; on the BALB/c background, it (Glant et al. 2001) is associated with high NK cell activity, high complement • Differences in fear responses between C57BL/6J activity, normal macrophage development, and normal and C57BL/6N substrains (Radulovic et al. 1998; antigen presenting cell functions (Custer et al. 1985; Shultz Stiedl et al. 1999) et al. 1995). • Differences in the effects of anesthetics on cardiac • Prkdcscid-associated leakiness (tendency to produce function between C57BL/6J and C57BL/6N substrains some functional B and T cells with age). Leakiness in (Roth et al. 2002) Prkdcscid mutants is generally high on the B6 and BALB/c • Differences in the electroconvulsive thresholds between backgrounds, low on the C3H background, and very low C57BL substrains (Yang et al. 2003) on the NOD background (Shultz et al. 1995). • IL10-deficiency. On the B6 background, IL10-deficiency only slightly increases susceptibility to inflammatory bowel disease (IBD); in the 129/SvEv, BALB/c, and C3H/HeJBir backgrounds, it greatly increases IBD susceptibility (Beckwith et al. 2005).

Diabetes db/db (Leprdb) Obesity ob/ob (Lepob)

• C57BL/6J (B6.Cg-m +/+ Leprdb/J) • C57BL/6J (B6.V-Lepob/J) obesity with transient diabetes obesity with transient diabetes

• C57BLKS/J (BKS.Cg-m +/+ Leprdb/J) • C57BLKS/J (BKS.V-Lepob/J) obesity with overt diabetes obesity with overt diabetes

Figure 1. On a B6 background, the diabetes (db) and obese (ob) mutations cause obesity and transient diabetes, but, on a C57BLKS/J (BKS) background, they cause obesity and overt diabetes.

The Jackson Laboratory 3 Genetic Background

The Influence of 129 Substrain Backgrounds on Targeted Mutations

In the last ten years, the influence of various 129 substrain Recently, Petkov and colleagues (Petkov et al. 2004b), backgrounds on gene expression has become particularly using a panel of SNPs, determined that the 129X1/SvJ apparent. Because embryonic stem (ES) cells derived from substrain likely has genetic contributions from C57BL/6J 129 mice colonize germlines so efficiently, they are the on chromosomes 5, 7, 14, 18, and 19, and from BALB/cJ on most widely used cell lines for producing targeted mutants. chromosomes 7, 8, 10, 18, 19, and X, suggesting that the “X” Simpson et al. (1997) found that protein markers, simple is an F1 hybrid between C57BL/6J and BALB/cJ. sequence length polymorphic marker alleles, tail skin graft Both Simpson and Threadgill found that ES cell lines from acceptance, and coat color alleles vary among seventeen 129 the numerous 129 substrains were being used with little substrains and twelve 129-derived ES cell lines. Threadgill attention to their genetic differences. Indeed, Threadgill and and colleagues (Threadgill et al. 1997) found evidence that his colleagues concluded that confusing results of their own strain 129X1/SvJ is significantly different from other 129 experiments involving targeted Egfr alleles are due to genetic substrains and suggested that it should be classified as a differences in the 129 substrains they used. In another recombinant congenic strain (129cX/Sv) derived from context, Hogan et al. (1994) invoked differences between 129 129/Sv and an unknown strain, “X.” The “X” was added to substrains to explain why 129X1/SvJ is a high ovulator in reflect this hypothesized contamination (Festing et al. 1999). response to exogenous gonadotropins, whereas 129P3/J and 129P1/ReJ are low ovulators.

Consequences of Using Inappropriate Backgrounds

As mentioned earlier, using mouse models with • Confounded results due to lack of awareness of 129 inappropriate genetic backgrounds can produce unreliable substrains (Hogan et al. 1994; Threadgill et al. 1997) results. This risk is more than theoretical, as several examples • Dubious results because of inattention to C57BL substrain have already been reported, including the following: differences (Specht et al. 2001; Wotjak 2003) • Wasted efforts because of a mix-up in AL/N substrains Many other instances have likely been unreported (Bailey 1982) or undetected.

4 The Jackson Laboratory Genetic Background

Minimizing the Confounding Effects of Genetic Background

Although you may not be able to eliminate the • Construct congenics and transgenics on well-defined confounding effects of genetic background in your research, backgrounds, such as B6 and FVB you can minimize them considerably by observing the (Silver 1995; Linder 2006) following practices: • Construct targeted mutation strains on well-defined ES cell lines, derived either from B6 or well-defined 129 • Use mutants with genetically well-defined backgrounds strains (Simpson et al. 1997; Threadgill et al. 1997; (Silver 1995; Linder 2001, 2006; Yoshiki and Linder 2006) Moriwaki 2006) • If possible, analyze mutations on several backgrounds. • Use appropriate controls. If a mutation arose If the mutation is maintained on a mixed genetic spontaneously or was induced on a well-characterized background, analyze it in hybrids of the two progenitor inbred strain, the inbred strain is likely coisogenic with strains (Silver 1995; Linder 2001, 2006) and therefore the best control for the mutant harboring • Consider the effects of environmental factors such as that mutation (Silver 1995; Linder 2001, 2006) noise, light, home cage environment, handling, and diet • When possible, construct congenic, targeted mutation, on gene expression and behavior (Crawley et al. 1997; transgenic, and other genetically altered strains that are Bailey et al. 2006) coisogenic to controls (Silver 1995; Linder 2006) • In all your research communications, describe your mouse models with correct nomenclature (Linder 2006)

How Substrains Arise Substrains are genetic variants of an inbred strain. They arise for the following reasons:

• Most commonly, genetic drift following separation • Undetected spontaneous mutations that become fixed in of a colony from its parent colony for more than 20 a colony (Radulovic et al. 1998; Sluyter et al. 1999; Stiedl generations (10 generations in the sub-colony plus the et al. 1999; Specht et al. 2001; Roth et al. 2002; Wotjak 10 that simultaneously pass in the parent colony) 2003) (Silver 1995) • Undetected genetic contamination (Naggert et al. 1995) • Residual heterozygosity or incomplete inbreeding at the • Deliberate and subsequent unrecorded and/or forgotten time of separation from progenitors (Bailey 1977, 1982; outcrossing of strains for specific experimental purposes Silver 1995) (Bailey 1977, 1982; Simpson et al. 1997; Threadgill et al. 1997; Wotjak 2003)

The Jackson Laboratory 5 Resources to Help You Choose Appropriate Models

Many Jackson Laboratory resources can help you choose the most appropriate genetic backgrounds and controls for your research.

The Mouse Phenome Database (www.jax.org/phenome)

The Mouse Phenome Database (MPD, www.jax.org/ • Detailed protocols on how the phenotypes were measured phenome) is the product of an international collaboration • Tools for manipulating, downloading, statistically (The Mouse Phenome Project) and is maintained at analyzing, and displaying the raw data The Jackson Laboratory. It is particularly useful for helping researchers select appropriate background strains. It contains The MPD is continually updated with data generated by the following: contributing scientists.

• Hundreds of baseline measurements of phenotypes for a set of 40 commonly used and genetically diverse inbred mouse strains

The JAX® Mice Database (www.jax.org/jaxmice)

The JAX® Mice Database is the most comprehensive The strain information page (www.jax.org/jaxmice/info) source of information on JAX® Mice strains. For each has links to detailed descriptions of inbred, hybrid, strain, there is a strain data sheet, which includes a genetically engineered, wild-derived, recombinant inbred, strain description, mating systems, colony maintenance, recombinant congenic, chromosomal substitution strains, H2 haplotype, generation number, strain development, and chromosomal aberrations. related strains, references, animal health reports, research applications, and suggested controls.

The JAX® Mice Catalog

The JAX® Mice Catalog has a great deal of information on Programs, and appendices on H2 haplotypes, JAX® Services, JAX® Mice strains and can help you select the strains and and literature. The JAX® Mice Catalog and Price Lists may be controls that best support your research. It features sections requested online at www.jax.org/jaxmice/literature. on Technical Knowledge Resources and Quality Control

JAX Technical Support

Our technical support personnel are the best in the field. Contact them at 1-800-422-6423 or via the website at They are eager to help you select the appropriate JAX® Mice micetech.jax.org. strains and controls for your research.

6 The Jackson Laboratory Correct Nomenclature

By using correct strain nomenclature, scientists can followed by a forward slash and a substrain symbol that accurately communicate with their colleagues, compare and may be a number and/or the Laboratory Registration Code draw conclusions across experiments, locate the institutions of either the individual or institution that maintains or or researchers that maintain strains, and accurately generated the substrain. For example, DBA/1J, DBA/1LacJ, document data in animal facilities, research laboratories, and and DBA/2J are substrains of DBA: numbers 1 and 2 identify publications. For these reasons, the International Committee the substrains, Lac is the Laboratory Registration Code for on Standardized Genetic Nomenclature for Mice established Laboratory Animal Centre at Carshalton, U.K., and J is the guidelines that accurately describe the following: Laboratory Registration Code for The Jackson Laboratory. When successive substrains arise, substrain symbols • Mouse models, including inbred, transgenic, targeted accumulate. For example, A/HeJ is a substrain held first by mutation, spontaneous mutation, congenic, wild-derived Heston and now maintained at The Jackson Laboratory. inbred, recombinant inbred, consomic, and recombinant Related inbred strains (strains with a common origin but congenic strains, strains with chromosome aberrations separated before F20) are given symbols that indicate this and hybrid mice relationship (for example, NZB and NZW; NOD and NON). • Mutations, including type, alleles, mode of inheritance, For more detailed information on mouse strain and transgenes (including the species of origin) and nomenclature, consult the following: • Genetic background, relation to other strains, and laboratory of origin • The Jackson Laboratory Mouse Nomenclature Home Page (www.informatics.jax.org/nomen) It is particularly important that substrains be properly • JAX® Mice Nomenclature Articles and Announcements named. An inbred mouse colony that is found to be (www.jax.org/jaxmice/info/nomenclature) genetically different or separated from its parent colony for • Interactive Nomenclature Tutorial (www.jax.org/jaxmice/ 20 or more generations should be given substrain status. request/nomenclature) It should be designated by the name of the parent strain

The Jackson Laboratory 7 How We Ensure Genetic Quality & Stability

Even if you choose the proper genetic background, the genetic quality of our mice through the following consider substrain differences, and clearly communicate two programs: the genetic background of the mouse models you use, your research results can still be confounded by genetic 1. A rigorous Genetic Quality contamination and genetic drift. As the repository for over Control (GQC) program 4,000 JAX® Mice strains and the distributor of these mice to biomedical researchers worldwide, we at The Jackson 2. An innovative Genetic Stability Laboratory are committed to supplying you with the most Program (GSP) genetically well-defined mouse models possible. We ensure

Our Genetic Quality Control Program

Our Genetic Quality Control (GQC) program is designed 2. Systematic screens of all stocks for phenotypic to detect and prevent the spread of genetic contamination. variations, including coat color, body size, Genetic contamination is the accidental introduction and behavior of genes from one inbred mouse strain into the genome of a second inbred strain. The most common cause of genetic contamination is human error, such as inadvertent 3. SNP-genotyping outcrossing or strain mislabeling. It can also result from deliberate outcrossing. In any case, it occurs quickly and is relatively easy to detect. Three examples follow: Foundation Stocks (Self Perpetuating) • Inadvertent outcrossing of C57BL/6J to DBA/2J, resulting in the C57BLKS strain (Naggert et al. 1995) [Pedigreed] • Deliberate outcrossing of the 129 strain (Simpson et al. 1997; Threadgill et al. 1997) • Genetic contamination (affecting several chromosomes) Pedigreed Expansion with either FVB or an FVB-like strain in two NIA contract Stocks

colonies of C57BL/6 (www.nia.nih.gov) [Pedigreed]

The purpose of our GQC program is to prevent such mistakes. Continually improved over the past 30 years, the program is founded on the following five components: Production Stocks

1. Rigorous breeding protocols Pooled Production Stocks • We limit the number of generations attained in our foundation and expansion stocks to less than 10 generations from the main pedigree line • We isolate foundation, expansion, and production stocks Investigators from each other • We maintain detailed pedigrees of our foundation and expansion stocks • We systematically refresh our production stocks with mice Figure 2. Relationship between foundation stocks, pedigreed from foundation stocks expansion stocks, and production stocks at The Jackson Laboratory. • We adhere to proven mouse husbandry practices

8 The Jackson Laboratory How We Ensure Genetic Quality & Stability

Our Genetic Quality Control Program (continued)

Between the 1960s and 2003, our GQC program used For details on other molecular markers we use, see the biochemical (isoenzyme) variants to distinguish among following website: different strains of mice. In 2003, after extensive testing, www.jax.org/jaxmice/geneticquality/monitoring we converted to single nucleotide polymorphisms (SNPs). Although SNPs are the most abundant type of 4. Verification of mutant alleles in mutant stocks polymorphism, until the mouse genome was sequenced, few Our molecular genotyping lab uses allele-specific mouse SNPs were mapped or available in public databases. assays, PCR-genotyping, and other assays to verify mutant Additionally, the feasibility of using them as genetic genotypes of genetically engineered and cloned spontaneous markers had not been established. Dr. Petko Petkov and his mutations. Experienced technicians verify the visible colleagues at The Jackson Laboratory demonstrated that phenotype of many spontaneous mutant colonies. SNP-genotyping with a panel of only 28 carefully selected SNPs can distinguish between virtually all JAX® Mice strains. 5. Phenotypic deviant search (Petkov et al. 2004a; Petkov et al. 2004b) The panel has the The Mouse Mutant Resource phenotypic deviant search following advantages: detects and prevents spontaneous mutations that cause visible phenotypes from becoming fixed in JAX® inbred • It is reliable, simple, quick, and inexpensive strains. Animal care technicians send mice from breeding • It is amenable to high throughput units with deviant phenotypes to a bi-weekly clinic. • It is suitable for both large and small scale animal facilities Scientists determine which deviant phenotypes are likely • It may be used to type mice before they are used caused by a mutation and test them for heritability. Breeding as breeders units with deviant mice are removed from the breeding colonies, and the parent strains are closely monitored to see Using this panel has greatly facilitated monitoring the if the phenotypes recur. genetic integrity of JAX® Mice. For example, we formerly used erythrocyte antigen (Ea) assays to distinguish strains like C57BL/6J and C57BL/10J, which type identically for 23 isoenzymes, are both black, have the same major histocompatibility (H2) haplotype, but have different Eas. However, we no longer use the Ea assay because several SNP Our Genome Scanning markers can distinguish between C57BL/6J, C57BL/10J, and other closely related strains. Service Can Confirm your Occasionally, we use other assays to either verify SNP results or to type closely related strains that SNP-genotyping Strain’s Genetic Identity cannot distinguish. For example, only a hemolytic complement (Hc) assay can distinguish between congenic By using a panel of over 2,000 single nucleotide strains B10.D2-Hc1 H2d H2-T18c/nSnJ and B10.D2-Hc0 H2d polymorphic (SNP) markers, our Genome Scanning H2-T18c/oSnJ (whereas B10.D2-Hc1 H2d H2-T18c/nSnJ Service can confirm and monitor your strain’s genetic expresses hemolytic complement, B10.D2-Hc0 H2d background. Service technicians can work with you H2-T18c/oSnJ does not). to design a protocol that meets your needs. Contact JAX® Services at [email protected].

The Jackson Laboratory 9 How We Ensure Genetic Quality & Stability

Our Genetic Stability Program

Our Genetic Stability Program (GSP) is designed to limit due to deliberate outcrossing, most are probably due genetic drift, a cumulative change in the genetic makeup to maintaining colonies separate from the originating of an organism over time. Genetic drift in inbred mouse colony for more than 10 generations colonies happens slowly, subtly, and is difficult to detect and • Histocompatibility variants exist within A, AKR, control. It is caused by the same factors that lead to substrain BALB/c, CBA, C3H, C57BL, C57L, DBA, and WG divergence: strains (Bailey 1982) • Separation of a sub-colony from its parent colony for • Substrains C57BL/6N, C57BL/6Nmg, and C57BL/6JKun more than 20 generations (10 generations in the parent are phenotypically different from each other and from colony plus the 10 that simultaneously pass in the the C57BL/6J founder line (Radulovic et al. 1998; sub-colony) Sluyter et al. 1999; Stiedl et al. 1999; Roth et al. 2002; • Undetected spontaneous mutations that become fixed Wotjak 2003) in a colony • C57BL/6JOlaHsd, a substrain of C57BL/6J, has a • Residual heterozygosity in or incomplete inbreeding of a spontaneous deletion encompassing part of the alpha- colony before it is separated from its progenitors synuclein (Snca) gene and the entire multimerin-1 (Bailey 1977, 1982) (Mmrn1) gene (Specht and Schoepfer 2001, 2004; Wotjak 2003) Following are seven examples of genetic drift: • A deletion of the killer cell lectin-like receptor, • At least 40 C57BL substrains develop between 1930 subfamily D, member 1 gene (Klrd1) on Chromosome 6 and 1970 (Fig. 3): although some of these substrains are is identified in JAX® Mice strain DBA/2J (Wilhelm et al. 2003)

Figure 3. At least 40 C57BL substrains develop between 1930 and 1970, some of which are due to genetic drift (adapted from Bailey 1977 by Dr. Michael V. Wiles, The Jackson Laboratory).

10 The Jackson Laboratory How We Ensure Genetic Quality & Stability

Our Genetic Stability Program (continued)

• A spontaneous deletion of two ion channel genes, Kcnq2 We have already cryopreserved embryos from JAX® Mice and Chrna4, in a C57BL/6J substrain generates a mouse strains 129S1/SvImJ (002448), C3H/HeJ (000659) C57BL/6J model of epilepsy (Yang et al. 2003). (000664), DBA/2J (000671), FVB/NJ (001800), NOD/ShiLtJ • C3H/HeJ mice are homozygous for a paracentric (001976), and NOD.CB17-Prkdcscid/J (001303) (Taft et al. inversion in Chromosome 6 (JAX® Notes 2003). 2006). Limiting genetic drift in these strains is particularly important because each of them, except NOD.CB17- “This insidious evolution of the inbred genotype is known as Prkdcscid/J (001303), has been resequenced. C57BL/6J was genetic drift. It is capable of subverting the conclusions reached sequenced by the Mouse Genome Sequencing Consortium about comparable research results coming from different in 2003 and FVB/NJ, BALB/cByJ, and 13 other JAX® Mice laboratories when each uses its own subline of the same inbred strains were resequenced by the National Institute of strain.” (Bailey 1977) Environmental Health Sciences, as part of their 15-strain Resequencing Project (JAX® Notes 2005). To further limit genetic drift in our mouse colonies, we implement a three-component Genetic Stability Program: Embryos derived • We minimize the number of generations attained in our from brother sister foundation and production stocks (see GQC section). matings • We use highly skilled and experienced technicians to oversee breeding in those stocks (see GQC section). • We use a unique cryopreservation approach to virtually stop genetic drift in the most commonly used inbred Establishment strains (see below). of frozen bank Re-establish Foundation The cryopreservation component of our GSP was initiated about every five in 2003 and entails cryopreserving supplies of embryos from generations broadly used strains (if technically feasible to do so) and Frozen Stock Foundation refreshing our foundation stocks with these embryos about Sufficient for every five generations (Fig. 4). To obtain enough mice for up to 25 Years Stock infusion that span only one or two generations, we rapidly Expansion expand the strain of interest by in vitro fertilization. Figure 4. The Jackson Laboratory cryopreserves supplies of embryos from several widely-used strains and refreshes foundation stocks with these embryos about every five generations.

The Jackson Laboratory: Pioneer in Cryopreservation for Over 30 Years

The technique of cryopreserving mouse embryos was first reported by Whittingham et al. (1972). Later, Whittingham and Dr. Bailey of The Jackson Laboratory (Whittingham 1974; Bailey 1977) suggested that cryopreservation could be used to solve the problem of genetic drift in inbred mouse colonies. Indeed, it has been shown to stop genetic drift in embryos of outbred stocks (Goto et al. 2002). During the last 30 years, the Jackson Laboratory has successfully cryopreserved over 6,500 mouse strains.

The Jackson Laboratory 11 How We Ensure Genetic Quality & Stability

Do Your Part to Lessen the Impact of Genetic Drift

You can lessen the impact of genetic drift on mouse-based biomedical research by practicing the following:

• Obtain mice from a reliable breeding source. • If you maintain your own private colonies of these mice, periodically obtain new breeding stock from your supplier. • Although colonies of inbred mice expanded from our breeding stock can be maintained either by sibling or non-sibling matings, they may develop into substrains if they are expanded beyond ten generations. • Avoid comparing results from substrains that either arose early in a strain’s inbreeding regimen or that have been long separated. • Use proper nomenclature to describe your mouse models (see Nomenclature section). • Include a detailed description of the genetic background of the mice you use in all your communications. • When possible, use a common genetic background so that your experiments can be replicated.

“. . . the constant tendency of genes to evolve even in the absence of selective forces. Genetic drift is fueled by spontaneous neutral mutations that disappear or become fixed in a population at random. Inbred lines separated from a common ancestral pair can drift rapidly apart from each other.” (Silver 1995).

12 The Jackson Laboratory References

Bailey DW. 1977. Genetic drift: the problem and its possible Glant TT, Bardos T, Vermes C, Chandrasekaran R, Valdez solution by frozen-embryo storage. Ciba Found Symp JC, Otto JM, Gerard D, Velins S, Lovasz G, Zhang J, 291-303. Mikecz K, Finnegan A. 2001. Variations in susceptibility to proteoglycan-induced arthritis and spondylitis among Bailey DW. 1982. How pure are inbred strains of mice? C3H substrains of mice: evidence of genetically acquired Immunol Today 3:210-14. resistance to autoimmune disease. Arthritis Rheum 44:682-92. Bailey KR, Rustay NR, Crawley JN. 2006. Behavioral phenotyping of transgenic and knockout mice: practical Goto K, Muguruma K, Kuramochi T, Shimozawa N, Hioki K, Itoh concerns and potential pitfalls. ILAR J 47:124-31. T, Ebuduro M. 2002. Effects of cryopreservation of mouse embryos and in vitro fertilization on genotypic frequencies Beckwith J, Cong Y, Sundberg JP, Elson CO, Leiter EH. 2005. in clolonies. Mol Reprod Dev 62:307-11. Cdcs1, a major colitogenic locus in mice, regulates innate and adaptive immune response to enteric bacterial antigens. Hogan B, Beddington R, Costantini F, Lacy E. 1994. Manipulating Gastroenterology 129:1473-84. the mouse embryo: a laboratory manual, 2nd ed. Cold Spring Harbor (NY). Coleman DL. 1978. Obese and diabetes: two mutant genes causing diabetes-obesity syndromes in mice. JAX® Notes. 2003. Chromosomal inversion discovered in Diabetologia 14:141-8. C3H/HeJ mice. JAX® Notes 491:15.

Coleman DL, Hummel KP. 1973. The influence of genetic JAX® Notes. 2005. NIEHS to Sequence 15 JAX® Mice Strains. background on the expression of the obese (Ob) gene in the JAX® Notes 496:3 mouse. Diabetologia 9:287-93. Linder CC. 2001. The influence of genetic background on Cormier RT, Bilger A, Lillich AJ, Halberg RB, Hong KH, Gould KA, spontaneous and genetically engineered mouse models of Borenstein N, Lander ES, Dove WF. 2000. The Mom1 AKR complex diseases. Lab Anim 30:34-9. intestinal tumor resistance region consists of Pla2g2a and a locus distal to D4Mit64. Oncogene 19:3182-92. Linder CC. 2006. Genetic variables that influence phenotype. ILAR J 47:132-40. Cormier RT, Hong KH, Halberg RB, Hawkins TL, Richardson P, Mulherkar R, Dove WF, Lander ES. 1997. Secretory MacPhee M, Chepenik KP, Liddell RA, Nelson KK, Siracusa LD, phospholipase Pla2g2a confers resistance to intestinal Buchberg AM. 1995. The secretory phospholipase A2 gene tumorigenesis. Nat Genet 17:88-91. is a candidate for the Mom1 locus, a major modifier of ApcMin-induced intestinal neoplasia. Cell 81:957-66. Crawley JN, Belknap JK, Collins A, Crabbe JC, Frankel W, Henderson N, Hitzemann RJ, Maxson SC, Miner LL, Moser AR, Dove WF, Roth KA, Gordon JI. 1992. The Min Silva AJ, Wehner JM, Wynshaw-Boris A, Paylor R. (multiple intestinal neoplasia) mutation: Its effect on gut 1997. Behavioral phenotypes of inbred mouse strains: epithelial cell differentiation and interaction with a modifier implications and recommendations for molecular studies. system. J Cell Biol 116:1517-26. Psychopharmacology (Berl) 132:107-24. Naggert JK, Mu JL, Frankel W, Bailey DW, Paigen B. 1995. Custer RP, Bosma GC, Bosma MJ. 1985. Severe combined Genomic analysis of the C57BL/Ks mouse strain. immunodeficiency (SCID) in the mouse. Pathology, Mamm Genome 6:131-3. reconstitution, neoplasms. Am J Pathol 120:464-77. NIH News. 2006. Mouse DNA to Aid Biomedical Research. Dietrich WF, Lander ES, Smith JS, Moser AR, Gould KA, Luongo NIEHS PR #06-17, October 25 C, Borenstein N, Dove W. 1993. Genetic identification (www.niehs.nih.gov/oc/news/snp2.htm). of Mom-1, a major modifier locus affecting Min-induced intestinal neoplasia in the mouse. Cell 75:631-9. Petkov PM, Cassell MA, Sargent EE, Donnelly CJ, Robinson P, Crew V, Asquith S, Harr RV, Wiles MV. 2004a. Development Festing MF, Simpson EM, Davisson MT, Mobraaten LE. 1999. of a SNP genotyping panel for genetic monitoring of the Revised nomenclature for strain 129 mice. Mamm Genome laboratory mouse. Genomics 83:902-11. 10:836. Petkov PM, Ding Y, Cassell MA, Zhang W, Wagner G, Sargent EE, Asquith S, Crew V, Johnson KA, Robinson P, Scott VE, Wiles ME. 2004b. An efficient SNP system for mouse genome scanning and elucidating strain relationships. Genome Res 14:1806-11.

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14 The Jackson Laboratory Acknowledgements

Senior Editor and Technical Writer: Ray Lambert, M.S. Many people helped to compile, write, and lay out this manual. Special thanks to Muriel Davisson, Ph.D., Beverly Day, B.A., Leah Rae Donahue, Ph.D., Christian Gilbert, B.S., Michael Greene, M.B.A., Mike Kirby, B.A., Chip Leighton, M.B.A., Linda Neleski, Mike Sasner, Ph.D., Laura Trepanier, M.S., Alicia Valenzuela, M.S., Ray VonderHaar, Ph.D., and Michael V. Wiles, Ph.D. “Just as the purity of the chemical assures the pharmacist of the proper filling of the doctor’s prescription, so the purity of the mouse stock can assure a research scientist of a true and sure experiment.”

— Dr. Clarence Cook Little founder of The Jackson Laboratory

Founded in 1929, The Jackson Laboratory is a nonprofit biomedical research institution dedicated to leading the search for tomorrow’s cures. Our mission: We discover the genetic basis for preventing, treating and curing human disease, and we enable research and education for the global biomedical community.

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