IDENTIFICATION AND CHARACTERIZATION OF HYDIN, A LARGE NOVEL GENE DISRUPTED IN A MURINE MODEL OF CONGENITAL HYDROCEPHALUS
DISSERTATION
Presented in Partial Fulfillment of the Requirements for
the Degree Doctor of Philosophy in the Graduate
School of The Ohio State University
By
Brian Davy
*****
The Ohio State University 2004
Dissertation Committee: Approved by Professor Michael L. Robinson, Advisor
Professor Gail E. Herman ______Professor Heithem El-Hodiri Advisor Graduate Program in Molecular, Professor Michael B. Weinstein Cellular and Developmental Biology
ABSTRACT
Congenital hydrocephalus is a frequent human birth defect, occurring with an estimated incidence of 1 in 1000 live births. While a significant portion of human hydrocephalus is genetic in origin, the molecular genetics of this disease remains poorly understood. The murine autosomal-recessive mutation hydrocephalus-3 (hy3) results in lethal communicating hydrocephalus with perinatal onset. A recently described transgenic insertional mutation, OVE459, represents a new allele of hy3. In previous work, the
OVE459 transgene insertion site was cloned, and a wild-type BAC clone encompassing the transgene insertion site was subsequently identified. Here, we describe the isolation of expressed sequences on this BAC clone by direct cDNA selection. Selected cDNAs facilitated the identification of two novel candidate genes, Hydin and Vac14. Hydin consists of at least 86 exons spanning more than 340 kb on mouse chromosome 8. The full-length Hydin transcript is nearly 16 kb and encodes a putative 5099 amino acid protein. Hydin is expressed in compartments of the brain that are directly involved in
CSF production and homeostasis. In situ hybridization revealed Hydin transcripts in the choroid plexus and ciliated ependymal cells lining the ventricles at various stages of development and in the adult animal. Outside the central nervous system, Hydin is specifically expressed in cell-types that possess 9+2 cilia or flagella. The OVE459
ii transgene insertion resulted in a rearrangement within Hydin, disrupting the order of
Hydin exons in these mice. Northern analysis revealed a marked reduction of Hydin mRNA levels in OVE459 and hy3 homozygotes relative to wild-type littermates.
Sequencing of all 87 Hydin exons from homozygous hy3 genomic DNA revealed a single
CG base-pair deletion in exon 15, causing a premature termination signal two codons downstream of the deletion. From this evidence, we conclude that Hydin is the disrupted gene in the OVE459/hy3 mouse model of congenital hydrocephalus. The Hydin gene product does not resemble any previously identified protein with the exception of a 314 amino acid region with homology to caldesmon, an actin-binding protein. To facilitate future investigations concerning Hydin function, a conditional targeting construct generated via recombineering was used to target Hydin exon 15 in ES cells.
iii
Dedicated to Mom, Dad and Jenn
iv
ACKNOWLEDGEMENTS
I would like to thank my advisor, Michael Robinson, for his inspiration, support
and enthusiasm toward this research project. Dr. Robinson has been both advisor and friend, and I am fortunate to have worked in his laboratory. I would like to thank two other members of the Robinson laboratory, Haotian Zhao and Nick Berbari. Haotian was very generous with his time, providing technical advice and expertise. It has been a pleasure to work with Nick Berbari, and I am grateful for his contributions concerning the Hydin expression pattern analysis.
I would also like to thank Gail Herman and David Cunningham for guidance,
intellectual input and stimulating discussions throughout the course of this project. I am
indebted to Michael Weinstein for his time and expertise regarding ES cell targeting. I
am thankful to Robert Munson and Huachun Zhong for providing DNA sequencing
services. I am also thankful to Neil Copeland, Nancy Jenkins and Donald Court for
supplying us with reagents and technical advice for recombineering.
This work was supported financially by Columbus Children’s Research Institute
and The March of Dimes Birth Defects Foundation.
v
VITA
02/18/1973...... Born – Wurzburg, Germany
1997...... B.A. Washington University, St. Louis, Mo.
1998 – 1999...... Teaching Asst. The Ohio State University.
1998 – present ...... Graduate Research Associate The Ohio State University.
PUBLICATIONS
Research Publications
1. Davy, B.E., and Robinson, M. L. 2003. Congenital hydrocephalus in hy3 mice is caused by a frameshift mutation in Hydin, a large novel gene. Human Molecular Genetics 12, 1163-1170.
2. Robinson, M. L., Allen, C. E., Davy, B. E., Durfee, W. J., Elder, F. F., Elliott, C. S., and Harrison, W. R. 2002. Genetic mapping of an insertional hydrocephalus-inducing mutation allelic to hy3. Mammalian Genome 13, 625-632.
FIELDS OF STUDY
Major Field: Molecular, Cellular and Developmental Biology
vi
TABLE OF CONTENTS
P a g e
Abstract...... ii
Dedication...... iv
Acknowledgments ...... v
Vita ...... vi
List of Tables...... xi
List of Figures ...... xii
Chapters
1 Introduction ...... 1
1.1 The ventricular system and cerebrospinal fluid homeostasis...... 1
1.2 Categories and treatment of hydrocephalus ...... 3
1.3 The genetics of human hydrocephalus ...... 4
1.4 Mouse models of congenital hydrocephalus ...... 5
1.5 Cilia and flagella formation, structure and motility...... 9
1.6 Previous Investigations by others concerning the hy3 mutation ...... 11
1.7 Genetic and physical mapping of OVE459, an allele of hy3 ...... 13
1.8 Purpose of this study ...... 14
vii
2 Identification and characterization of candidate genes near the OVE459 transgene insertion site ...... 19
2.1 Introduction ...... 19
2.2 Materials and methods ...... 21 2.2.1 Direct cDNA selection ...... 21 2.2.2 Reverse Transcription-Polymerase Chain Reaction ...... 24 2.2.3 BAC DNA isolation ...... 24 2.2.4 Pulsed-field gel electrophoresis ...... 26 2.2.5 Candidate 1 northern analysis ...... 26 2.2.6 Northern analysis for Candidate 2/3b ...... 27 2.2.7 Mouse and human Hydin cDNA sequencing ...... 28 2.2.8 Mouse genotyping and generation of OVE459 and hy3 congenic lines . . . . . 29
2.3 Results ...... 30 2.3.1 Direct cDNA selection ...... 30 2.3.2 Analysis of selected cDNAs ...... 32 2.3.3 Characterization of Candidate 1 ...... 33 2.3.4 Characterization of Candidate 2 (Hydin) ...... 34
2.4 Discussion ...... 40
3 Hydin expression pattern analysis by in situ hybridization ...... 53
3.1 Introduction...... 53
3.2 Materials and methods ...... 54 3.2.1 Tissue preparation and sectioning ...... 54 3.2.2 In situ hybridization ...... 55
3.3 Results ...... 55 3.3.1 Hydin expression in the central nervous system ...... 55 3.3.2 Hydin expression in the reproductive system ...... 57 3.3.3 Hydin expression in the respiratory system ...... 58 3.3.4 Hydin expression in the inner ear ...... 58
3.4 Discussion ...... 58 3.4.1 The role of ependymal cilia in hydrocephalus ...... 59 3.4.2 Hydin expression in the choroid plexus ...... 60 3.4.3 Hydin expression beyond the central nervous system ...... 61
viii 4 Quantitative analysis of Hydin mRNA levels in hy3/OVE459 mice and identification of a frameshift mutation in the hy3 allele of Hydin ...... 74
4.1 Introduction ...... 74
4.2 Materials and methods ...... 75 4.2.1 Hydin northern analysis ...... 75 4.2.2 Sequencing of Hydin exons from hy3 genomic DNA ...... 76 4.2.3 Allele-specific PCR ...... 77
4.3 Results ...... 77 4.3.1 Testis Hydin mRNA levels are markedly reduced in hy3 and OVE459 mice .77 4.3.2 The hy3 allele of Hydin contains a frameshift mutation in exon 15 ...... 78 4.3.3 Development of the first molecular test for the hy3 mutation ...... 80
4.4 Discussion ...... 80
5 Generation of a Hydin conditional knock-out allele ...... 88
5.1 Introduction ...... 88
5.2 Materials and methods ...... 93 5.2.1 RPCI-22(129S6/SvEvTac) BAC library screening ...... 93 5.2.2 Pulsed field electrophoresis of BAC RP22-193L17 ...... 94 5.2.3 Transformation of BAC RP22-193L17 into recombineering strain EL350 . . 94 5.2.4 Cloning of the pL253-based BAC retrieval plasmid ...... 95 5.2.5 BAC retrieval ...... 96 5.2.6 Cloning of the intron 14 minitargeting vector ...... 97 5.2.7 Cloning of the intron 15 minitargeting vector ...... 98 5.2.8 Intron 14 recombineering ...... 98 5.2.9 CRE-mediated excision of Neo ...... 99 5.2.10 Intron 15 recombineering ...... 99 5.2.11 Determining the targeting cassette functionality by treatment with Cre and Flpe ...... 100 5.2.12 ES cell targeting ...... 100 5.2.13 ES cell DNA isolation and screening ...... 100
5.3 Results ...... 102 5.3.1 Identification and characterization of a suitable BAC clone for retrieval . . .102 5.3.2 Retrieval of BAC DNA into plasmid pL253 ...... 103 5.3.3 Cloning of the intron 14 and intron 15 minitargeting plasmids ...... 104 5.3.4 Minitargeting of introns 14 and 15 ...... 105 5.3.5 ES cell targeting and screening ...... 107
ix 5.4 Discussion ...... 108
6 Conclusions ...... 124
6.1 Hydin encompasses the OVE459 transgene insertion site ...... 124
6.2 Hydin is expressed specifically in compartments responsible for CSF homeostasis and cell types that contain 9+2 cilia and flagella ...... 126
6.3 Hydin mRNA levels are reduced in OVE459 and hy3 mice, and the hy3 allele of Hydin contains a frameshift mutation in exon 15 ...... 128
6.4 ES cells harboring a conditional Hydin allele have been generated ...... 129
6.5 Future investigations concerning Hydin function ...... 130
Appendix ...... 135
Bibliography ...... 147
x
LIST OF TABLES
Table Page
5.1 Summary of 129 BAC library screening ...... 115
A.1 Mouse Hydin RT-PCR primers...... 135
A.2 Human Hydin RT-PCR primers...... 139
A.3 Mouse Hydin exon genomic primers...... 141
xi
LIST OF FIGURES
Figure Page
1.1 CSF production, circulation and absorption pathways ...... 16
1.2 Hydrocephalus and runting in OVE459 homozygotes...... 16
1.3 The OVE459 transgene inserted at the distal end of mouse chromosome 8 . . . . 17
1.4 OVE459 and hy3 are non-complementing mutations …… ...... 17
1.5 Lambda clones BAA and CAA represent opposite sides of the transgene insertion site ...... 18
1.6 Mapping the transgene insertion site by interspecific backcross ...... 18
2.1 Schematic illustration of the direct cDNA selection procedure ...... 42
2.2 cDNAs after successive rounds of selection ...... 43
2.3 Specific selection of polα- and Arx-specific cDNAs using BAC 123D15 as bait ...... 43
2.4 PCR amplification of 54 BAC 218P4-selected cDNAs ...... 44
2.5 Alignment of several 218P4-selected cDNAs to lambda clones BAA and CAA reveals the presence of exons near the OVE459 transgene insertion site...... 45
2.6 Initial designation of three candidate genes near the OVE459 insertion site. . . . 46
2.7 Verification of Candidate 1 by RT-PCR...... 46
2.8 Verification of Candidate 2 by RT-PCR...... 47
xii
2.9 Verification of Candidate 3a by RT-PCR...... 47
2.10 Pulsed field electrophoresis of BAC RPCI-21B7...... 48
2.11 Sources of information used to characterize the genomic structure of Candidate 2...... 48
2.12 Northern analysis for Candidates 3b and 2 revealed that they are part of a single gene...... 49
2.13 The genomic organization of Candidate 2...... 50
2.14 Strategy for sequencing the mouse and human Candidate 2 transcripts...... 50
2.15 Human Candidate 2 northern analysis...... 51
2.16 Genomic structure of wild-type Hydin and the OVE459 transgene insertion locus...... 51
2.17 The OVE459/hy3 candidate region on mouse chromosome 8 and the corresponding region on human chromosome 16...... 52
3.1 Hydin expression in the E12.5 brain...... 64
3.2 Hydin expression in the E15.5 brain and spinal cord...... 65
3.3 Hydin expression in the newborn brain...... 66
3.4 Hydin expression in the newborn cerebral aqueduct...... 67
3.5 Hydin expression in the newborn central canal...... 67
3.6 Hydin expression in the adult brain...... 68
3.7 Hydin expression in the adult cerebral aqueduct...... 69
3.8 Hydin expression in the adult testis...... 69
3.9 Localization of Hydin transcripts to developing spermatocytes...... 70
3.10 Hydin expression in the developing and immature testis...... 71
3.11 Hydin expression in the adult oviduct...... 71
xiii
3.12 Hydin expression in the respiratory system...... 72
3.13 Hydin expression in the inner ear...... 73
4.1 Northern analysis of Hydin and Vac14 in hy3 and OVE459 homozygous mutant and wild-type littermates...... 84
4.2 Procedure for sequencing Hydin exons from hy3 -/- genomic DNA...... 85
4.3 The hy3 allele of Hydin contains a single base-pair deletion in exon 15...... 86
4.4 The wild-type and hy3 alleles of Hydin can be distinguished by allele-specific PCR...... 87
5.1 The lambda-prophage recombination system and DH10B-based recombineering strains...... 111
5.2 The completed Hydin exon 15 targeting plasmid...... 112
5.3 Southern strategy for ES cell screening...... 113
5.4 Identification and characterization of BAC 193L17...... 114
5.5 Retrieval of the targeting region from BAC 193L17 into plasmid pL253. . . . . 116
5.6 Confirmation of clones harboring the retrieved targeting locus...... 117
5.7 Minitargeting plasmid construction...... 118
5.8 Minitargeting plasmid screen...... 119
5.9 Intron 14 minitargeting...... 120
5.10 Confirmation of CRE-mediated excision of NEO in intron 14 by sequence analysis...... 121
5.11 Intron 15 minitargeting and completion of the Hydin CKO plasmid...... 122
5.12 Confirmation of targeted ES cell clones by southern hybridization...... 123
xiv CHAPTER 1
INTRODUCTION
1.1 The ventricular system and cerebrospinal fluid homeostasis
During development, the lumen of the anterior neural tube expands to form the
ventricular system of the brain (1). The ventricular system consists of the lateral, third
and fourth ventricles, which are lined by an epithelial layer of ependymal cells.
Circumventricular organs, such as the choroid plexus and subcommisural organ, reside
within the brain ventricles. The choroid plexus, a leaf-like cluster of capillaries surrounded by specialized ependymal cells, resides on the floor of the lateral ventricles and the roof of the third and fourth ventricles.
The central nervous system is entirely surrounded by cerebrospinal fluid (CSF).
CSF is similar to blood plasma, but the concentrations of ions such as sodium, chloride
and magnesium is slightly greater in CSF. CSF renders the brain and spinal cord buoyant,
providing the CNS protection against rapid movements and trauma. CSF provides a
means for removing waste products of cellular metabolism from the CNS. The CSF also 1 provides nutrients to the CNS, and the presence of biologically active molecules such as hormones and neurotransmitters suggests that CSF functions as a medium through which different compartments of the brain communicate. Finally, CSF provides diagnostic capabilities to clinicians, as its composition can indicate a pathological state of nervous system function.
The majority of CSF is produced by the choroid plexus by a combination of active transport and filtration processes. While the choroid plexus produces 70-80% of CSF, it is predicted that 10-30% of CSF is produced by bulk flow of extracellular fluid from the brain parenchyma across the ependymal layer into the ventricular lumen (2). Production, circulation and absorption of CSF is a continuous process (Figure 1.1). The majority of
CSF is produced by the choroid plexus of the lateral ventricles. From the lateral ventricles, CSF flows through the interventricular foramen into the third ventricle. From the third ventricle, CSF flows into the fourth ventricle via the long, narrow cerebral aqueduct. From the fourth ventricle, the majority of CSF exits the ventricular system through the lateral and median apertures that open into enlargements of the subarachnoid space. A small portion of CSF flows from the fourth ventricle into the central canal of the spinal cord.
The pia and dura layers reside beneath and above the subarachnoid space, respectively. Within the subarachnoid space lies the arachnoid membrane, composed of two membranes held apart by trabeculae. Arachnoid villae extend from the outer arachnoid layer into the dura where they interface with the sagital sinuses. It is at this interface where CSF is reabsorbed into the venous system.
2 1.2 Categories and treatment of hydrocephalus
In adult humans, the choroid plexus produces approximately 500 ml of CSF each day. This volume is four times greater than the capacity of the ventricular lumen, central canal and subarachnoid space. Therefore, the balance between CSF production and reabsorption is critical in maintaining an appropriate intracranial pressure. In addition, efficient communication between the brain ventricles is required to ensure the flow of
CSF from the lateral ventricles to the fourth ventricle. Hydrocephalus results from an excess accumulation of CSF in the ventricles of the central nervous system.
Hydrocephalus can result from the overproduction of CSF by the choroid plexus, defects in reabsorption of CSF in the subarachnoid space or the loss of communication between the ventricles (3). Congenital hydrocephalus results from developmental errors or dysfunction of the ventricular system that is present at birth. Congenital hydrocephalus is subdivided into syndromal (contributing to a typical constellation of malformations) or non-syndromal (isolated hydrocephalus). Acquired hydrocephalus is non-genetic in origin and is typically caused by post-natal infections or injuries that perturb CSF homeostasis.
Obstructive hydrocephalus results from the blockage of CSF flow between the brain ventricles. A common site of blockage in obstructive hydrocephalus is the cerebral aqueduct, the narrow channel through which the third and fourth ventricles normally communicate. Blockage of the cerebral aqueduct results in increased pressure and expansion of the lateral and third ventricles. Communicating hydrocephalus occurs when the volume of CSF and the ventricular lumen increases in the absence of a blockage
3 between the ventricles. The causes of communicating hydrocephalus are typically the overproduction of CSF from the choroid plexus or defects in the reabsorption of CSF into the dural sinuses. In the case of communicating hydrocephalus, all of the ventricles become enlarged and intracranial pressure increases. In infants and young children, where the cranial sutures are not yet closed, hydrocephalus results in an enlargement of the head. In older children and adults, where the size of the skull is fixed, ventricular dilatation results in the compression and thinning of the brain tissue (1).
Untreated, progressive hydrocephalus invariably results in brain damage and death (4). Postnatal progressive hydrocephalus is normally treated by the surgical insertion of a shunt into the ventricular lumen (5). Shunts are typically used to drain excess CSF into the peritoneal cavity (ventriculoperitoneal shunt) or into the right atrium of the heart (ventriculoatrial shunt). The use of shunts has dramatically improved the prognosis of progressive hydrocephalus (6). However, CSF shunts must be maintained throughout the life of the individual and are susceptible to infections and other potentially lethal complications (7, 8). Attempts have been made to treat fetal progressive hydrocephalus in utero using ventriculoamniotic shunts (9). Despite these attempts, the prognosis of fetal hydrocephalus remains poor. Fetuses surviving to birth typically die during the first year of life or suffer from severe mental and physical handicaps (10-12).
1.3 The genetics of human hydrocephalus
Congenital human hydrocephalus occurs with an incidence of approximately 1 in every 1000 live births (4, 11). Using information provided in the U.S. Vital Statistics report for 2002, this rate translates into 4,021 hydrocephalic births per year in the United
4 States alone. While a significant portion of human congenital hydrocephalus is genetic in origin, the molecular genetics of human hydrocephalus remains poorly understood.
L1CAM, residing on the X chromosome, is currently the only gene known to be involved in human congenital hydrocephalus (13, 14). L1CAM, a neural cell adhesion molecule belonging to the immunoglobulin family, is expressed in neurons and Schwann cells (15).
Mutations in L1CAM result in obstructive hydrocephalus attributed to stenosis of the cerebral aqueduct (16). L1CAM mutations appear to be responsible for most cases of X- linked hydrocephalus, which overwhelmingly affects males and occurs at a frequency of
1 in 30,000 male births (17). Autosomal recessive hydrocephalus would be expected to affect males and females equally. Several examples of mixed sex siblings or multiple female siblings having hydrocephalus have been reported. In these cases, autosomal recessive hydrocephalus is suspected. Some of these cases have presented with obstructive hydrocephalus involving stenosis of the aqueduct (18), third ventricle (19), interventricular foramen (20) or the foramina of the fourth ventricle (21). In other families, hydrocephalus appeared to be communicating as obstructions were not observed
(22-24). A particularly interesting group of Palestinian Arab families exhibit autosomal recessive hydrocephalus with a high frequency (25, 26). While these reports demonstrate that autosomal recessive hydrocephalus exists, the causative genes or chromosomal locations associated with these cases remain entirely unknown.
1.4 Mouse models of congenital hydrocephalus
Mice are exceptional models for many human genetic diseases. Mice are easily maintained and propagated, and the genetic resources available for mice is unparalleled
5 by any non-human vertebrate. In addition, the high conservation of gene order between
mouse and human genomic segments allows one to reasonably predict the human
chromosomal location of nearly any gene based on its position in the mouse genome. A
number of genetic mouse models of both obstructive and communicating hydrocephalus
currently exist. Some of these models arose from spontaneous mutations, while others
have resulted from transgenic insertions or gene targeting. Identifying specific genes
involved in hydrocephalus in mice will undoubtedly facilitate our current understanding
of both genetic and non-genetic mechanisms underlying human hydrocephalus.
Obstructive hydrocephalus, typically involving aqueductal stenosis, is a feature of several mouse mutations. The SUMS/NP mouse model exhibits obstruction of the cerebral aqueduct (27). The genetic lesion and mechanism of stenosis in these mice remains unknown. Recent evidence suggests that Reissner’s fiber, an aggregation of glycoproteins (secreted by the subcommisural organ) that runs from the entrance of the cerebral aqueduct into the central canal, plays an important role in maintaining communication between the third and fourth ventricles (2). In some mouse models, it is unclear whether aqueductal stenosis is the primary cause of hydrocephalus, or a secondary event resulting from another underlying defect. Hydrocephaly with hop gait
(hyh) mice exhibit stenosis of the aqueduct, but this is likely to be a secondary defect as it
is preceded by a denudation of both the dorsal and ventral ependymal layers (28, 29).
Two independent groups recently identified the mutation underlying hydrocephalus in
hyh mice (30, 31). The hyh mutation resides in the gene encoding αSNAP, which is
required for apical protein localization and cell fate in neuroepithelial cells (30). An
6 obstructive mechanism may be operating in mice harboring a mutation in non-muscle myosin II-B (32). However, abnormalities of the ventricular surface and communicating hydrocephalus precede aqueductal stenosis in these mice.
Clinically, defects of CSF reabsorption in the subarachnoid space have been emphasized as the prominent cause of communicating hydrocephalus (33). The spontaneous mouse mutation congenital hydrocephalus (ch) resides in a gene on chromosome 13 encoding the transcription factor Foxc1 (34). In the absence of Foxc1, the mesenchyme surrounding the brain fails to differentiate properly, resulting in the absence of a bony skull calvarium and an accompanying absence of the subarachnoid space where CSF is normally reabsorbed (35). Subarachnoid hemorrhage also leads to defective CSF reabsorption and communicating hydrocephalus (36), and transgenic mice over-expressing TGFβ1 develop communicating hydrocephalus attributed to defective
CSF reabsorption. Furthermore, it has been suggested that defective CSF reabsorption in the subarachnoid space causes communicating hydrocephalus in hydrocephalus-3 (hy3) mice.
Many of the current mouse models of communicating hydrocephalus suggest that the primary cause of hydrocephalus may lie in defects of the choroid plexus or ependymal cells. Among these are several genes known or suspected to be involved in cilia function or ciliogenesis including Spag6 (37), Foxj1 (38), orpk (39), Mdnah5 (40) and DPCD (41) (42). The gene underlying hydrocephalus in the mouse mutant hop- sterile (hop) has not been identified, but cilia dysfunction is also the likely cause of hydrocephalus in these mice (43).
7 The ependymal cells lining the brain ventricles are densely coated with motile
9+2 cilia. Cilia are sparse but also present on the specialized ependymal cells covering the choroid plexus, where microvillae are the more prominent apical structures. A genetic association between cilia dysfunction and hydrocephalus has also been reported in rats
(44), dogs (45) and humans (46-51). Primary ciliary dyskinesia (PCD), a term describing human diseases that directly result from congenital cilia defects, is strongly associated with frequent respiratory tract infections, left-right body axis randomization and male infertility. Hydrocephalus is found in only a subset of PCD patients. Similarly, cilia dysfunction in mice does not always manifest in hydrocephalus. Targeted disruption of
MDHC7, encoding a component of the inner arm dynein, reduces cilia beat frequency and male fertility but does not cause situs abnormalities, susceptibility to respiratory infection or hydrocephalus (52). The spontaneous inversus viscerum (iv) allele of left- right dynein (lrd, also known as Dnah11) disrupts cilia motility at the embryonic node, resulting in situs inversus (53). However, cilia function at later development stages appears normal, and iv homozygotes do not exhibit defects in respiratory clearance, fertility or CSF homeostasis (54). The genetic association between cilia dysfunction and hydrocephalus is likely to be complex.
The mechanism by which cilia dysfunction disrupts CSF homeostasis is unclear.
The beating of ependymal cilia is sufficient to create local currents close to the
ventricular surface toward the predicted direction of bulk CSF flow (55). Currents at the
ventricular surface may assist in the clearance of metabolites and toxins from the brain,
as they would create a diffusion gradient between the brain parenchyma and ventricular
8 lumen. However, it is improbable that the beating of ependymal cilia alone significantly contributes to the bulk flow of CSF through the ventricular system. Rather, the pulsing of
CSF from the choroid plexus vasculature during systole appears to serve as the primary driving force behind bulk CSF flow (56). It has been proposed that cilia motility is especially important to facilitate laminar flow of CSF through the narrow channels (i.e. the cerebral aqueduct) of the ventricular system (57). This hypothesis remains to be established and is not easily tested.
Mice with targeted mutations in E2F5 (58) and Nfia (59) also exhibit communicating hydrocephalus. E2F5 is highly expressed in ependymal cells covering the choroid plexus and the ependymal layer lining the brain ventricles, and E2F5 mutant mice exhibit ultrastructural abnormalities in choroid plexus ependymal cells (58). Defects in the choroidal ependyma suggest that overproduction of CSF is responsible for hydrocephalus in E2F5 null mice. The mechanism of hydrocephalus in Nfia mutants is unclear. However, it is notable that surviving mutant males, but not females, appear to be infertile (59).
1.5 Cilia and flagella formation, structure and motility
Cilia and flagella are ancient organelles that initially served to provide motility to unicellular organisms in water. Formation of a cilium and flagellum is initiated by the movement of a centrosome to the cell surface, where it is subsequently referred to as the basal body (for review see 60). The basal body consists of nine triplet microtubules and nucleates the formation of axonemal microtubule filaments. Tubulin dimers consisting of
9 α and β tubulin subunits are subsequently transported to the distal (positive) end of the growing cilium/flagellum by the kinesin family of molecular motor proteins.
The classical 9+2 structure of motile cilia and flagella consists of nine peripheral microtubule doublets surrounding a central pair of microtubules (for review see 61). Each peripheral doublet is composed of an A and B tubule, each with 13 and 11 protofilaments, respectively. In 9+0 primary cilia, which function predominantly as sensory organelles, the central microtubule pair is absent. In 9+2 cilia, each outer doublet is connected to the central pair by a complex of proteins collectively referred to as the radial spoke. Cilia with 9+2 ultrastructure are found on the ependymal surface, respiratory epithelium, the luminal surface of the oviduct and the efferent ducts of the testis. Primary cilia with 9+0 ultrstructure are found on the embryonic node, rod photoreceptors of the retina, olfactory epithelium and tubular cells of the kidney. With the exception of a specific population of nodal cilia, 9+0 cilia serve a sensory rather than motile function.
Cilia and flagella motility is achieved by the ATP-dependent attachment of the dynein arm of an A tubule to the B tubule of an adjacent doublet. Once attached, the dynein arm moves along the B tubule toward the minus (proximal) end of the axoneme.
Inter-doublet sliding is prevented by nexin links that connect the peripheral doublets at regular intervals along the axoneme. Thus, the movement of the dynein arm down the B tubule results in the bending of the axoneme and movement of the cilium or flagellum.
10 1.6 Previous investigations by others concerning the hy3 mutation
The spontaneous autosomal recessive mutation hy3 was originally identified by
Hans Gruneberg in 1941 (62). It was named hydrocephalus-3 because it was the third hydrocephalus mutation identified in mice. Unfortunately, the first two hydrocephalus mutations (hydrocephalus-1 and hydrocephalus-2) are extinct, and their relationship to hy3 or other hydrocephalus mutations cannot be determined. Homozygous hy3 mice are indistinguishable from wild-type littermates at birth but develop progressive hydrocephalus that is externally apparent by 3 to 5 days after birth. hy3 homozygotes do not survive beyond six weeks of age, and most die prior to weaning. In the original hy3 stock, Gruneberg described a hydrocephalus-inducing mutation with incomplete penetrance that was accompanied by a nasal discharge consisting of epithelial cells and polymorphonuclear leukocytes (62). In subsequent generations, the penetrance and severity of hydrocephalus increased, while the accompanying nasal discharge was lost
(63). A study where dye was injected into the ventricles of hydrocephalic hy3 mice indicated that there were no obstructions of CSF flow between the ventricles, but the dye never entered the subarachnoid space (63). This study indicated that hy3-induced hydrocephalus is communicating, and it was concluded that defects in CSF reabsorption are the likely cause of hydrocephalus in these mice. In the later stages of hydrocephalus, stenosis of the cerebral aqueduct was observed (64), but this is clearly a secondary defect rather than an initiating event in hy3 pathogenesis. Adhesion of the skull to the brain, characteristic of alterations in the meninges, has been observed in hy3 homozygotes (63,
65). Raimondi and McLone published several reports in the 1970s concerning the
11 pathogenesis of hydrocephalus in hy3 homozygotes (64-67). These investigators noted a significant increase in the extracellular space of the white matter and increased vacuolar size in the cytoplasmic sheath surrounding many axons. These abnormalities are presumably the result of edema. Severe edema in the subependymal layer and an increase in clear vacuoles of the ependyma was also observed. Bannister examined the ependymal surface of hy3 homozygotes in two reports (68, 69). Examinations moving from the basal portions to the lateral walls to the roof of the lateral ventricles revealed a progressive loss of ependymal cilia and eventually the ependymal cells. Bannister suggested that the loss of cilia and ependymal cells in hy3 homozygotes was the result of, rather than the cause of hydrocephalus in these mice (69). A potential problem with the reports of Raimondi and McLone, as well as Bannister, is that examinations on hy3 mice were conducted well after the onset of hydrocephalus. No examinations were performed on hy3 mice prior to
10 days of age, while hydrocephalus in these animals begins around the time of birth. We believe it is difficult to interpret the primary versus secondary aspects of hydrocephalus after 10 days of age in homozygous hy3 mice due to the severity of ventricular dilatation.
Prior to work performed in the Robinson laboratory, the consensus linkage map of mouse chromosome 8 placed hy3 at 58 centiMorgans (cM) from the centromere (Mouse
Genome Informatics, The Jackson Laboratory). The assignment of this map position was based on an unpublished three point test cross performed by M. C. Green and communicated to the Jackson Laboratory in 1970. This cross placed hy3 17 cM distal to oligosyndactylism (Os) placed at 40 cM, and 11 cM proximal to e (the melanocortin 1
12 receptor) placed at 68 cM on mouse chromosome 8 (Jeff Ceci, Mouse Chromosome 8
Committee, personal communication).
1.7 Genetic and physical mapping of OVE459, a new allele of hy3
Our interest in the hy3 mutation was sparked by the transgene-induced insertional mutation OVE459 (70). The OVE459 transgenic construct was designed to express brain derived neurotrophic factor (BDNF) in the developing lens via the murine αA-crystallin promoter. Ocular abnormalities were not observed in either the transgenic founder or any subsequent transgenic mice in line OVE459. However, mice homozygous for the transgene developed hydrocephalus with perinatal onset that was most often fatal prior to weaning (Figure 1.2). Fluorescence in situ hybridization (FISH) analysis revealed that the
OVE459 transgene inserted at a single site near the distal end of mouse chromosome 8
(Figure 1.3). The homozygous phenotype and the physical location of the transgene insertion site suggested that the OVE459 and hy3 mutations might be allelic. This hypothesis was confirmed by the failure of the OVE459 mutation to complement the hy3 mutation in double heterozygous animals (Figure 1.4). Of the 355 double heterozygotes examined, 79 (22.3%) exhibited lethal hydrocephalus that was indistinguishable from that found in homozygotes of either mutation.
To clone the transgene insertion site, a lambda phage genomic library was constructed from OVE459 homozygous mutant DNA and screened using a transgene- specific probe. Cloned genomic inserts from each end of the transgene array were identified and completely sequenced. These clones, arbitrarily named BAA and CAA,
13 contained inserts of approximately 11 kb and 13 kb, respectively (Figure 1.5). A PCR
polymorphism on the CAA side, designated D8Mlr1, was used to screen DNA from 188
animals from the combined Jackson BSS and BSB interspecific backcross mapping panel
from the Jackson Laboratory Backcross DNA Panel Mapping Resource. D8Mlr1 mapped unambiguously to mouse chromosome 8 and did not recombine with the genetic marker
D8Mit151 (Figure 1.6). This position corresponds to 54 cM on the consensus linkage map for mouse chromosome 8 and corresponds to human chromosome 16q21-23.
Genomic DNA flanking each end of the transgene array was also used to screen a wild- type mouse bacterial artificial chromosome (BAC) library from Research Genetics. One clone, BAC 9N1, was positive for the genomic sequence on the BAA side of the transgene insertion site. A second clone, BAC 218P4, contained sequences from both sides of the insertion site. The genomic insert size of BAC 218P4 is approximately 120 kb, physically linking the BAA and CAA sides of the transgene insertion site within this interval.
1.8 Purpose of this study
While autosomal recessive hydrocephalus clearly exists in human populations
(22-26), no causative genes have been identified. The purpose of this research project is to identify the gene, and corresponding genetic lesion, responsible for autosomal recessive congenital hydrocephalus in OVE459 and hy3 mice. Furthermore, it is our goal to understand the functional role of this gene in CSF homeostasis. As mouse models have provided valuable insights into human disease mechanisms, it is anticipated that the
OVE459/hy3 model of autosomal recessive congenital hydrocephalus will shed light on
14 the etiology of human hydrocephalus. Ultimately, we would like this study to contribute to diagnostic capabilities and therapeutic strategies for the early identification and effective treatment of a subset of hydrocephalus in humans.
15 Figure 1.1. CSF production, circulation and absorption pathways. A schematic illustration of the adult rat brain is shown (saggital view). The choroid plexus within each ventricle is boxed in blue. The lateral, 3rd and 4th ventricles, as well as the cerebral aqueduct (Aq) are shown in red. Black arrows indicated the direction of CSF flow through the ventricles. Double arrows (in opposite orientation) indicate the exchange of CSF between the brain parenchyma and ventricular lumen. Bulk CSF flow through the ventricles occurs in a rostrocaudal direction. After leaving the 4th ventricle, CSF either enters the central canal or the subarachnoid space, where it is drained into the venous system through the arachnoid villi (boxed in green).
Figure 1.2. Hydrocephalus and runting in OVE459 homozygotes. Heterozygous (top) and homozygous (bottom) OVE459 liitermates are shown. Mice homozygous for the transgene are runted and exhibit progressive, lethal hydrocephalus.
16 Figure 1.3. The OVE459 transgene inserted at the distal end of mouse chromosome 8. (A) A portion of a metaphase spread from a hydrocephalic OVE459 homozygous transgenic animal. (B) Hybridization using a fluorescent-labeled transgene-specific probe reveals the location of a single transgene insertion site on chromosome 8D3.
Figure 1.4. OVE459 and hy3 are non-complementing mutations. (A) A coronal section of an OVE459 hemizygous brain with normal appearance. (B and C) OVE459 and hy3 homozygotes exhibit dilatation of the lateral (asterisks) and third (arrow) ventricles characteristic of hydrocephalus. (D) Hydrocephalus is present in OVE459/hy3 double heterozygotes, indicating that these two mutations fail to complement each other.
17 Figure 1.5. Lambda clones BAA and CAA represent opposite sides of the OVE459 transgene insertion site. Diagrammatic representation of the lambda phage clones BAA and CAA representing opposite ends of the OVE459 transgene insertion site. The location of the T3 primer used for sequencing and the PCR primers B3/B4 and C1/C2 are indicated with arrowheads. Each phage clone contains tandem copies of transgene (broken gray line) linked to the 23-kb phage arm, and mouse genomic DNA (solid black line) linked to the 9-kb phage arm. Restriction enzyme sites shown are SalI (S) and NotI (N).
Figure 1.6. Mapping the transgene insertion site by interspecific backcross. Haplotype figure combining data from The Jackson BSB and BSS backcrosses showing part of Chr 8 with loci linked to D8Mlr1. Loci are listed in order with the most proximal at the top. The black boxes represent the C57BL6/JEi allele, and the white boxes the SPRET/Ei allele. The number of animals with each haplotype is given at the bottom of each column of boxes. The percentage recombination(R) between adjacent loci is given to the right of the figure, with the standard error (SE) for each R.
18 CHAPTER 2
IDENTIFICATION AND CHARACTERIZATION OF CANDIDATE GENES NEAR THE OVE459 TRANSGENE INSERTION SITE
2.1 Introduction
The preliminary objective of this research project was to identify the gene responsible for hydrocephalus in OVE459 and hy3 mice. Investigations performed prior to my entrance in the lab laid the groundwork for achieving this goal (70). Fluorescence in situ hybridization using a transgene-specific probe determined that the OVE459 transgene array integrated at a single site on the distal portion of mouse chromosome 8
(see Figure 1.3). A homozygous transgenic genomic phage library was generated and screened for the presence of the transgene. Lambda clones BAA and CAA, encompassing the transgene insertion site, were positively identified and sequenced (see Figure 1.4).
Utilizing a polymorphism between FVB/N and SPRET/Ei genomic DNA derived from clone CAA (D8Mlr1), high resolution mapping of the transgene insertion site was carried out using the Jackson Laboratory Backcross DNA Panel Mapping 19 Resource (see Figure 1.6) (71). This work placed the OVE459 transgene insertion site at
54 cM on the mouse genome informatics Chromosome 8 consensus linkage map (72).
This position is 3 cM distal to the mapped position of the hy3 mutation by three-point test cross (Jeff Ceci, personal communication). A breeding experiment confirmed that
OVE459 and hy3 are alleles of the same gene. To isolate wild-type genomic DNA encompassing the transgene insertion site, the Research Genetics 129/Sv CITB-CJ7 BAC library was screened by PCR using primers specific to lambda clone BAA. Two BAC clones, CITB-CJ7-218P4 (BAC 218P4) and CITB-CJ7-9N1 (BAC 9N1), were positive for the BAA-specific amplification product. BAC 218P4 was subsequently found to contain genomic DNA present on both phage clones, indicating that this clone contained a wild-type insert encompassing the transgene insertion site.
When I joined the Robinson laboratory, the initial goal was to identify genes in the vicinity of the transgene insertion site. At this time, however, the sequencing and annotation of the mouse genome was incomplete. Furthermore, the EST databases were sparse, and no ESTs corresponding to sequences on either lambda clone BAA or CAA existed in these databases.
We hypothesized that hydrocephalus in OVE459 mice was the result of a disrupted gene near the OVE459 transgene insertion site. All or parts of this gene were likely present on BAC 218P4. In order to identify genes near the transgene insertion site, a method to isolate expressed sequences on BAC 218P4 was needed. One such method, direct cDNA selection, had been used successfully by other investigators to isolate expressed sequences on large genomic clones (73, 74). We decided to employ direct
20 cDNA selection using BAC 218P4 as the driver. This chapter describes the direct cDNA
selection experiment and subsequent identification of the two candidate genes closest to the site of the OVE459 transgene integration locus.
2.2 Materials and Methods
2.2.1 Direct cDNA Selection
The protocol we employed for cDNA selection was adapted from Segre et al.
(73). Total RNA was isolated from E17.5, P0 and P2 heads using Trizol reagent
(Invitrogen). PolyA+ RNA was generated from 250 µg of pooled total RNA using the
Oligotex mRNA Purification Kit (Qiagen). cDNA was generated using the cDNA
Synthesis System (Invitrogen) and digested with AluI, HaeIII and RsaI in 20 µL reactions
(0.33 µg cDNA per reaction). The three digests were combined, purified using QIAquick
PCR purification columns (Qiagen) and eluted in 50 µL TE pH 8.0.
Oligonucleotide linkers were ligated to the pooled brain cDNAs and have the
following sequence: cDNA-1: 5’-CTGAGCGGAATTCGTGAGACC-3’, cDNA-2: 5’-
Phosphate -GGTCTCACGAATTCCGCTCAGTT-3’. The two oligos were diluted to 10
µM. Equal amounts of each oligo were combined to make a 10X cDNA linker mix. The
oligos were heated at 65OC for 5 minutes and allowed to anneal at RT. Two microliters of
10X cDNA linker mix was ligated to 5 µL digested cDNAs in an 85 µL reaction for 2
hours at RT. Ligation products were purified using a QIAquick spin PCR purification
column and eluted in 50 µL TE pH 8.0. One microliter of linkered cDNA was PCR-
amplified using 10 µL cDNA oligo 1 (20 µM) in a 100 µL reaction. The PCR program
21 was as follows: 94O 3’ for 1 cycle, 94O 45 sec, 64O 60 sec, 72O 90 sec for 14 cycles, 72O 3 min, 12O hold.
To prepare BAC 218P4 and BAC 123 for hybridization, 100 ng of BAC DNA
was digested with AluI, HaeIII and RsaI in individual 20 µL reactions. BAC digests were
heat inactivated, pooled and purified using QIAquick PCR purification columns.
Biotinylated oligonucleotide linkers were ligated to the digested BAC DNA and have the
following sequence: BIOBlunt-1: 5’- Biotin - GCGGTGACCCGGGAGATCTGAATTC
-3’, Blunt-2: 5’- Phosphate - GAATTCAGATC -3’. Linkers for BAC DNA were diluted,
combined and annealed in an identical manner as the cDNA linkers. 300 ng of BAC
DNA was used in a 100 µL ligation reaction containing 1.6 µL BIOblunt linkers.
Linkered BAC DNA was purified using a QIAquick column and eluted in 50 µL. Five
microliters of linkered BAC DNA was PCR-amplified using 5 µL BIOblunt-1 oligo (20
µM) in a 100 µL reaction. The PCR program was as follows: 65O 5 min for 1 cycle, 94O
45 sec, 72O 2.5 min for 14 cycles, 4O hold.
For prehybridization of PCR-amplified, linkered cDNAs, 2 µg mouse Cot-1 DNA
(Invitrogen) and 10 ng empty BAC vector (pBeloBac11) was added to 1 µg cDNA mix.
The resulting mixture was ethanol precipitated and resuspended in 5 µL double distilled water (ddH2O). The cDNA mix was denatured at 95O for 5 minutes and snap frozen. 5
µL of 2X hybridization buffer (1.5 M NaCl, 40 mM NaPhosphate pH 7.2, 10 mM EDTA,
10X Denhardt’s Solution, 0.2% SDS) was added to the denatured cDNA mix and incubated for 4 hours at 65O. For hybridization, 5 µL containing 100 ng BAC DNA was denatured for 5 minutes at 95O and snap frozen. Five microliters of 2X hybridization 22 solution and the 10 uL cDNA/blocker mix were added to the denatured BAC DNA.
Hybridization was performed at 65O for 54 hours. Hybridization reactions were
terminated by adding 100 µL of bead binding buffer (10 mM Tris pH 7.5, 1 mM EDTA,
1 mM NaCl).
The cDNA/BAC mixture was transferred to a fresh tube containing streptavidin-
conjugated Dynabeads (Dynal) in 100 µL bead binding buffer. The resulting mixture was
vortexed, incubated for 15 minutes at RT and concentrated for 1 minute using a magnetic
particle concentrator. Following removal of the supernatant, beads were washed twice at
room temperature in 1X SSC/0.1% SDS. The beads were then washed three times at 65O in 0.1X SSC/0.1% SDS. cDNAs were eluted by adding 50 µL 50 mM NaOH and incubating for 10 minutes at RT. After concentrating the beads, the cDNA-containing supernatant was transferred to a fresh tube and neautralized with 50 µL 1M Tris pH 7.5.
Eluted cDNAs were purified using a QIAquick column and eluted in 50 µL TE pH 8.0.
Five microliters of eluted cDNAs were PCR-amplified in a 100 µL reaction using 5 µL cDNA-1 oligo as the primer. The PCR program was as follows: 94O 45 sec, 64O 45 sec
72O 75 sec for 25 cycles. The amplified selected cDNAs were then subjected to a second round of hybridization/amplification as performed previously.
Two microliters of doubly-selected cDNAs were subcloned into 50 ng pT7Blue
vector (Novagen) in a 10 µL ligation reaction. Subcloned cDNAs were transformed into
XL1Blue E. coli. The insert sizes of isolated clones were checked by PCR using 1.25 µL
of the cDNA-1 oligo (20 µM) as the primer in 25 µL reactions. The PCR program was as
follows: 94O 5 min for 1 cycle, 94O 1 min, 64O 1 min, 72O 90 sec for 30 cycles.
23 2.2.2 Reverse Transcription-Polymerase Chain Reaction (RT-PCR)
An RT mix was prepared containing 3 µL 10X PCR buffer (Invitrogen), 6 µL 5 mM dNTP mix, 1 µL RNase inhibitor (10 U/µL), 1.5 µL 50 µM random hexamers, 3 µL
50 mM MgCl2 and 3 µL 100 mM DTT. To denature RNA, 10.5 µL of DEPC-treated
ddH2O containing 5 µg total RNA was heated at 95OC for 5 minutes and snap frozen.
RNA and 2 µL Superscript II reverse transcriptase (Invitrogen) was added to the RT mix, vortexed and spun briefly. Reactions were carried out at 40 OC for 1 hour and stored at -
20 OC prior to use for PCR. PCR amplification of cDNAs was carried out by adding 1.5
µL RT reaction to a mix containing 5 µL 10X PCR buffer, 3 µL DMSO, 4 µL 5 mM dNTP mix, 1.5 µL 50 mM MgCl2, 1 µL forward primer (20 µM stock), 1 µL reverse primer (20 µM stock), 0.3 µL Taq polymerase (Invitrogen) and 32.7 µL ddH2O. Standard cycling parameters for PCR were as follows: Denaturation at 95O for 2 min, 34 cycles of
O O O O [94 40 sec, TA 40 sec, 72 45 sec], 72 7 min, 4 hold. Usually, 5 uL of PCR reaction was used for analysis by gel electrophoresis.
2.2.3 BAC DNA Isolation
This protocol is a modified version of the Qiagen plasmid midiprep protocol.
Three ml LB supplemented with 20 µg/ml chloramphenicol was inoculated with a single colony and grown O/N at 37OC, 250 rpm. The next day, 100 ml of LB-chloramphenicol in a 1 liter flask was inoculated with 100 µl of culture and shaken O/N at 37OC, 250 rpm.
The next day, cells were pelleted at 6000 x g for 15 minutes at 4OC in a 250 ml centrifuge bottle. The supernatant was thoroughly drained, and the cell pellet was resuspended in 15 mL Qiagen buffer P1 by vortexing and pipetting. Fifteen ml of Qiagen buffers P2 and P3 24 were added sequentially according to the Qiagen protocol and lysates were incubated for
15 minutes at 4OC. The cell debris was pelleted at 20,000 x g for 30 minutes at 4 OC. The supernatant was carefully poured through a Nucleobond filter (Clontech) into two fresh
50 ml conical tubes.
A Qiagen Tip-100 column was equilibrated with 10 mL Qiagen buffer QBT, and the filtered lysate was applied to the column. The column was washed with 3 x 10 ml Qiagen buffer QC, and BAC DNA was subsequently eluted with 2 x 2.5 ml 65OC Qiagen buffer
QF. To precipitate BAC DNA, 3.5 ml isopropanol was added to the eluent, mixed immediately and aliquoted into six 1.5 ml eppendorf tubes. The tubes were immediately spun at 21,000 x g in a 4OC microcentrifuge for 30 minutes. The supernatant was
carefully poured off, and 750 µL of 70% EtOH was added. Tubes were inverted gently
several times and spun at 21,000 x g in a 4OC microcentrifuge for 30 minutes. The
supernatant was carefully poured off, and tubes were spun at 21,000 x g in a
microcentrifuge for 30 seconds. The remaining supernatant was discarded, and pellets
were dried for no more than 5 minutes (avoid over-drying) on the benchtop. Pellets were
gently resuspended in 15 µL 10 mM Tris-Cl pH 7.5 using a wide-bore pipette tip. The
DNA was incubated at 65OC for 5 minutes with the caps open, followed by a 20 minute
incubation at 65OC with the caps closed. DNA was pipetted gently up and down using a wide bore pipette tip, pooled into a single 1.5 mL eppendorf tube and quantitated using a spectrophotometer.
25 2.2.4 Pulsed-Field Gel Electrophoresis
250 ng of the NotI-digested BAC 21B7 was loaded onto a 1% agarose gel in 0.5X
TBE buffer (without ethidium bromide). DNA was electrophoresed in 14OC 0.5X TBE
running buffer using the CHEF MAPPER system (Bio Rad). The Auto Algorithm
program with a size range between 7 and 300 kilobases was used. On the following day,
the gel was stained for 30 minutes in 0.5 mg/ml ethidium bromide in 0.5X TBE and
destained in 0.5X TBE for 20 minutes.
2.2.5 Candidate 1 Northern Analysis
Total RNA was isolated from five P0 brains using Trizol reagent. mRNA was
generated by passing 1.5 mg of total RNA in 0.5 M LiCl through an oligo(dT) cellulose
column equilibrated with bead binding buffer (0.5 M LiCl, 10 mM Tris-Cl pH 7.5, 1 mM
EDTA, 0.1% SDS). Columns were washed with bead binding buffer, and the eluant was
passed through the column twice more. mRNA was eluted with 2 mM EDTA/0.1% SDS,
ethanol precipitated and resuspended in 50 µL DEPC-treated ddH2O. Fifteen ml of
formaldehyde loading buffer was made, containing 7.2 mL formamide, 1.6 mL 10X
MOPS buffer, 2.6 mL 37% formaldehyde, 1.8 mL DEPC ddH2O, 1.0 mL glycerol and
0.8 mL saturated bromophenol blue. Formaldehyde loading buffer was added to five
micrograms of Poly(A)+ RNA to a total volume of 20 µL.
Electrophoresis of the mRNA/loading buffer mixture was performed using a
MOPS/formaldehyde 1% agarose gel. To prepare the gel, 1 g of agarose was boiled in 85 mL DEPC ddH2O and cooled to 55OC. Ten mL of 10X MOPS buffer and 5.5 mL of 37%
formaldehyde were added to the molten agarose, mixed by swirling and poured into a
26 mini electrophoresis apparatus. Note: 500 ml of 10X MOPS buffer is made by adding
20.9 g MOPS to 400 mL DEPC ddH2O, adjusting the pH to 7 with NaOH, adding 10 mL
of 0.5 M EDTA pH 8.0 and bringing the volume up to 500 ml. The mRNA/loading buffer
mixture was loaded and electrophoresed at 60 V for 3 hours. mRNA was transferred to
Hybond N+ (Amersham) membrane overnight in DEPC-treated 10X SSC, dried and UV
irradiated.
The Candidate 1-specific probe was derived from a 426 bp RT–PCR product
amplified from newborn FVB/N total brain RNA using the primer pair C1-2S (5’-
GGCCTATGATGACCGCAAGAAAAG-3’) and C1-2A (5’-
TGAAGTAAGATGGGGAAGAGGCT-3’). Probes were labeled with 32P-dCTP using the Random Primers DNA Labeling System (Invitrogen) and purified using Mini Quick
Spin DNA columns (Roche). For hybridization, purified probe was added to 8 mL
ExpressHyb solution (Clontech) to a final concentration of 1,000,000 cpm/ml.
Hybridization was carried out for 2 hours at 65OC. The membrane was rinsed several times with room temperature 2X SSC/0.05% SDS, washed for 40 minutes at 25OC in 2X
SSC/0.05% SDS and washed once more at 50OC in 0.1X SSC/0.1% SDS for 40 minutes.
To detect the Candidate 1 transcript, autoradiography film was exposed to the membrane for 4 hours.
2.2.6 Northern Analysis for Candidate 2/3b
Total RNA was isolated from four adult FVB/N testes using Trizol reagent.
PolyA+ RNA was generated as described for Candidate 1 northern analysis. Five micrograms of polyA+ or total testis RNA was electrophoresed under identical conditions
27 as those for Candidate 1. The probe used for detection of Candidate 3b was a 32P-labeled
RT-PCR product generated using the primer set C3-2a/2b (444 bp) (C3-2a: 5’-
ACCCCGTCAGGATGGAGTTGTA-3’, C3-2b: 5’-CCCAGTTGTTCAGGTCGTTTCT-
3’). The probe used for detection of Candidate 2 was a 32P-labeled cocktail of two RT-
PCR products generated using the primer sets C2-12a/12b (385 bp) and 3’-1a/1b (428 bp)
(C2-12a: 5’-GAGAACAAGGTCCTATTTTG-3’, C2-12b: 5’-
AGTCCAGAACTCTTCCGTG -3’, 3 prime-1a: 5’-TCTACGAGGTGGAGTTGAAT-
3’, 3 prime-1b: 5’-GTGTGGGGTGTTGGTGTA-3’). Labeling and purification of probe, hybridization and washes were performed as described for Candidate 1. For autoradiography, film was exposed to the membrane for various lengths of time. The optimal exposure time was 1 hour and 15 minutes.
2.2.7 Mouse and Human Hydin cDNA Sequencing
Amplification of Hydin cDNA was carried out in 50 µl reactions using Pfu Turbo polymerase (Stratagene). Each reaction contained 5 µl 10X Pfu buffer (supplied with
polymerase), 5 µl 5mM dNTPs, 4 µl DMSO, 1.5 µl of RT reaction, 1 µl each primer (20
uM stock), 0.5 µl Pfu polymerase and 32 µl ddH2O. The sequences of each primer used
for amplification of the mouse and human transcripts are provided in Tables A.1 and A.2,
respectively. Following gel electrophoresis of each 50 µl reaction, amplification products
were isolated using Montage gel purification columns (Millipore).
For sequencing, 1.5 µl of purified PCR product was used as the template in a 10
µl reaction containing 0.5 µl DMSO, 0.8 µl 2 µM primer (1.6 pmoles), 4 µl Big Dye v1.1
sequencing mix (Applied Biosystems) and 3.2 µl ddH2O. The sequencing program was
28 as follows: 96O 1 min, 96O 10 sec, 50O 5 sec, 60O 4 min, repeat steps two through four 24 more times, 4O hold. Sequencing products were purified using Performa DTR Gel
Filtration Cartridges (Edge Biosystems), lyophilized in a speed vac and submitted to the core sequencing facility at Columbus Children’s Research Institute.
2.2.8 Mouse Genotyping and Generation of OVE459 and hy3 Congenic Lines
Tail or toe DNA was prepared using the HotShot method (75). For detection of the OVE459 transgene, the PCR primer pair PR4/11421 is used. Primer sequences are as follows: PR4: 5’-GCATTCCAGCTGCTGACGGT-3’, 11421: 5’-
ACACCTGGGTAGGCCAAGCCACCTT-3’. The reaction mix for OVE459 genotyping
consists of 5 µL 10X PCR buffer, 3 µL DMSO, 4 µL 5 mM dNTP mix, 1.5 µL 50 mM
MgCl2, 1 µL PR4 primer (20 µM), 1 µL 11421 (20 µM), 0.3 µL Taq polymerase
(Invitrogen) and 32.7 µL ddH2O. Each reaction contained 1.5 µL of HotShot DNA. The
PCR program is as follows: Denaturation at 95O for 3 min, 32 cycles of [94O 40 sec, 58O
40 sec, 72O 40 sec], 72O 7 min, 4O hold. Ten microliters of the PCR reaction was used for
analysis by gel electrophoresis.
Lines OVE459 and hy3 are on FVB/N and B6CBACa Aw-J genetic backgrounds, respectively. To generate congenic OVE459 and hy3 lines, mice were bred onto C57BL/6
and FVB/N backgrounds, respectively. The region of chromosome 8 harboring the
mutation was maintained utilizing the flanking polymorphic markers D8Mit248 and
D8Mit215, located at 93.9 Mb and 117.6 Mb (Ensemble mouse genome assembly version
20.32b.1), respectively. Primer sequences are as follows: D8Mit248-F: 5’-
CTAGATCCCTCAAGCAGTACCCCTAC-3’, D8Mit248-R: 5’-
29 AGCCAGTGAGCAGAGGACCACACCTTACA-3’, D8Mit215-F: 5’-
AATACACAAGGTTGGCCTCA-3’, D8Mit215-R: 5’-
ATGTGTGGATATTCATGTGCTC-3’. The PCR reaction mix and cycling parameters
were identical to those used for the OVE459 transgene, except the optimal annealing
temperature for D8Mit248 is 60OC. For both markers, the FVB/N amplification product is slightly smaller than the C57BL/6 or hy3 product. Heterozygous mice for each
successive generation were bred back to pure FVB/N or C57BL/6 mice. Both lines were
bred beyond generation N10 before continued maintenance by intercrossing.
2.3 Results
2.3.1 Direct cDNA Selection
Direct cDNA selection was performed to isolate expressed sequences on BAC
218P4. Selection of cDNAs using another clone, BAC 123D15, was also performed in parallel. BAC 123D15 served as a positive control for cDNA selection, as this clone harbors two known genes that are expressed in the neonatal brain, Polymerase-α (Polα) and Arx (Dr. Gail Herman, personal communication). A schematic illustration of the direct cDNA selection procedure is presented in Figure 2.1. The essence of direct cDNA selection is the specific hybridization of cDNAs to expressed sequences (exons) on large genomic clones from which those cDNAs are derived. cDNAs that hybridize to expressed sequences on the genomic clone (selected cDNAs) are then purified from non-selected cDNAs, eluted from the genomic DNA and cloned.
The cDNA selection procedure began by isolating total head RNA (brain and
overlying tissues, including skull) from embryonic day 17.5, newborn and two day old
30 mice. These time-points were chosen because they either precede or coincide with the
onset of hydrocephalus in OVE459 and hy3 homozygotes. Entire heads were used, rather
than brain alone, because a previous report suggested that defective reabsorption of CSF
in the subarachnoid space is the cause of hydrocephalus in these mice (63). Poly(A)+
RNA from each time- point was isolated, pooled and reverse transcribed to generate the
cDNA population. cDNAs were digested into smaller fragments using the frequent
cutters AluI, HaeIII and RsaI. Oligonucleotide linkers were ligated to the digested
cDNAs to facilitate amplification by PCR. To prepare BACs 218P4 and 123D15 for
selection, these clones were digested with the same enzymes (AluI, HaeIII and RsaI) used
to digest the cDNAs. Biotinylated linkers were ligated to the digested BAC fragments to facilitate the retrieval of BAC-cDNA hybrids following hybridization.
The actual selection of cDNAs occurred during a hybridization reaction between the cDNA and BAC fragments. Following hybridization, BAC fragments (some of which
consisted of BAC-cDNA hybrids) were purified from non-hybridizing cDNAs using
streptavidin-conjugated magnetic beads and washed. cDNAs were eluted, PCR amplified
using linker-specific primers and subjected to a second round of selection. An expected
decrease in cDNA abundance was observed following the second selection (Figure 2.2).
To assess specificity, cDNAs selected with BAC 218P4 and BAC 123D15 were tested by
PCR using primer sets specific to Polα and Arx (Figure 2.3). As expected if the
procedure was effective, amplification products were not present using 218P4-selected
cDNAs as template but were present using 123D15-selected cDNAs as template. This
result demonstrated the successful isolation of expressed sequences specific to the
31 genomic clone driving the selection. The cDNA pool selected by BAC 218P4 was cloned to facilitate characterization of individual selected cDNAs.
2.3.2 Analysis of selected cDNAs
Initially, cDNA clones were characterized in terms of insert size and primary sequence. To assess the insert size range of 218P4-selected cDNAs, inserts were amplified from liquid cultures (Figure 2.4) using primers specific to the oligonucleotide linkers flanking the cloned cDNAs. Insert sizes ranged between 150 bp and 800 bp, with the majority of cDNAs residing between 200 and 600 bp.
All 60 clones were sequenced from one end using a T7 primer and sequences
were Blasted (76) against an assembled sequence of λ clones CAA and BAA (Figure
2.5A). Sequence alignment of several cDNAs to the phage clone assembly identified
three putative exons near the transgene insertion site. cDNA clone 5 aligned with 100%
identity to a proximal portion of phage clone CAA. Several cDNA clones (clones 17, 31
and 22) aligned with 100% identity to phage clone BAA. Interestingly, the sequence of
clones 17 and 31 split into two different regions on the BAA sequence. The presence of
splice donor and acceptor consensus sequences flanking the regions of alignment
provided strong evidence that these regions were indeed exons on the BAA side (Figure
2.5B). cDNA clone sequences were blasted against the dbEST database. A human EST,
T47371, showed significant sequence homology with cDNA 17 (Figure 2.5C). Nucleic
and amino acid conservations of 88% and 96%, respectively, between selected cDNA 17
and the human EST indicated that the mouse gene residing near the OVE459 transgene
insertion site has a human homologue.
32 An additional 384 selected cDNA clones were sequenced to determine whether additional exons resided on phage clones BAA and CAA. One clone, cDNA 340, split into 3 regions and identified two additional exons on the CAA side. At this point, we designated three clusters of exons, represented by selected cDNAs, as candidate genes
(Figure 2.6). All three candidate genes were novel at this time. Candidate 1, represented by cDNAs 11, 12 and 47 (among others), resided on the BAA side of the transgene insertion site beyond the end of the BAA insert. Assignment of the positions of Candidate
1 exons relative to the transgene insertion site was based on the association of these exons with a BAC clone specifically residing on the BAA side, BAC RPCI-9N1.
Candidate 2, represented by cDNAs 17, 22, 31 and 3 (among others), resided on the BAA insert itself. The third candidate gene was represented by cDNA 340 and resided on the
CAA side of the OVE459 transgene. For reasons to be addressed later, the gene represented by the cDNA 340-specific exons will be referred to as Candidate 3a. The exons of all three candidate genes were confirmed by RT-PCR using P16 brain RNA as starting material (Figures 2.7, 2.8 and 2.9). For each candidate gene, RT-PCR products of the appropriate size and abundance were amplified from homozygous OVE459 brain
RNA. In addition, homozygous hy3-derived RT-PCR products were indistinguishable from wild-type products (data not shown).
2.3.3 Characterization of Candidate 1 (Vac14)
Previously, a probe consisting of the entire BAC 218P4 insert was used to screen a high density array of mouse IMAGE cDNA clones (Genome Systems, (77)) to identify expressed sequences on this BAC clone. A single clone, 704m09 (IMAGE ID 312752),
33 exhibited a strong hybridization signal after high stringency washing of the filters. After
the cDNA selection procedure was completed, the entire insert of this clone was
sequenced (GenBank accession no. AY220476). Several selected cDNAs representing
Candidate 1 aligned with the 704m09 insert sequence. This clone was later removed from
the mouse IMAGE clone set because it was discovered to be of rat origin. However,
clone 704m09 appeared to contain the full-length rat homologue of Candidate 1.
Candidate 1 is highly conserved, as we identified homologues from fission and
budding yeast to human. In Saccharomyces cerevisiae, this gene was designated VAC14,
as mutations in this gene cause defects in vacuolar morphology, inheritance and size
regulation in response to osmotic stress (78). Due to the homology between yeast VAC14
and Candidate 1, Candidate 1 was subsequently designated Vac14 and VAC14 in the
mouse and human genomes, respectively. Vac14 consists of 19 exons covering 102 kb in
the mouse genome and expresses a ubiquitous 3.1 kb transcript. While Vac14 begins 12.7
kb distal to the transgene insertion site, we have determined by northern analysis that
Vac14 expression is unaltered in both OVE459 and hy3 homozygous mice (data presented in Chapter 4).
2.3.4 Characterization of Candidate 2 (Hydin)
From the sequence of phage clones CAA and BAA, in conjunction with extensive
sequencing of selected cDNAs, we identified a cluster of exons near the transgene
insertion site designated Candidate 2. This exon cluster did not appear to constitute a
complete gene, as consensus splice acceptor and donor sites flanked each identified exon.
In addition, transcription start sites and polyadenylation signals were noticeably absent.
34 The utility of the selected cDNA sequences to further characterize Candidate 2 had been exhausted in the absence of additional genomic sequence flanking the BAA and CAA inserts. At this time, an effort to sequence mouse BAC clones was initiated by the Trans-
NIH Mouse Sequencing Consortium. To identify a larger BAC clone encompassing the transgenic locus, we screened the RPCI-23 C57BL/6 mouse BAC library using BAA- and CAA-specific probes. BAC clone RP23-21B7 (BAC 21B7) was identified as harboring sequences from both phage clones. Pulsed field gel electrophoresis of NotI- digested BAC 21B7 revealed an insert size of approximately 240 kb (Figure 2.10), roughly twice the size of the BAC 218P4 insert. The large insert of BAC 21B7 and its potential relevance to hydrocephalus made this clone an excellent candidate for sequencing by the consortium. Our petition to have this clone sequenced was granted.
During the following year, a dramatic influx of sequence information greatly facilitated the characterization of Candidate 2 (Figure 2.11). As the sequence of BAC
21B7 was incrementally released (Genbank accession no. AC069308), we identified additional regions of alignment between the BAC sequence and selected cDNAs not represented on either phage clone. Furthermore, web-based gene and splice site prediction algorithms assisted in exon identification. Coincident with the sequencing of
BAC 21B7, efforts to sequence the human and mouse genomes in the public and private
(Celera) sectors were nearing completion (assembly of the mouse genome into scaffolds of meaningful size came a bit later). Furthermore, the EST databases were significantly expanded during this period. All of these sources of information were implemented to
35 predict the structure of Candidate 2. During this process, predicted exons were either confirmed or dismissed by RT-PCR.
Using the sequence information described in the previous paragraph, several milestones were reached in the process of characterizing the structure of Candidate 2.
First, we determined by human chromosome 16/BAC 21B7 sequence homology and RT-
PCR that Candidates 2 and 3a (Figure 2.6) represented a single gene, subsequently designated Candidate 2. This discovery indicated that Candidate 2 encompasses the
OVE459 transgene insertion site. In addition, we realized that Candidate 2 is extraordinarily large, consisting of at least 63 exons covering more than 131 kb in the mouse genome. At this time, we predicted the Candidate 2 transcript to be approximately
12 kb.
A second milestone was the discovery of a partial gene at the proximal end of
BAC 21B7. This gene, which we will refer to as Candidate 3b, was identified by sequence homology between human chromosome 16 and BAC 21B7. In addition, a human 3652 bp cDNA clone was described for this gene (GenBank accession no.
AK022933). In the human genome, AK022933 splits into 20 exons spanning more than
204 kb. Exons 12 thru 20 are present on BAC 21B7. Exon 20 of AK022933 includes a 3’ untranslated region and polyadenylation signal. The polyadenylation signal was not detected in the mouse genome at the corresponding location. Furthermore, a termination codon present in human genomic DNA is not conserved in mice, and a perfect consensus splice donor resides 10 bp upstream of this codon. Human ESTs facilitated the identification of four additional exons downstream of exon 20, with the fourth exon
36 appearing to represent the final exon in the human genome. However, RT-PCR using antisense primers specific to exon 24 consistently failed to amplify a product from mouse brain cDNA. The status of exon 24 in the mouse genome remains to be established. It remained a possibility, however, that Candidate 3b was an upstream portion of Candidate
2, as the direction of transcription (centromeric to telomeric on mouse chromosome 8) was the same for both genes.
Northern analysis of Candidates 2 and 3b marked a third milestone in the characterization of Candidate 2 (Figure 2.12). Previous attempts to detect Candidate 2 and 3b transcripts by northern using brain mRNA were unsuccessful. Several ESTs for
Candidate 2 were derived from testis, and RT-PCR amplification of Candidate 2-specific exons from testis consistently yielded robust amplification products. Our attempt to use testis RNA for northern analysis was successful. This analysis provided two important pieces of information. First, it confirmed our prediction that the Candidate 2 transcript was extremely large. Two high molecular weight transcripts (much larger than the 9 kb
RNA marker) were detected using a cocktail of two probes specific to a 3 prime portion of the transcript. Second, this analysis provided strong evidence that Candidate 3b was actually the 5 prime portion of Candidate 2. A Candidate 3b-specific probe detected a transcript of identical size to the higher molecular weight transcript of Candidate 2.
Candidate 3b exon 23 was subsequently joined to exon 2 of Candidate 2 by RT-PCR, confirming that this was a single gene. We designated this gene Candidate 2.
Prior to the northern results, we believed that the entire Candidate 2 gene was present on BAC 21B7. The discovery that Candidates 3b and 2 were the same gene
37 indicated that BAC 21B7 did not include the 5’ end of the current version of Candidate 2.
Fortunately, the mouse and human genome assemblies were complete at this time.
Alignment of the mouse genome upstream of BAC 21B7 to the corresponding location in the human genome allowed us to identify the remaining 11 Candidate 2 exons. In addition, clone AK022933 assisted in characterizing the 5’ end of Candidate 2. Our final version of Candidate 2 consists of 87 exons covering 340 kb in the mouse genome
(Figure 2.13). The status of exon 2 remains inconclusive and this exon is not included in the 86 exon Candidate 2 mRNA sequence submitted to GenBank. The evidence for exon
2 is its presence in a mouse lung EST (GenBank accession no. BB664150), but we have been unable to confirm its inclusion experimentally.
To obtain primary sequence data for the full-length Candidate 2 transcript, the entire transcript was sequenced (Figure 2.14). First, overlapping brain-derived RT-PCR products spanning the entire transcript were generated. Next, each RT-PCR product was sequenced from both ends in individual reactions. The chromatograms for each sequencing reaction were assembled using the SeqMan program (Lasergene) to generate a full-length consensus sequence for Candidate 2. The transcription start site of Candidate
2 was ascertained by 5’ RACE, completing the characterization of the 15,783 bp mouse
Candidate 2 mRNA (GenBank accession no. AY173049). This transcript encodes a putative 5099 amino acid protein. Despite the presence of several upstream AUG codons, the length of the putative protein is based on choosing an AUG codon in exon 3 that is both consistent with translation initiation (79) and conserved in the human Candidate 2 homologue on chromosome 16.
38 Currently, 97% (15.25 kb out of 15.73 kb) of the predicted human Candidate 2
transcript has been sequenced using the same strategy employed in the mouse. Only the
extreme 5’ and 3’ ends of the human transcript are absent from the 15.25 kb contiguous primary mRNA sequence. Two attempts to clone the human 5’ and 3’ ends by RACE have been unsuccessful. Northern analysis on total human testis RNA was performed using a human probe analogous to that used for the 5’ end of mouse Candidate 2 (Figure
2.15). In mouse, this probe detects a single high molecular weight transcript consistent with the predicted full-length mRNA. Interestingly, the analogous human probe detects a transcript that is larger than 9 kb, but substantially smaller than the detected mouse transcript. It is possible that the observed size difference reflects splicing events in the human transcript that are absent in the mouse.
The transgenic locus was reconstructed using the phage clones BAA and CAA
and compared to the wild-type organization of Candidate 2 (Figure 2.16). This
comparison revealed that the order of Candidate 2 exons is disrupted in line OVE459.
Exons 80 thru 85 lie immediately upstream of the transgene insertion site, while exons 46
thru 53 reside immediately downstream of the transgene. Interestingly, we were able to
amplify each overlapping Candidate 2 RT-PCR product from homozygous OVE459 brain
RNA (data not shown), indicating that a complex chromosomal rearrangement (possibly
involving a duplication) resulted from transgene integration. Nonetheless, the disruption
of Candidate 2 by the transgene makes Candidate 2 the primary suspect for
hydrocephalus in line OVE459. For this and a number of other reasons detailed in the
following chapters, we have named this gene Hydin (for Hydrocephalus-inducing). A
39 complete map of the mouse and human OVE459 candidate regions, including Hydin and
Vac14, is presented in Figure 2.17.
2.4 Discussion
In this chapter, we demonstrate the successful use of cDNA selection to isolate
expressed sequences near the OVE459 transgene insertion site. Identification of exons
present on phage clones BAA and CAA by cDNA selection marked the beginning of an
arduous journey toward characterizing the remarkably large, novel candidate gene,
Hydin. In the chapters to follow, we show that our efforts to identify and characterize the
structure of this gene were not in vain. In addition to discovering Hydin, this work is also
responsible for the identification of mouse and human Vac14. The high amino acid
conservation of Vac14 in all sequenced eukaryotes implies an ancient, essential function
for this gene.
Reconstructing the transgenic locus using the phage clone sequences revealed a
transgene-induced chromosomal rearrangement within Hydin. Assembly of the phage
clones suggests that a complete, uninterrupted copy of Hydin does not exist in the mutant
genome. It is difficult to reconcile the appropriate amplification of every portion of the
Hydin transcript in OVE459 homozygotes by RT-PCR. Multiple attempts to demonstrate
a splicing event from exon 85 to exons on the opposite side of the transgene array have
failed. The possibility of such a splicing event cannot be excluded. If the transgene is
included in the nascent transcript and subsequently spliced out, we would predict the
protein encoded by the mature transcript to be non-functional. Despite the apparent complexity of the rearrangement in line OVE459, the value of the transgene as a
40 molecular tag for the region most likely responsible for hydrocephalus in these mice cannot be understated.
Like Vac14, Hydin exhibits considerable evolutionary conservation as we have identified complete (or portions of) Hydin homologues from Chlamydomonas reinhardtii and Ciona intestinalis to humans. Despite the remarkable size of the putative 5099 amino acid mouse Hydin protein, few conserved functional domains have been credibly identified. A stretch of amino acids encoded by exon 7 exhibits weak homology to the
Major Sperm Protein domain. In nematodes, major sperm proteins (MSPs) functionally replace actin during pseudopod extension and facilitate the amoeboid-like movement of nematode sperm (80). A predicted transmembrane domain resides between amino acids
675–697. Amino acids 2258–2572 share significant similarity with caldesmon (e value of
4e-12 (76)) compared with human caldesmon (GenBank accession no. Q05682) amino acids 258–566. Caldesmon is a widely expressed actin-binding protein thought to be important in cytoskeletal assembly and stabilization as well as regulation of smooth muscle contraction (81). A cytoskeleton-related function for Hydin is conceivable, as its large size appears amenable to serve a structural role in the cell. Additional clues to the function of Hydin come from its expression pattern and will be discussed in the following chapter.
41 Figure 2.1. Schematic illustration of the direct cDNA selection procedure. (Top) BAC 218P4 was prepared for hybridization by digestion with frequent cutting restriction enzymes and ligation to biotinylated (yellow circles) oligonucleotide linkers. (Bottom) Pooled cDNAs derived from E17.5, P0 and P2 head mRNA were digested with frequent cutting restriction enzymes and ligated to oligonucleotide linkers. (Middle) Selection of cDNA sequences represented on the BAC was carried out by hybridization. Following purification of BAC- cDNA hybrids using streptavidin-conjugated magnetic beads, cDNAs were eluted, amplified by linker-mediated PCR and subjected to a second round of selection.
42 Figure 2.2. cDNAs after successive rounds of selection. (Left) Electrophoresis of cDNAs (*) after one round of selection using BAC 218P4 or BAC 123D15 indicates a predominant size range between 200 and 500 bp. (Right) Electrophoresis of cDNAs (*) indicating a decrease in cDNA abundance following a second round of selection.
Figure 2.3. Specific selection of Polα- and Arx-specific cDNAs using BAC 123D15 as bait. PCR amplification of Polα- and Arx-specific cDNAs from BAC 123D15- selected cDNAs (yellow asterisks), but not BAC 218P4-selected cDNAs (white asterisks) indicates the success of this approach in isolating cDNAs specific to the genomic clone driving the selection.
43 Figure 2.4. PCR amplification of 54 BAC 218P4-selected cDNA clone inserts. Primers specific to the cDNA linkers were used to amplify the inserts of 54 (out of the initial 60) individual cDNA clones selected by BAC 218P4. Each of the three panels contains 18 amplified selected cDNA inserts flanked by 100 bp DNA ladder. The size range of amplified inserts is between 150 bp and 800 bp, with the majority of cDNAs residing between 200 and 600 bp.
44 Figure 2.5. Alignment of several 218P4-selected cDNAs to λ clones BAA and CAA reveals the presence of exons near the OVE459 transgene insertion site. (A) A map of assembled phage clones BAA and CAA with the transgene array indicated (red box). The sequence of selected cDNA clone 5 (orange) is represented on the proximal portion of phage clone CAA. On phage clone BAA, selected cDNA clones 17, 31 and 22 are shown. The sequence of clones 17 and 31 split into two locations on phage clone BAA, representing two putative exons (green vertical bars) near the transgene insertion site. (B) Consensus splice donor and acceptor sites flank the putative exons on the BAA side. (C) Significant sequence homology between cDNA 17 and human EST T47371 (both boxed in blue with asterisk in Figure 2.5A) indicates that the gene near the OVE459 transgene insertion site has a human homologue.
45 Figure 2.6. Initial designation of three candidate genes near the OVE459 insertion site. Three candidate genes near the transgene insertion site were designated following analysis of additional selected cDNA clones. Candidate genes 1 and 2 (represented in red and green, respectively), are situated on the BAA side of the insertion site. Candidate 3a, represented in orange, resides on the CAA side of the insertion site. Exons for each candidate gene are indicated by horizontal boxes. Representative selected cDNA clones for each candidate are indicated below the exons.
Figure 2.7. Verification of Candidate 1 by RT-PCR. Primers specific to exons represented by cDNAs 11 and 12 (red boxes) were used to amplify a 600 bp product from P16 brain-derived cDNA. Amplification products were present using both OVE459 heterozygous and homozygous cDNA (white asterisks).
46 Figure 2.8. Verification of Candidate 2 by RT-PCR. Three exons represented by cDNAs 3 and 17 (green boxes) were amplified from P16 brain-derived cDNA. A 190 bp product specific to the two exons represented by cDNA 17 was observed using both OVE459 heterozygous and homozygous cDNA (white asterisks). A 280 bp product joining the cDNA 3-specific exon and the distal-most cDNA 17-specific exon was also observed using both OVE459 heterozygous and homozygous cDNA (yellow asterisks).
Figure 2.9. Verification of Candidate 3a by RT-PCR. Three exons represented by cDNA 340 (orange boxes) were amplified from P16 brain-derived cDNA. Using primers specific to the outer exons, a 310 bp product was amplified from both OVE459 heterozygous and homozygous cDNA (white asterisks).
47 Figure 2.10. Pulsed field gel electrophoresis of BAC RPCI-21B7. The BAC 21B7 insert was separated from the BAC vector by digestion with NotI and subjected to pulsed field gel electrophoresis. The BAC insert ran just below the 242 kb marker, indicating an insert size of approximately 240 kb.
Figure 2.11. Sources of information used to characterize the genomic structure of Candidate 2. The sequence of BAC 21B7 was analyzed using a variety of sequence information. Among these were selected cDNAs not present on the phage clones, gene and splice site prediction, sequence homology to human chromosome 16 and ESTs from mouse and human. Predicted exons were confirmed or dismissed by RT-PCR.
48 Figure 2.12. Northern analysis for Candidates 3b and 2 revealed that they are part of a single gene. Adult testis mRNA was hybridized to probes specific to Candidate 3b (blue) or Candidate 2 (green). The blot on the right, hybridized to the Candidate 2 probe, exhibits two high molecular weight transcripts running well above the 9 kb RNA marker. The blot on the left, hybridized to the Candidate 3b probe, exhibits a single transcript of equal size to the higher molecular weight Candidate 2 transcript. These results strongly suggested that Candidates 2 and 3b represent a single gene, which was subsequently confirmed by RT-PCR (data not shown).
49 Figure 2.13. The genomic organization of Candidate 2. The final version of Candidate 2 consists of 87 exons covering more than 343 kb in the mouse genome. Not all of the exons, indicated by vertical lines, can be resolved at this scale. The location of the putative translation initiation codon (ATG) in exon 3 is indicated.
Figure 2.14. Strategy for sequencing the mouse and human Candidate 2 transcripts. Overlapping RT-PCR products were amplified from P0 mouse brain RNA or adult human testis RNA. Each product was sequenced from both ends. The resulting trace files were assembled to generate a consensus sequence for each transcript.
50 Figure 2.15. Human Candidate 2 northern analysis. Human and mouse testis RNA were electrophoresed in adjacent lanes and hybridized to probes specific to the analogous exons of each transcript. A high molecular weight (> 9kb) human transcript is detected. This transcript is substantially smaller than the corresponding mouse transcript.
Figure 2.16. Genomic structure of wild-type Hydin (top) and the OVE459 transgene insertion locus (bottom). (Top) The 87 exons of Hydin are indicated by vertical lines. (Bottom) The phage clones BAA and CAA, which flank the multi-copy transgene (TG) insertion site, are separated by 51 kb in the wild-type genome. The transgene array in line OVE459 is situated between exons 85 (proximal) and 46 (distal), indicating a genomic rearrangement at the transgene insertion site.
51 Figure 2.17. The OVE459/hy3 candidate region on mouse chromosome 8 (A) and the corresponding region on human chromosome 16 (B). (A) Four genes located at 8D3 are situated near the OVE459 transgene insertion site. Genes,arranged on the chromosome from centromere (cen) to telomere (tel), are represented by boxes with transcriptional direction indicated by arrows. BAC clones described in the text are represented as solid horizontal bars beneath the chromosome, and solid bars above the chromosome represent the locations of the genomic DNA present on phage clones CAA and BAA that flank the transgene insertion site in OVE459 mice. Upward pointing arrows indicate the positions of the polymorphic genetic markers D8Mlr1 and D8Mit151. Calretinin (CalB2) and a gene defined by its putative protein (accession BC025546) lie upstream of Hydin (Candidate 2) and Vac14 (Candidate 1) lies downstream of Hydin. (B) The hy3/OVE459 candidate region corresponds to human chromosome 16q22. The order and orientation of candidate genes through this region are conserved in the human genome. BAC clones are represented as solid horizontal bars beneath the chromosome.
52 CHAPTER 3
HYDIN EXPRESSION PATTERN ANALYSIS BY IN SITU HYBRIDIZATION
3.1 Introduction
Hydin surfaced as the primary candidate gene for hydrocephalus in OVE459 mice
due to the transgene-induced rearrangement. It was also established that Hydin is
expressed in the brain at a time coincident with the onset of hydrocephalus, as the Hydin-
specific cDNAs identified by direct cDNA selection were derived from brain mRNA present at E17.5, P0 and P2. Furthermore, PCR amplification of P0 brain-specific, reverse transcribed mRNA confirmed that Hydin expression in the brain precedes the appearance of hydrocephalus by several days.
Precisely determining where Hydin expression occurs in the neonatal brain is an important step toward validating Hydin as the relevant hydrocephalus gene. Hydin expression in structures responsible for CSF homeostasis would further implicate this gene as the primary candidate and contribute to our understanding of the mechanism 53 underlying hydrocephalus in Hydin homozygous mutants. In addition, determining the expression pattern of Hydin in cellular compartments outside the CNS would provide important information regarding the biological function of this gene. A number of approaches may be employed to determine the expression pattern of a particular gene of interest. In some instances, it is sufficient to perform RT-PCR or northern analysis using
RNA harvested at a various time-points from a range of tissues. Furthermore, if an antibody specific to the gene product is available, western blotting or immunohistochemistry may be employed. In the specific case of Hydin, an antibody was not available, and it was necessary to determine in which specific compartments of the brain and other tissues Hydin is expressed. The best approach for our needs was in situ hybridization, where a Hydin-specific antisense RNA probe was used to precisely detect the locations of active Hydin transcription within a particular tissue.
3.2 Materials and Methods
3.2.1 Tissue Preparation and Sectioning
Anesthetized mice were perfused with 4% PFA. Briefly, the right atrium was torn, followed by sequential perfusion with 2 ml room temperature 1X PBS and 2 ml ice-cold
4% paraformaldehyde using a 26 gauge needle inserted into the left ventricle. Tissues were extracted and fixed in 4% paraformaldehyde at 4O C overnight on a nutator. On the following day, the tissue was transferred to 70% EtOH in DEPC-treated ddH2O. Tissue samples were then processed overnight for paraffin embedding in the CCRI histology core facility. Paraffin-embedded tissue was sectioned at a thickness of 5 µm using a Leica microtome.
54 3.2.2 In Situ Hybridization
In situ hybridization was carried out as described (82). Tissue sections were
subjected to hybridization using a cocktail of two 35S-labeled antisense or sense RNA probes. The probes were derived from RT-PCR products amplified using the primer sets
C2-12a/12b (385 bp) and 3 prime-1a/1b (428 bp), representing exons 71 to 73 and 75 to
78, respectively. Primer sequences are as follows: C2-12a: 5’-
GAGAACAAGGTCCTATTTTG-3’, C2-12b: 5’-AGTCCAGAACTCTTCCGTG -3’, 3 prime-1a: 5’-TCTACGAGGTGGAGTTGAAT-3’, 3 prime-1b: 5’-
GTGTGGGGTGTTGGTGTA-3’. Following amplification, RT-PCR products were TA- cloned into pCRII-TOPO (Invitrogen) and sequenced to determine the orientation of the inserts. For in vitro transcription of antisense RNA, the T7 or SP6 polymerases were used for plasmids C2-12a/12b and 3 prime-1a/1b, respectively. For in vitro transcription of sense RNA, the SP6 or T7 polymerases were used for plasmids C2-12a/12b and 3 prime-
1a/1b, respectively. Prior to in vitro transcription, the plasmids were digested with SacI or
NotI for transcription with the T7 and SP6 polymerases, respectively.
3.3 Results
3.3.1 Hydin expression in the central nervous system
During the process of identifying Hydin as a candidate hydrocephalus-inducing
gene, it was determined that Hydin is expressed in the brain. To identify the specific
regions of the brain that express Hydin, in situ hybridization was performed on brain
sections at various stages of development. Currently, we have examined Hydin
expression in the brain at several time-points from E12.5 thru adulthood.
55 The earliest embryonic stage at which Hydin expression has been analyzed is
E12.5, where Hydin is expressed exclusively in cells that contribute to the ependymal lineage (Figure 3.1). In situ hybridization using and antisense probe on horizontal sections of an E12.5 mouse brain revealed Hydin expression in cells comprising the first
evidence of the choroid plexus (Figure 3.1A-B). The specificity of this result was verified
by the lack of signal using a sense Hydin probe on an adjacent tissue section (Figure
3.1C-D). In addition to the choroid plexus expression, Hydin transcripts were also
detected in the cell layer adjacent to the choroid plexus and at the roof of the third
ventricle. As the choroid plexus is the major source of CSF production in the brain, this
result established Hydin as an excellent candidate gene for association with
hydrocephalus. It is not precisely clear at which stage the ependymal lineage is
established, but a true ependymal layer is not evident at E14 (83). The expression of
Hydin observed at E12.5 is likely in the germinal matrix from which ependymal cells
differentiate at a later time-point (84).
The next stage analyzed was E15.5, where Hydin expression in the brain is exclusive to the choroid plexus (Figure 3.2A-B). Expression in the ependymal cells lining the ventricles at E15.5 was not detected. Hydin transcripts are also present in portions of the spinal cord, presumably the central canal (Figure 3.2C-D), indicating that expression in the CNS at this stage is not restricted to the brain.
On the day of birth, Hydin expression appears to diminish in the choroid plexus but is strikingly specific to the ependymal cells lining the lateral, third and fourth ventricles (Figure 3.3A-F). In addition, Hydin is expressed intermittently in the
56 ependymal layer lining the cerebral aqueduct (Figure 3.4), through which CSF flows from the third to the fourth ventricle. Strong expression was also observed in the fourth
ventricle choroid plexus. Ependymal cells also line the central canal, a space within the
spinal cord through which CSF circulates. The ependymal cells of the central canal
express Hydin at P0 (Figure 3.5).
In the adult brain, Hydin expression persists in the ependymal cells lining the
brain ventricles (Figure 3.6). In addition, Hydin expression resumes in the choroid plexus.
Hydin is also expressed on all surfaces of the adult cerebral aqueduct (Figure 3.7).
3.3.2 Hydin expression in the reproductive system
Two Hydin-specific ESTs, AK006604 and AK016044, are derived from testis
mRNA. We therefore examined Hydin expression in the testis by in situ hybridization. In
the adult testis, high-level expression of Hydin was observed within the seminiferous
tubules (Figure 3.8). In bright-field images at higher magnification, careful examination
of the silver grains corresponding to Hydin transcripts revealed that Hydin mRNA is
localized to the region of spermatocyte development within the tubule (Figure 3.9). The
silver grains are generally absent at the location of the spermatogonia at the basal lamina
and spermatids at the lumen of the tubule, but present in between these regions. As
cytoplasmic projections of the sertoli cells are also present in this region, it cannot be
ruled out that that the observed signal is derived from this cell type. Hydin expression in
the testis is initiated early in development, as Hydin mRNA is detected at E15.5 (Figure
3.10A-B). Hydin transcripts are sparse but present in the P16 testis (Figure 3.10C-D),
where expression correlates with tubules having a more mature appearance. We have not
57 yet determined when Hydin expression initiates in the testis. Lastly, Hydin is expressed in
the ciliated epithelial cells lining the adult oviduct (Figure 3.11).
3.3.3 Hydin expression in the respiratory system
Two human Hydin ESTs, AK074472 and AK02688, are derived from lung mRNA. We have subsequently determined that Hydin is expressed at various levels of the
respiratory system (Figure 3.12). Strong expression of Hydin is observed in the nasal
respiratory epithelium at E15.5 (data not shown) and P0 (Figure 3.12A-D). In addition,
Hydin transcripts are detected in epithelial cells lining the trachea at P16 (Figure 3.12E-
F). Finally, Hydin transcripts are present in cells lining the bronchi in the adult lung
(Figure 3.12G-H). Currently, we have not performed in situ hybridization on each of these three tissues at various stages of development to determine the onset of Hydin expression in the respiratory epithelia.
3.3.4 Hydin expression in the inner ear
In addition to the brain, reproductive organs and respiratory system, we have
observed Hydin expression in the vestibular system of the inner ear. Hydin is expressed in
the sensory epithelia of the saccule and semicircular canal at P0 (Figure 3.13A-D). At
40X magnification, kinocilia are readily apparent on the cupula of the semicircular canal
(Figure 3.13E), a sensory structure where Hydin is specifically expressed.
3.4 Discussion
This preliminary expression pattern analysis has provided two very important
pieces of information regarding Hydin as a candidate gene for the phenotype observed in
OVE459 and hy3 homozygotes. First, Hydin expression in the brain is exclusive to
58 cellular compartments directly involved in CSF production and homeostasis. The choroid plexus produces the majority of CSF in the brain, while the ependymal layer lining the ventricles serves as a selective barrier between the brain parenchyma and the CSF-filled ventricular lumen. Second, an emerging theme from this study is that Hydin is exclusively expressed in cells possessing cilia or flagella. This study lends support to the hypothesis that Hydin is the relevant hydrocephalus-inducing gene. Furthermore, the expression pattern of Hydin suggests a role for this gene in the function and/or maintenance of cilia and flagella. Notably, Hydin expression was not observed in the
meninges (data not shown), a site previously suspected to play a role in the pathogenesis
of hydrocephalus in hy3 homozygotes (63).
Previous investigations by others have firmly established an association between
cilia dysfunction and hydrocephalus. In mice, cilia dysfunction and hydrocephalus are
features of null mutations in Spag6 (37), Hfh4 (38), Polaris (39), Mdnah5 (40) and Pol λ
(41). Furthermore, ciliary dyskinesia is associated with hydrocephalus in rats (44), dogs
(45) and humans (46) (50) (51) (47) (48) (49). These reports support the notion that cilia function in the brain is required for proper CSF homeostasis.
3.4.1 The role of ependymal cilia in hydrocephalus
The ependymal cells lining the ventricular surfaces are densely coated with 9+2 motile cilia (85). The functional significance of these cilia is poorly understood. The beating of ependymal cilia is sufficient to create local currents close to the ventricular surface toward the predicted direction of bulk CSF flow (55). Currents at the ventricular surface may assist in the clearance of metabolites and toxins from the brain, as they
59 would create a diffusion gradient between the brain parenchyma and CSF. However, it is
improbable that the beating of ependymal cilia alone significantly contributes to the bulk flow of CSF through the ventricular system. Rather, pulses of CSF emanating from the choroid plexus vasculature during systole appear to serve as the primary driving force behind bulk CSF flow (56). Currently, it is not understood how cilia motility contributes to CSF homeostasis, if it does at all.
It is also possible that ependymal cilia regulate CSF homeostasis by a sensory-
based, rather than motility-based, mechanism. Primary renal cilia act as mechanosensors,
bending in response to shear forces that ultimately results in Ca2+ influx (86). Shear forces created by CSF currents could conceivably illicit a similar response in ependymal cilia. In some tissues, the cilium itself is the site at which signal transduction is initiated
(87, 88). These and other reports clearly show that the utility of cilia for certain cell-types goes well beyond motility. However, a study by Nakamura and Sato provides evidence that specifically disrupting the motility of ependymal cilia (by ventricular infusion of metavanadate, a potent inhibitor of cilia movement) is sufficient to cause hydrocephalus in rats (89). A role for ependymal cilia as sensors of CSF flow or composition remains to be established.
3.4.2 Hydin expression in the choroid plexus
The choroid plexus expression of Hydin in both the embryo and adult raises several questions. In the embryo, mutations in Hydin may perturb the development of the
choroid plexus, resulting in dysregulated CSF production that manifests in perinatal
hydrocephalus. Hydin expression in the choroid plexus from its conception through
60 adulthood suggests that Hydin provides an essential function in this tissue that must be
maintained. Alternatively, despite its expression in the choroid plexus, Hydin may not be
functionally relevant in this compartment. In the brain and beyond, exclusive expression
of Hydin in ciliated epithelia suggests a functional role specific to cilia or ciliated cells.
Cilia are only sparsely present on the specialized ependymal cells of the choroid plexus
(90), especially compared to the high density of cilia (>40 per cell) present on the ependymal cells comprising the ventricular surfaces (85). If the hypothesis concerning
Hydin and cilia function is correct, the site of pathogenesis in OVE459/hy3 mice is more
likely to be the non-choroidal ependyma lining the ventricular lumen.
3.4.3 Hydin expression beyond the central nervous system
On its own, Hydin expression in the ciliated ependymal cells is not sufficient to
conclude a cilia-specific functional role for Hydin. The anatomical locations of Hydin
expression outside of the central nervous system, however, provide compelling evidence
for a role in the function or maintenance of cilia and flagella. Interestingly, the cell-types
that express Hydin possess cilia/flagella with a 9+2 ultrastructure. Hydin expression in
organs possessing primary 9+0 cilia has not been observed. We have performed in situ
hybridization in the kidney, which possesses a large population of primary 9+0 cilia, at
various developmental stages and have failed to detect even low level Hydin expression.
However, it is premature to conclude that Hydin expression is absent in all cells possessing primary cilia. Primary cilia are a prominent feature of the embryonic node, which has not yet been analyzed for Hydin expression by in situ hybridization. Situs abnormalities have not been observed in OVE459 or hy3 homozygous mutants.
61 Expression of Hydin in the adult testis supports the proposed connection between
Hydin and 9+2 axoneme-containing structures. Hydin expression in developing spermatocytes is likely due to the pending requirement for Hydin protein once sperm tail assembly is initiated during spermiogenesis. The expression of genes throughout spermatocyte development that encode flagellar-specific proteins is commonly observed
(91).
It is intriguing that Hydin is expressed in the E15.5 and P16 testis. Primordial germ cells populate the developing gonad by day E12.5 (92), but only undifferentiated type A1 spermatogonia are present in the testis on the day of birth. Spermatocytes are not present in the testis until post-natal day 8, and the earliest post-meiotic spermatids are not present until 20 days after birth. During the next 13 days, spermiogenesis occurs where spermatid elongation, nuclear condensation and sperm tail formation occurs. Given this timeline for testis development, it is unclear why Hydin is expressed at E15.5. In addition to a cilia/flagella-related function, Hydin may serve an important function in certain stem cell populations. Preliminary support for this notion is the finding that HYDIN is expressed in cultured human neural stem cells (personal communication with Richard C.
Krueger, M.D.). Hydin expression in the P16 testis is sparse, but correlates with seminiferous tubules that are more mature in appearance (see Figure 3.10C-D).
We have also shown that Hydin is expressed in epithelia at multiple levels of the respiratory system. Motile 9+2 cilia are present on each of the three respiratory epithelia
(nasal, trachea and lung) analyzed thus far. Notably, affected mice in the original mixed background hy3 colony exhibited abnormal nasal discharge (62). This phenotype was lost
62 in subsequent generations (63), most likely due to a change in genetic background.
However, it is interesting that the hy3 mutation was associated with abnormal nasal discharge, as this is often the result of defective mucociliary clearance.
Expression in the vestibular epithelia of the inner ear suggests a functional role for Hydin in the maintenance of balance and acquisition of kinetic information. The vestibular epithelia of the inner ear possess 9+2 kinocilia. However, recent studies concerning the ultrastructure of vestibular kinocilia suggest that the central pair of microtubules is not continuous (93). Extracellular filaments connect a single vestibular kinocilium to an adjacent stereocilia hair cell bundle (94), which transduces kinetic information to the brain. We observed Hydin expression in the cupula of the semicircular canal, a structure responsible for sensing angular velocity (i.e. head tilting). In addition,
Hydin is expressed in the saccule, which detects linear acceleration (i.e. gravity). It is notable that late-stage hy3 and OVE459 homozygotes display a marked impairment in balance (unpublished observation). A typical mutant older than two weeks of age has difficulty walking a single length of the cage and frequently falls on its side while continuing to try to walk. Prior to observing Hydin expression in multiple vestibular organs, we presumed that this balance disruption was a secondary effect of severe ventricular dilatation in these animals. However, the expression of Hydin specifically in the sensory epithelia of the semicircular canals and saccule suggests the possibility that balance impairment in homozygous animals is a primary defect resulting from Hydin loss
of function in these compartments.
63 Figure 3.1. Hydin expression in the E12.5 brain. (A-B) In a horizontal brain section at E12.5, Hydin transcripts are detected in the first evidence of the choroid plexus (CP) and neighboring ependymal cells lining the lateral ventricles (LV) and roof of the 3rd ventricle (*). (C-D) A serial section was subjected to hybridization using a sense Hydin RNA probe as a negative control. No transcripts were detected, validating the signal observed in panel B.
64 Figure 3.2. Hydin expression in the E15.5 brain and spinal cord. (A-B) Saggital sections of the E15.5 brain reveal Hydin transcripts exclusively in the choroid plexus (CP). (C-D) Saggital sections through the spinal cord (SC) reveal Hydin expression in the cells lining the central canal (CC). V=vertebrae, PSV=posterior spinal vein.
65 Figure 3.3. Hydin expression in the newborn brain. (A-B) A coronal section through an anterior portion of the newborn brain reveals the specific localization of Hydin transcripts to the ependymal cells lining the medial layer of the lateral ventricles. (LV). A horizontal section where Hydin transcripts are present in the ependymal cells lining the third (C-D) and fourth (E-F) ventricles.
66 Figure 3.4. Hydin expression in the newborn cerebral aqueduct. A saggital section of the newborn brain reveals intermittent Hydin expression (white arrows) in the ependymal cells lining the cerebral aqueduct (Aq). CBM=cerebellum, 4th= fourth ventricle.
Figure 3.5. Hydin expression in the newborn central canal. A transverse section through the spinal cord (SC) showing specific expression of Hydin in the ependymal cells lining the central canal.
67 Figure 3.6. Hydin expression in the adult brain Coronal brain sections reveal the persistent expression of Hydin in the adult brain. (A-B) Hydin transcripts are present in ependymal cells surrounding the entire lateral ventricle (LV). In addition, expression in the choroid plexus (CP) of the lateral ventricle is observed. (C-D) Expression of Hydin in the third ventricle, with low level expression in the choroid plexus. (E-F) A coronal view showing continuous Hydin expression in the ependymal layer connecting the lateral and third ventricles (*).
68 Figure 3.7. Hydin expression in the adult cerebral aqueduct. A saggital section through the adult brain reveals Hydin expression in the ependymal cells lining the cerebral aqueduct (Aq) and fourth ventricle (4th). CBM=cerebellum.
Figure 3.8. Hydin expression in the adult testis. (A-B) A section through the adult testis showing strong expression of Hydin within the seminiferous tubules. (C-D) The absence of signal using a sense Hydin probe validates the signal observed in panel B.
69 Figure 3.9. Localization of Hydin transcripts to developing spermatocytes Careful examination of silver grains corresponding to Hydin mRNA indicates the presence of transcripts between the spermatogonia (SG) and mature sperm. This region correlates to the site of spermatocyte development. For clarity, the basement membrane of the tubule has been outlined in black.
70 Figure 3.10. Hydin expression in the developing and immature testis Hydin transcripts are present in the E15.5 (A-B) and P16 (C-D) testis.
Figure 3.11. Hydin expression in the adult oviduct Hydin is strongly expressed in the ciliated epithelium lining the lumen of the oviduct (Ov), extending out to the fimbrae (F). U = uterus.
71 Figure 3.12. Hydin expression in the respiratory system. (A-B) Hydin transcripts are present in the epithelial cells lining the nasal cavity (NC) on the day of birth. (C-D) A second section, more dorsal to that shown in panels A and B, reveals exclusive expression in the respiratory epithelium lining the nasal cavity. Hydin mRNA is also observed in the respiratory epithelium of the trachea at P16 (E-F) and the bronchi (Br) in the adult lung (G-H).
72 Figure 3.13. Hydin expression in the inner ear. Horizontal sections through the vestibular system at P0 reveal Hydin expression in the epithelia lining the saccule (Sa, A-B) and the semicircular canal duct (SC, C-D). (E) At higher magnification, vestibular kinocilia are apparent on the surface of the semicircular canal duct that expresses Hydin. Two representative kinocilia are indicated by the arrows.
73 CHAPTER 4
QUANTITATIVE ANALYSIS OF HYDIN mRNA LEVELS IN hy3/OVE459 MICE AND IDENTIFICATION OF A FRAMESHIFT MUTATION IN THE hy3 ALLELE OF HYDIN
4.1 Introduction
The location of the transgene insertion site and the expression pattern of Hydin
provided convincing, but circumstantial, evidence that Hydin is the gene responsible for autosomal recessive hydrocephalus in hy3 and OVE459 mice. Identifying defects in the expression or consequential mutations in the primary sequence of this gene in both mutant strains would demonstrate that Hydin is the causative hydrocephalus gene in these mice. One might predict that Hydin transcription in homozygous hy3 and OVE459 mice is abolished due to genetic lesions in this gene. Previous experiments, however, indicated that Hydin mRNA is present in homozygous mice from both lines. Amplification of
Hydin by standard reverse transcription-PCR and detection of Hydin mRNA by in situ
74 hybridization in OVE459 homozygous mutants (data not shown) demonstrated that Hydin is transcribed at levels sufficient for detection by these methods. However, neither of these methods is truly quantitative. It was clear that a quantitative assessment of Hydin transcription in hy3 and OVE459 homozygotes was necessary. Furthermore, the size of the Hydin message may be altered in these mutants. A smaller or larger Hydin transcript would indicate the absence or addition, respectively, of certain exons (or introns) that could interfere with the function of the gene product. We chose to perform northern analysis, a suitable approach to determine the relative abundance and size of a particular message between RNA samples.
Regardless of the results obtained by Northern analysis, it was also imperative to sequence Hydin in hy3 mice. If hy3 homozygotes transcribe Hydin at wild-type levels, it could not be ruled out that the hy3 allele of Hydin contains a point mutation that does not affect mRNA stability but results in a non-conservative amino acid change that alters protein function. Alternatively, if Northern analysis reveals a decreased abundance of
Hydin transcripts in hy3 mice, it would be necessary to identify the genetic lesion underlying this difference.
4.2 Materials and Methods
4.2.1 Hydin Northern Analysis
Northern blotting and hybridization were performed as described in Chapter 2, except 15 µg of total testis RNA was used here for detection of the Hydin transcript. The probe template used for this analysis was the RT-PCR product C3-2a/2b, corresponding to nucleotides 3232-3675 in the complete mouse Hydin mRNA sequence (GenBank
75 accession number AY173049). The sequences of the primers C3-2a and C3-2b are
provided in Chapter 2, as this probe was used for detection of the candidate 3 transcript,
which we now know to be the 5 prime portion of Hydin. This probe is derived from
Hydin exons 21-24, which produces a single, high-molecular weight signal thought to
represent the full-length Hydin transcript. To detect the Vac14 transcript, 5 µg of polyA+ newborn brain RNA per lane was run under standard formamide denaturing conditions.
The Vac14-specific probe consisted of a 426 bp RT-PCR product amplified from newborn FVB/N total brain RNA using the primer pair C1-2S (5’-
GGCCTATGATGACCGCAAGAAAAG-3’) and C1-2A (5’-
TGAAGTAAGATGGGGAAGAGGCT -3’).
4.2.2 Sequencing of Hydin exons from hy3 genomic DNA
Amplification of Hydin exons from homozygous hy3 genomic DNA was carried out in 50 µl reactions using Pfu Turbo polymerase (Stratagene). Each reaction contained
5 µl 10X Pfu buffer (supplied with polymerase), 5 µl 5mM dNTPs, 4 µl DMSO, 2 µl of hy3 genomic DNA (diluted 1:500), 1 µl each primer (20 uM stock), 0.5 µl Pfu polymerase and 31.5 µl ddH2O. The sequences of each primer used for amplification are provided in Table A.3. Following gel electrophoresis of each 50 µl reaction, amplification products were isolated using Montage gel purification columns (Millipore).
For sequencing, 1.5 µl of purified PCR product was used as the template in a 10
µl reaction containing 0.5 µl DMSO, 0.8 µl 2 µM primer (1.6 pmoles), 4 µl Big Dye v1.1 sequencing mix (Applied Biosystems) and 3.2 µl ddH2O. The sequencing program was as follows: 96O 1 min, 96O 10 sec, 50O 5 sec, 60O 4 min, repeat steps two through four 24
76 more times, 4O hold. Sequencing products were purified using Performa DTR Gel
Filtration Cartridges (Edge Biosystems), lyophilized in a speed vac and submitted to the core sequencing facility at Columbus Children’s Research Institute.
4.2.3 Allele-specific PCR
Tail DNA from wild-type, heterozygous and homozygous hy3 mice was prepared
as described (75). Two µL of each sample was used as PCR template using the primer
pairs Cand2 wt sense (5’- CTTCCAGGATTTCCCCAT-3’) and Cand2 genotype anti (5’-
TGTGATCTCAGAGGCTTAGT-3’) and independently using primers Cand2 hy3 sense
(5’- CTTCCAGGATTTCCCATA-3’) and Cand2 genotype anti. The final concentration
of each primer was 0.8 µM. To increase the specificity of the reaction, an oligonucleotide
antisense to the wild-type or hy3 sense primer was added to each reaction at a final
concentration of 0.8 µM (95). PCR conditions were 94°C for 40s, 57°C for 40s, 65° for
40s for 33 cycles. Amplifications were carried out using HotMaster Taq polymerase
(Eppendorf) which has an optimum elongation temperature of 65O C. The amplification
products are 410 and 409 bp in length for the wild-type and hy3 alleles, respectively.
4.3 Results
4.3.1 Testis Hydin mRNA levels are markedly reduced in hy3 and OVE459 mice
For a quantitative assessment of Hydin and Vac14 mRNA levels between wild-
type and homozygous hy3 and OVE459 mice, Northern analysis was performed. The
Hydin-specific probe used for this analysis was derived from the RT-PCR product C3-
2a/2b, corresponding to nucleotides 3232-3675 in the full-length mouse Hydin mRNA
sequence (GenBank accession number AY173049). This probe was previously used to
77 detect the candidate 3 transcript, which we now know represents exons 21-24 of Hydin.
This probe detects a single, high-molecular weight message thought to represent the full- length Hydin transcript.
Northern analysis for Hydin was performed on total testis RNA (Figure 4.1A), as multiple attempts to detect the Hydin message from brain RNA were unsuccessful. In wild-type hy3 and non-transgenic OVE459 RNA, the high-molecular weight Hydin transcript was present (lanes 2 and 4, respectively). However, in homozygous hy3 and
OVE459 RNA, this transcript was not detected (lanes 3 and 5, respectively). This result
revealed that the levels of this Hydin transcript are markedly reduced, if not completely
absent, in the testis of both homozygous mutants. Subsequent hybridization with a beta
actin control probe confirmed that the total RNA loaded in each lane was equivalent. The
relative abundance and size of the Vac14 transcript was equivalent between wild-type
and homozygous mutant mice (Figure 4.1B). This result indicates that Vac14 expression
is unaltered in the homozygous mutant mice and that the altered levels of Hydin mRNA are a specific feature of the hydrocephalus-inducing mutations.
4.3.2 The hy3 allele of Hydin contains a frameshift mutation in exon 15
Northern analysis had established that the expression of Hydin is altered in hy3 and OVE459 homozygous mice, but the sources of these differences remained to be identified. The genomic rearrangement that characterizes the OVE459 allele of Hydin is likely responsible for the reduction of Hydin mRNA in OVE459 homozygotes. Rather than attempting to fully characterize the OVE459 allele of Hydin, which had the potential to be extremely complex and time-consuming, we chose to sequence the hy3 Hydin gene.
78 As a spontaneous mutation, the hy3 allele was more likely to possess an easily
identifiable genetic lesion to which altered Hydin expression could be attributed.
To sequence the hy3 allele of Hydin, each of the 87 Hydin exons were amplified
from homozygous hy3 genomic DNA using exon-flanking intronic primers (Table A.3).
These primers are not only useful for detecting mutations in Hydin exons, but also
capable of detecting splice-site mutations at the intron-exon boundaries. The sequencing
procedure and a representative agarose gel containing amplified Hydin exons are
presented in Figure 4.2. While most of the PCR primer sets allowed amplification of the
exon of interest, some did not work. In these cases, new primer sets were designed and
used successfully for exon amplification. Amplified exons were gel-purified and
sequenced using both the forward and reverse primers used in the initial PCR
amplification. Exon (and flanking intron) sequences from hy3 were aligned with the
Ensembl consensus Hydin genomic map to detect potential sequence discrepancies.
Eighty-six of the eighty-seven exons showed no differences in primary nucleotide
sequence between hy3 and wild-type genomic DNA. However, sequencing of exon 15
revealed the deletion of a single CG base-pair in the hy3 allele of Hydin. This result was
reconfirmed in archival hy3 genomic DNA obtained from Jackson Laboratory, indicating
that this primary sequence difference was not acquired during the maintenance of the hy3
line in our mouse facility. Furthermore, sequencing of a hy3-/- RT-PCR product spanning exon 15 indicated that this mutant allele is transcribed (Figure 4.3A). The deletion of this
CG base-pair is predicted to have a devastating effect on the Hydin gene product, as it shifts the reading frame and creates a premature termination signal just two codons
79 downstream of the deletion. The premature stop codon results in a truncation of more
than 88% of the predicted full-length Hydin protein (Figure 4.3B).
4.3.3 Development of the first molecular test for the hy3 allele
We reasoned that the exon 15 deletion specific to the hy3 allele could be exploited
in a molecular assay to distinguish the various hy3 genotypes prior to the appearance of hydrocephalus. We attempted allele-specific PCR using a common antisense primer specific to intron 15 and a sense primer specific at its 3 prime end to the wild-type or hy3 exon 15 sequences. Initial attempts using standard PCR techniques failed to achieve the desired specificity. We modified the assay by adding an equivalent amount of an oligonucleotide antisense to the allele-specific sense primer (95). This modification improved the assay’s specificity significantly (Figure 4.4). Reactions containing the wild- type sense primer yielded an amplification product exclusively from wild-type and heterozygous hy3 genomic DNA. Similarly, reactions containing the hy3-specific sense primer only gave rise to an amplification product from heterozygous and homozygous hy3 genomic DNA. This assay represents the first molecular test specific for the hy3 allele.
4.4 Discussion
Here, we have demonstrated a quantitative reduction of Hydin mRNA in
homozygous hy3 and OVE459 mice. Furthermore, we have identified a mutation in the
hy3 allele of Hydin that likely explains the marked reduction of Hydin transcripts in hy3 mice. One would reasonably predict that the premature termination signal early in the hy3 transcript destabilizes the message through a nonsense-mediated decay mechanism (96).
80 Not only are the levels of Hydin mRNA decreased, but the CG base-pair deletion is
predicted to have a severe consequence on any Hydin protein that is translated from messages that escape decay. We predict that the loss of more than 88% of the full-length
Hydin gene product makes the hy3 allele of Hydin a null allele.
While this data convincingly demonstrates altered expression of Hydin and a
specific genetic lesion in hy3 mice, several qualifications must be made. First, we have
yet to demonstrate the reduction of Hydin mRNA in hy3 and OVE459 homozygous brain
RNA. The highly restricted expression of Hydin in the brain has prevented the detection
of Hydin transcripts by Northern analysis. However, Hydin transcripts in the brain are
easily amplified by RT-PCR. It may be worthwhile to perform real-time RT-PCR using
total brain RNA from wild-type and homozygous mutants. This approach may reveal
quantitative differences in brain-specific Hydin transcripts between wild-type and mutant
animals.
As previous Northern analysis has demonstrated (see Chapter 2), at least two
splice-variants of the Hydin transcript exist. Exon 15 may not be represented in every
Hydin isoform. In this scenario, Hydin transcripts in hy3 homozygotes that do not include
exon 15 would presumably be stable and encode functional Hydin protein. However,
exon 15 appears to be an important component to the Hydin message. Exon 15 cannot
easily be skipped, as the next exon to which exon 14 can splice and still preserve the ORF
is exon 31. Therefore, the absence of exon 15 prohibits the representation of 15 additional
exons (>2.5 kb) in Hydin mRNA. Such an analysis, however, assumes the inclusion of
exon 14 in each of the putative Hydin isoforms. Northern analysis using a battery of
81 probes may help answer questions regarding the number of Hydin isoforms that are present in a particular tissue. Furthermore, the importance of exon 15 could ultimately be addressed by real-time RT-PCR using wild-type RNA. The relative abundance of an exon
15-specific amplification product could be compared to a product specific to the 3’ UTR.
If the exon 15-specific product is less abundant, one could conclude that Hydin isoforms
that exclude exon 15 exist. Such a result would suggest that the hy3 allele is not likely to
be a true null allele.
Lastly, we have developed the first molecular test for the hy3 mutation, initially identified more than 60 years ago. This assay has been useful for several reasons. First, it has revolutionized our ability to track the hy3 allele in our mouse colony. Wild-type, heterozygous and homozygous hy3 mice are easily distinguished using this two-step PCR approach. Second, this assay is invaluable in its ability to unambiguously detect homozygosity for the hy3 allele prior to the onset of hydrocephalus. Previously, we developed a method of distinguishing heterozygous from homozygous hy3 mice before the appearance of hydrocephalus using the polymorphic markers D8Mit248 and
D8Mit215. These markers flank the Hydin locus but cannot distinguish genotypes unambiguously because of their separation by more than 23 Mb. This highly specific assay based on the actual hy3 mutation enables studies regarding the pathogenic mechanism of hydrocephalus in these animals to be carried out prior to severe increases in intracranial pressure and ventricular dilatation. Increased intracranial pressure and enlargement of the ventricles result in secondary effects that have complicated previous
82 investigations into the pathogenesis of these mice. The hy3 allele-specific PCR assay has eliminated this problem.
83 Figure 4.1. Northern analysis of Hydin and Vac14 in hy3 and OVE459 homozygous mutant and wild-type littermates. (A) Total testis RNA was blotted and hybridized with a radiolabeled Hydin-specific probe spanning exons 21-24. A single transcript much larger than the 9 kb RNA marker (M) is detected in both hy3 age 21 days (P21) and OVE459 (P20) wild-type animals, but is undetectable in hy3 and OVE459 homozygous mutants. The β-actin RNA loading control is shown below the Hydin blot. (B) PolyA+ brain RNA was blotted and hybridized to a Vac14-specific probe. No difference in Vac14 mRNA size or abundance was observed between FVB/N, and homozygous mutant OVE459 or hy3 animals.
84 Figure 4.2. Procedure for sequencing Hydin exons from hy3 -/- genomic DNA. Exon-flanking, intronic primers (red and blue for forward and reverse, respectively) were used to amplify all 87 Hydin exons from hy3 genomic DNA. A representative agarose gel containing PCR products for exons 3 thru 10 is shown. Following PCR, amplified exons were gel purified and sequenced independently with the forward and reverse primers used in the initial amplification. The two sequences for each PCR product were combined to generate a consensus sequence, which was subsequently aligned to the relevant region in the Ensemble mouse genome assembly.
85 Figure 4.3. The hy3 allele of Hydin contains a single base pair deletion in exon 15. (A) Sequence comparison of Hydin exon 15 between wild-type (+/+) and hy3 homozygous mutant (-/-) littermates. An RT–PCR product spanning exon 15 was generated from wild-type and hy3 homozygous mutant brain RNA and sequenced. The hy3 allele (bottom) is missing a cytosine between positions 2163 and 2166 of the Hydin transcript, resulting in a premature termination signal two codons downstream of the mutation. (B) The premature termination signal occurs at codon 576 of the 5099 codon Hydin open reading frame (ORF).
86 Figure 4.4. The wild-type and hy3 alleles of Hydin can be distinguished by allele-specific PCR. Using a wild-type-specific sense primer, amplification products are generated from wild-type (+/+) and hy3 heterozygous (+/-) genomic DNA, but not from homozygous mutant hy3 (-/-) genomic DNA. Using an hy3-specific sense primer, amplification products are generated from hy3 +/- and -/- genomic DNA, but not hy3 +/+ genomic DNA.
87 CHAPTER 5
GENERATION OF A HYDIN CONDITIONAL KNOCK-OUT ALLELE
5.1 Introduction
We have demonstrated that Hydin is the gene responsible for congenital
hydrocephalus in hy3 mice. Loss of Hydin function in the ciliated ependymal cells lining the brain ventricles is the most likely cause of hydrocephalus in these mice. However, the
function of the Hydin gene product remains unknown. The expression of Hydin in other
ciliated cell-types, in addition to the ependyma, suggests a role for Hydin in cilia function
(motility or signaling) and/or maintenance. hy3 and OVE459 homozygous mutants do not
survive beyond weaning due to rapid progression of hydrocephalus, making it difficult to
assess the phenotypic consequences of Hydin disruption in other ciliated/flagellated
cellular compartments.
88 In situ hybridization analysis revealed Hydin expression in developing spermatocytes.
The hallmark phenotypic outcome of motility defects in sperm is male infertility (41). An important question to address is whether sperm in mice lacking Hydin have motility defects that cause infertility. Currently, this question cannot be answered due to the mortality of Hydin homozygous mutants prior to sexual maturity. To circumvent this, we have chosen to make a conditional null allele of Hydin in mice using the Cre-loxP system
(for review see 97). The ability to control when and where Hydin is disrupted should facilitate the elucidation of Hydin function. For example, the motility of mature sperm in
Hydinflox/flox mice expressing Cre in the spermatogonial stem cells could be easily assessed as sperm are readily isolated from adult male mice and amenable to functional analysis.
Our conditional targeting strategy for Hydin was influenced by several factors.
First, it was not feasible to flank the entire gene with loxP sites in a single targeting step due to the large size of Hydin. Second, splice variants, including alternative transcription start sites, in Hydin mRNA have not been characterized and are potentially extensive.
Targeting exons that are not represented in the majority of Hydin mRNA species would not likely result in the desired null allele. Third, the absence of characterized functional domains in the Hydin protein did not permit us to target a particular portion of the gene according to known function. With these considerations in mind, we chose to target
Hydin exon 15. The hy3 allele of Hydin, which contains a single base-pair deletion in exon 15, is sufficient to cause a marked reduction in Hydin mRNA levels, severe runting and rapid progression of disease. These results provide evidence that disruptions in exon
89 15 can interfere with normal Hydin expression/function. Furthermore, exon 15 cannot easily be skipped, as the next exon to which exon 14 can splice and preserve the ORF is exon 31. Therefore, the absence of exon 15 prohibits the representation of 15 additional exons (>2.5 kb) in Hydin mRNA. Exons 14 and 31 are separated by more than 98.6 kb in the mouse genome.
Over the past several years, recombineering has emerged as a powerful approach
to create specific genomic alterations (98). Recombineering involves the use of phage-
encoded proteins to facilitate cloning via homologous recombination in bacteria (99). We
decided to employ a recombineering-based approach to generate a conditional targeting
construct for Hydin, as recombineering offers distinct advantages over traditional cut-
and-paste cloning. First, recombineering allows the desired alteration to be made at any
location in the genome via homologous recombination, obviating the need to find
restriction sites that are compatible between the targeting vector and the genomic region
of interest. Second, recombineering approaches generally achieve the desired genomic
modification in less time than traditional cut-and-paste cloning methods.
Reagents for efficient recombineering have been developed in which DH10B-
derived E. coli carry an integrated λ−prophage recombination system (100). The
prophage encodes three gene products: gam, bet and exo (Figure 5.1A). Gam inhibits
RecBCD nuclease activity of the host cell, thereby preventing the active degradation of linearized targeting constructs post-electroporation. Exo is a 5’Æ3’ exonuclease that generates single-stranded 3’ ends from linear double-stranded targeting constructs. Beta binds to the single-stranded end created by Exo and facilitates the annealing of this strand
90 with a complementary strand in the target DNA. The expression of these genes is under
the control of the λ−PL promoter. This promoter is tightly regulated by the temperature
sensitive λ−cI857 repressor. At 32O C, the λ−cI857 repressor is active and shuts down expression of the λ-prophage to undetectable levels. The repressor is inactivated when the cells are shifted to 42O C, and gam, bet and exo are expressed at sufficient levels for efficient recombinogenic cloning.
Three DH10B-derived strains carrying the λ−prophage recombination system have been generated (Figure 5.1B) (100). The original strain, DY380, contains the
λ−prophage system and a tetracycline (Tet) selectable marker. Strains EL250 and EL350 are DY380 derivatives, where the Tet marker was replaced by an arabinose-inducible
Flpe or Cre cassette, respectively. As DH10B-derived strains are capable of propagating
large genomic clones, transforming the DY380, EL250 or EL350 strains with BAC
clones is relatively efficient.
We adapted an innovative method developed in Neil Copeland’s laboratory (101)
to generate a conditional targeting construct for Hydin exon 15 via recombineering. In the first step of this strategy, a BAC clone encompassing Hydin exon 15 was identified and transformed into a DY380-based recombineering strain. Second, the relevant portion of this BAC was retrieved (subcloned) by recombineering into plasmid pL253, a pBluescript
(pBS)-based plasmid containing the MC1 TK negative selection cassette. Next, a single loxP site was inserted into intron 14 via recombinogenic targeting of a homology arm- flanked loxP-Neo-loxP cassette, followed by subsequent excision of Neo via transient expression of Cre recombinase in strain EL350. The final recombineering step was the
91 recombinogenic targeting of a homology arm-flanked frt-Neo-frt-loxP cassette into intron
15.
The end result of these recombineering events is a plasmid containing exon 15
with a loxP site in intron 14 and an frt-Neo-frt-loxP cassette in intron 15 (Figure 5.2).
This entire plasmid was linearized by digestion with NotI and used for gene targeting in
ES cells. The size of the homology arms (for gene targeting in ES cells) of the final targeting construct is governed by the position of the homology arms used during BAC retrieval. We chose to retrieve an 11 kb fragment containing 6.6 kb upstream and 4.2 kb downstream of exon 15. After completion of the targeting construct, the 5’ homology arm
for ES cell targeting (between the 5’ end of the retrieved BAC DNA and the intron 14
loxP site) was 5.2 kb in length. The 3’ homology arm (between the loxP site in intron 15
and the 3’ end of the retrieved BAC DNA) was 2.2 kb in length.
The loxP sites in introns 14 and 15 are marked by NdeI restriction enzyme sites.
These sites were engineered into the homology arms used for recombineering of introns
14 and 15. NdeI was a convenient restriction enzyme for ES cell screening, as the entire
exon 15 targeting region resides on a 13.8 kb NdeI fragment (Figure 5.3). The addition of
an NdeI-marked loxP site in intron 14 results in a shift from 13.8 kb in the wild-type
allele to 7.1 kb in the targeted allele using the 5’ southern probe. Using the 3’ southern
probe, the NdeI-marked loxP site in intron 15 results in a 13.8 kb to 4.9 kb shift in the
wild-type and targeted alleles, respectively.
Once the targeting construct is complete and targeted ES cells have been
identified, two methods to excise the Neo selectable marker can be used. First, Flpe
92 recombinase can be transiently expressed in ES cells prior to blastocyst injection. Second, mice carrying the conditional allele can be bred to mice that express Flpe in the germline
(102). We have chosen to use the Flpe deleter mice to excise the Neo cassette after mice carrying the conditional allele have been generated, as additional manipulations of ES cells prior to blastocyst injection may adversely affect the competence of these cells for germline transmission. After excision of the intron 15 Neo cassette via Flpe-mediated recombination, the 3’ NdeI fragment will decrease in size from 4.9 to 3.1 kb.
5.2 Materials and Methods
5.2.1 RPCI- 22(129S6/SvEvTac) BAC library screening
Three PCR products were amplified from 129 ES cell genomic DNA using the primer sets 5’EST-F/R, 243454-F/R and Exon64 5’-F/R, representing the 5 prime end, exon 15 and the 3 prime end of Hydin, respectively. The sizes of the PCR products are
370 bp, 408 bp and 500 bp, respectively. Primer sequences are as follows: 5’EST-F: 5’-
GGCAGTGCAGCAAAAAGTGG-3’, 5’EST-R: 5’-
AGACGGAAAGGGCTGGAACAAG-3’, 243454-F: 5’-
TGTTGATCAAGGGAGGCTACTGG-3’, 243454-R: 5’-
ACGGCATTCCCTATCACTGTCCTT-3’, Exon64 5’-F: 5’-
GGTTCCCCGATGGTCTCTTTAGG-3’, Exon64 5’-R: 5’-
AGGCCGCCAGTTGACACAGTTG-3’. PCR products were labeled using the Random
Primers Labeling System (Invitrogen) and purified using Mini Quick Spin DNA columns
(Roche). A cocktail containing each probe at a concentration of 2,000,000 cpm/ml in FBI hybridization buffer (10% PEG 8000, 7% SDS, 1.5X SSPE) was used to screen eight
93 gridded filters constituting the RPCI-22(129S6/SvEvTac) mouse BAC library. The filters
were washed three times (20 minutes each wash) in 0.1X SSC/0.1% SDS.
Autoradiography film was exposed to the filters for 2 hours and 16 hours for short and
long exposures, respectively. Identification of positive clones was carried out according
to instructions provided with the filters.
5.2.2 Pulse-field gel electrophoresis of BAC RP22-193L17
BAC RP22-193L17 was isolated using the Qiagen Midiprep kit. Size
determination by pulse-field gel electrophoresis was carried out as described for BAC
RP23-21B7 in Chapter 2.
5.2.3 Tranformation of BAC RP22-193L17 into recombineering strain EL350
A single EL350 colony was picked and grown overnight at 32 O C in 5 ml of low salt LB (5 g of NaCl per liter) broth in a 15 ml round bottom tube until the OD600 reached
1.2 to 1.4. Cells were spun in a Sorvall RT7 tabletop centrifuge at 3200 rpm in an RTH-
250 rotor (Sorvall) for 10 minutes at 0O C and placed on ice prior to washing. The LB supernatant was discarded and the cell pellet was resuspended in 900 uL ice-cold sterile ddH2O using a wide-bore pipette tip. Cells were transferred to a pre-chilled 1.5 ml eppendorf tube and spun at maximum speed in a microfuge for 25 seconds, followed by discarding of the supernatant. Cells were washed two more times. Following the third wash, the cell pellet was resuspended in 75 uL ice-cold sterile ddH2O containing 5 ug of freshly prepared 193L17 BAC DNA. This mixture was placed in a pre-chilled 0.1 cm gap cuvette and electroporated at 1.8 kV, 25 uF and 200 ohms using a BTX ECM 630 electroporator. Contents of the cuvette were immediately transferred to a 2 mL microfuge
94 tube containing 1 ml of 32 O C pre-warmed LB. Cells were shaken horizontally (220 rpm) at 32 O C for 1 hour. The entire contents of the 2 mL tube were plated on five LB plates containing 25 ug/mL chloramphenicol (CAM). Hundreds of CAMR colonies were
obtained, and the presence of intact BAC DNA was verified using PCR primer sets
specific for Hydin exons 13, 15 and 18.
5.2.4 Cloning of the pL253-based BAC retrieval plasmid
Homology arms for the 5’ and 3’ retrieval boundaries were PCR-amplified using
the primer sets Retrieve 5’-F/R and Retrieve 3’-F/R, respectively. Primer sequences are
as follows, with spacer regions and restriction sites underlined: Retrieve 5’-F (NotI): 5’-
ATAAGCGGCCGCGGAGGCCCAATGAGACAGT-3’, Retrieve 5’-R (HindIII): 5’-
GTCAAGCTTAACGGAATGAGACGAGGTAATGAT-3’, Retrieve 3’-F (HindIII):
5’-GTCAAGCTTTGGGAAGCAAAGGAATAG-3’, Retrieve 3’-R (SpeI): 5’-
TCTACTAGTGTGCACAGCCAGAGGGGATGAG-3’). PCR products were gel-
purified using Montage gel purification columns (Millipore) and digested with NotI and
HindIII (Retrieve 5’-F/R) or HindIII and SpeI (Retrieve 3’-F/R). Digests were terminated
by adding 5X volume of Qiagen buffer PB. The digested PCR products were purified
using Qiagen miniprep columns and eluted in 30 uL of Qiagen buffer EB. PCR products
were ligated into NotI/SpeI-digested pL253 plasmid (3:1 molar ratio of each PCR product
to 100 ng pL253 vector) using the LigaFast Rapid DNA Ligation System (Promega).
Ligation reactions were carried out for 8 minutes at 25O C, and 2.5 uL of each reaction
was used for transformation of chemically competent TOP10 E. coli (Invitrogen). Correct
clones were identified by screening via PCR and restriction digestion.
95 5.2.5 BAC Retrieval
Electrocompetent EL350 cells containing BAC RP22-193L17 were prepared as
follows. A single colony was picked and grown O/N at 32O C in 5 mL low-salt LB broth.
On the following day, 50 mL of low-salt LB in a 500 mL flask was inoculated with 1 mL
O of the overnight culture and shaken at 32 C until the OD600 reached 0.5. For induction of the lambda RED system, 10 mL of this culture was placed in a pre-warmed 125 mL flask and shaken at 42O C for exactly 15 minutes. For the un-induced control, 10 mL of the 50
mL culture was transferred to a pre-warmed 125 mL flask and shaken at 32O C for 15 minutes. Following this 15 minute period, the induced and un-induced cells were immediately transferred to an ice-water slurry and shaken by hand for 5 minutes to rapidly cool the cells. Cells were transferred to pre-chilled 15 mL conical tubes and spun in a Sorvall RT7 tabletop centrifuge at 3200 rpm in an RTH-250 rotor (Sorvall) for 10 minutes at 0O C and placed on ice prior to washing. The supernatants were discarded, and the pellets were resuspended in 1 mL of ice-cold sterile ddH2O and transferred to pre- chilled 1.5 mL eppendorf tubes. Cells were washed 3 times as described and resuspended in 100 uL of sterile ddH2O containing 150 ng of HindIII-digested, gel purified retrieval plasmid. The cells were transferred to 0.1 cm gap cuvettes and electroporated at 1.8 kV,
200 ohms and 25 µF. Cells were transferred to 2 mL eppendorf tubes containing 1 mL of pre-warmed LB medium, shaken horizontally at 32O C for 1 hour and plated on LB plates
containing 100 µg/mL ampicillin. Clones carrying the desired portion of BAC RP22-
193L17 were identified by PCR and restriction digestion. To select against plasmid
96 multimers created during retrieval (103), 1 ng of plasmid was retransformed into
electrocompetent DH10B cells (Invitrogen).
5.2.6 Cloning of the intron 14 minitargeting vector
Homology arms for the loxP-Neo-loxP intron 14 targeting cassette were PCR-
amplified using the primer sets 5’floxedNEO-F/R and 3’floxedNEO-F/R. Primer
sequences are as follows, with spacer regions and restriction sites underlined:
5’floxedNEO-F (NotI): 5’-ATAAGCGGCCGCTAGGCATAGCATTCATACCA-3’,
5’floxedNEO-R (EcoRI/NdeI): 5’-
GTCGAATTCCATATGCTCTTCAGATCTTGCCCAGTG-3’, 3’floxedNEO-F
(BamHI): 5’-ATAGGATCCCTAAGAGAAGGTGGTTTGAGG, 3’floxedNEO-R
(SalI): 5’-GTCGTCGACGAGTTAGATCCAGCCAGAGAAGAC-3’. PCR products
were gel-purified using Montage gel purification columns (Millipore) and digested with
NotI and EcoRI (5’floxedNEO-F/R) or BamHI and SalI (3’floxedNEO-F/R). Digests
were terminated by adding 5X volume of Qiagen buffer PB. The digested PCR products
were purified using Qiagen miniprep columns and eluted in 30 µL of Qiagen buffer EB.
PCR products were ligated to both the EcoRI/BamHI-excised loxP-NEO-loxP cassette
from plasmid pL452 and NotI/SalI-digested pBluescriptII KS+ plasmid (3:1 molar ratio
of each PCR product and pL452 fragment to 100 ng pBSII vector) using the LigaFast
Rapid DNA Ligation System (Promega). Ligation reactions were carried out for 8
minutes at 25O C, and 2.5 µL of each reaction was used for transformation of chemically
competent TOP10 E. coli (Invitrogen). Correct clones were identified by screening via
PCR and restriction digestion.
97 5.2.7 Cloning of the intron 15 minitargeting vector
Homology arms for the frt-Neo-frt-loxP intron 15 targeting were PCR-amplified
using the primer sets 5’frtNEO-F/R and 3’frtNEO-F/R. Primer sequences are as follows,
with spacer regions and restriction sites underlined: 5’frtNEO-F (NotI): 5’-
ATAAGCGGCCGCTGCACTTTCAGCCAGGTCTATGT-3’, 5’frtNEO-R
(EcoRI/NdeI): 5’-GTCGAATTCCATATGAAGGTCCAAAGCCAGGTCCAC-3’,
3’frtNEO-F (BamHI): 5’-ATAGGATCCGCCCGTGCAAGTTTCTA-3’, 3’frtNEO-F
(SalI): 5’-GTCGTCGACCATTTCTGGGGTGACATTTAT-3’. PCR products were gel-
purified using Montage gel purification columns (Millipore) and digested with NotI and
EcoRI (5’frtNEO-F/R) or BamHI and SalI (3’frtNEO-F/R). Digests were terminated by
adding 5X volume of Qiagen buffer PB. The digested PCR products were purified using
Qiagen miniprep columns and eluted in 30 µL of Qiagen buffer EB. PCR products were ligated to both the EcoRI/BamHI-excised frt-NEO-frt-loxP cassette from plasmid pL451 and NotI/SalI-digested pBluescriptII (pBSII) KS+ plasmid (3:1 molar ratio of each PCR product and pL451 fragment to 100 ng pBSII vector) using the LigaFast Rapid DNA
Ligation System (Promega). The ligation reaction and transformation were carried out as described for the intron 14 targeting plasmid. Correct clones were identified by screening via PCR and restriction digestion.
5.2.8 Intron 14 Recombineering
Induced and un-induced electrocompetent DY380 cells were prepared as described for
EL350 cells during BAC retrieval. The intron 14 targeting cassette was excised from
pBSII by digestion with NotI/SalI and gel-purified using the Qiaex II gel purification kit
98 (Qiagen). Recombineering in EL350 cells was performed by co-electroporation of 150 ng
of purified targeting cassette and 10 ng of the pL253-based retrieval plasmid.
Electroporation parameters and outgrowth were performed as described previously. Cells
were plated on LB plates containing 25 µg/ml kanamycin (Kan). DNA from a correctly recombineered clone was isolated and retransformed (500 pg) into electrocompetent
DH10B cells to eliminate residual untargeted retrieval plasmids.
5.2.9 CRE-mediated excision of Neo
Frozen aliquots of electrocompetent CRE-induced EL350 cells were prepared as
described (101) and transformed with 1 and 10 ng of intron 14-targeted retrieval plasmid.
Electroporation parameters and outgrowth were performed as described. Twenty, 50 and
100 µL of each electroporation was plated on LB-Amp (100 µg/ml) plates while 100 µL of each electroporation was plated on LB-Kan (25 µg/ml) plates as a negative control.
Plates were incubated O/N at 320 C. Colonies were only present on the LB-Amp plates.
5.2.10 Intron 15 Recombineering
The procedure for recombineering intron 15 with the frt-Neo-frt-loxP cassette
containing the intron 15-specific homology arms was performed as described for intron
14 recombineering. For reduction of background KanR colonies due to contaminating minitargeting plasmid, the minitargeting plasmid was digested with PvuI in addition to
NotI and SalI. PvuI cuts the pBSII backbone, allowing better separation of the targeting cassette from vector-derived DNA. Following co-electroporation of intron 15 targeting cassette and the intron 14-targeted pL253-based retrieval plasmid, correctly recombineered KanR clones were identified by digestion with NotI and NdeI. DNA from
99 a correctly recombineered clone was isolated and retransformed (500 pg) into
electrocompetent DH10B cells to eliminate residual untargeted retrieval plasmids. The
resulting retransformed plasmid was designated Hydin CKO.
5.2.11 Determining the targeting cassette functionality by treatment with Cre and
Flpe
Frozen aliquots of electrocompetent Flpe-induced EL250 cells were prepared as described for Cre-induced EL350 cells. 1 ng of the Hydin CKO plasmid was electroporated into Flpe-induced EL250 cells or Cre-induced EL350 cells. 100 µL of electroporated cells were plated on LB-Amp or LB-Kan plates. No colonies were present on LB-Kan plates, as was expected if Flpe- and Cre-induced recombination occurred.
Two colonies from the Flpe-treated and Cre-treated LB-Amp plates were screened by digestion with NotI and NdeI.
5.2.12 ES Cell Targeting
100 µg of the Hydin CKO plasmid was linearized by digestion with NotI.
Following digestion, the DNA was extracted with phenol:chloroform and then chloroform, ethanol precipitated and resuspended in 70 µL of sterile ddH20. 50 µg of linearized targeting cassette was electroporated into TC1 129 ES cells (104).
Approximately 150 G418/gancyclovir resistant colonies were isolated and maintained in the laboratory of Dr. Michael Weinstein.
5.2.13 ES Cell DNA Isolation and Screening
ES cell clones in 24 well plates were lysed with 400 µL ES cell lysis buffer (0.5%
SDS, 0.1 M NaCl, 0.01 M EDTA, 0.02 M Tris-Cl pH 7.6, 100 µg/mL proteinase K) for
100 several minutes at 370 C. Lysates were transferred to eppendorf tubes and incubated at
500 C for 2 hours. After addition of 200 µL saturated (6 M) NaCl, tubes were shaken
vigorously for 1 minute and incubated on ice for 10 minutes. Tubes were spun at
maximum speed for 10 minutes, and 500 µL of the supernatant was transferred to clean
eppendorf tubes. For precipitation, 1 mL of EtOH was added, and tubes were inverted
several times and spun at maximum speed for 10 minutes. The DNA pellet was
resuspended in 60 µL of Qiagen buffer EB. 10 µL of each DNA sample was digested
with NdeI or BglII and run on a 0.8% TBE agarose gel and transferred to Hybond XL
nylon membrane (Amersham).
5’ and 3’ southern probe templates were amplified using the primer sets 5’CKO Probe-
F/R and 3’CKO Probe-F/R, respectively. Primer sequences are as follows: 5’CKO
Probe-F: 5’-CACTTTAAATTGCCTCTC-3’, 5’CKO Probe-R: 5’-
TCTAAACTCTTGTGACTCC-3’, 3’CKO Probe-F: 5’-
CACCCAAGCAGTACAGCCAGTTA-3’, 3’CKO Probe-R: 5’-
AGCGCCCAAAGTCCACCTC-3’. PCR products were labeled using the Random
Primers Labeling System (Invitrogen) and purified using Mini Quick Spin DNA columns
(Roche). Blots containing NdeI-digested genomic DNA were hybridized with the 5’ and
3’ probes at a concentration of 2,000,000 cpm/ml in FBI hybridization buffer (10% PEG
8000, 7% SDS, 1.5X SSPE). In addition, blots containing BglII-digested DNA were hybridized with the 3’ probe under the same conditions. Blots were washed 2 times for 20 minutes each in 0.1X SSC/0.1% SDS.
101 5.3 Results
5.3.1 Identification and characterization of a suitable BAC clone for retrieval
As ES cell targeting efficiency is generally higher when the homology arms of the
targeting cassette are isogenic with the ES cell strain used, it was necessary to identify a
BAC cloned derived from mouse strain 129 that encompasses the region flanking Hydin exon 15. The mouse BAC library RPCI-22(129S6/SvEvTac), consisting of eight arrayed filters, was screened by hybridization using a cocktail of probes specific for the extreme
5’ end, exon 15 and extreme 3’ end of the Hydin gene. Eighteen BAC clones exhibiting high levels of hybridization were obtained and subsequently screened using the same probes as before, but individually rather than in a cocktail (Figure 5.4A). A summary of this second round of BAC screening using individual probes is presented in Table 5.1.
One clone, BAC 193L17, was positive for both the 5’ and exon 15 probes. BAC 193L17 was screened by PCR for Hydin exons 13, 15 and 18 to verify that this clone encompassed the entire region to be subcloned by gap repair (Figure 5.4B). This PCR screen indicated that BAC 193L17 was suitable for use in the construction of the exon 15 targeting construct.
To determine the size of the BAC 193L17 genomic insert, this clone was digested with NotI and subjected to pulse-field electrophoresis (Figure 5.4C). The insert was approximated to be 180 kb, as it ran slightly faster than the 194 kb PFG marker. For a definitive confirmation that BAC 193L17 encompassed the entire genomic region of interest, Southern analysis was performed to ascertain that the 13.8 kb NdeI fragment spanning introns 13 thru 17 was present in the insert (Figure 5.4D). The 5’ and 3’ probes
102 to be used for screening in ES cells were hybridized to blots containing NdeI-digested
FVB genomic and BAC 193L17 DNA. For both probes, the expected 13.8 kb fragment was observed on the BAC and was identical in size to the fragment observed in total
genomic DNA. BAC 193L17 was subsequently transformed into EL350 cells for use in
the retrieval of genomic DNA spanning exon 15.
5.3.2 Retrieval of BAC DNA into plasmid pL253
The first recombineering step toward generating a conditional targeting construct for Hydin was the retrieval of an 11 kb portion of the BAC 193L17 insert into the MC1
TK-containing plasmid pL253 (Figure 5.5). Prior to retrieval, homology arms specific to the boundaries of BAC DNA to be retrieved must be cloned into pL253 to generate the retrieval plasmid. To accomplish this, an intron 13-specific 5’ homology arm and an intron 16-specific 3’ homology arm were amplified by PCR. The forward and reverse primers used for amplification of the 5’ arm contained NotI and HindIII restriction sites, respectively. The forward and reverse primers used for amplification of the 3’ arm contained HindIII and SpeI restriction sites, respectively. After amplification and digestion with the appropriate enzymes, the two homology arms were simultaneously ligated into NotI/SpeI-digested pL253, generating the BAC retrieval plasmid.
For retrieval, expression of the λ-prophage recombination system was induced in
EL350 cells harboring BAC 193L17. These cells were then made electrocompetent and transformed with HindIII-linearized retrieval plasmid. Selection for cells containing the subcloned BAC DNA was achieved using ampicillin (Amp) resistance. Hundreds of
AmpR colonies were obtained. Of the 23 clones that were picked and screened by PCR,
103 all 23 clones carried the retrieved BAC DNA. Plasmids from 4 clones were isolated and
digested with NotI and SpeI. Each of the four clones exhibited the expected restriction pattern (Figure 5.6).
5.3.3 Cloning of the Intron 14 and Intron 15 Minitargeting Plasmids
Intron 14 was targeted with a loxP-Neo-loxP cassette which is present on plasmid pL452 (101). Intron 15 was targeted with an frt-Neo-frt-loxP cassette, which is present on plasmid pL451 (101). In both the pL452 and pL451 plasmids, Neo expression is under the control of a hybrid PGK-EM7 promoter. The PGK promoter is functional in mammalian cells, while the EM7 promoter is functional in bacteria (conferring resistance
to Kanamycin). Digestion with EcoRI and BamHI excises the Neo-containing cassettes
from plasmids pL452 and pL451.
The Neo cassettes to be targeted to introns 14 and 15 must first be flanked by
PCR-amplified homology arms specific to those introns and cloned into an empty pBluescript vector (Figure 5.7). To accomplish this, the primers used for homology arm amplification contain the necessary restriction sites for ligation to both the Neo cassette and a linearized pBluescript vector. In the case of the homology arm upstream of the Neo cassette, the forward and reverse primers contain NotI and EcoRI sites, respectively. For the homology downstream of the Neo cassette, the forward and reverse primers contain
BamHI and SalI sites, respectively. In addition, the reverse primer used for amplification of the homology arm upstream of the Neo cassette contains an NdeI site internal to the
EcoRI to facilitate ES cell screening by Southern hybridization.
104 The intron 14 and intron 15 minitargeting plasmids were generated
simultaneously. After amplification of the homology arms, the upstream arms were
digested with NotI and EcoRI and the downstream arms were digested with BamHI and
SalI. The Neo cassettes from plasmids pL452 and pL451 were excised with EcoRI and
BamHI. pBluescriptII KS+ (pBSII) was linearized using NotI and SalI. All four elements
(the upstream and downstream homology arms, the Neo cassette and pBSII) were ligated
together and transformed into chemically competent E. coli. Transformants were initially screened by whole-cell PCR and none of the clones produced amplification products of the expected sizes. Two clones each from the intron 14 and intron 15 minitargeting ligations were digested with NdeI/SalI or NdeI/BamHI (Figure 5.8). Contrary to the PCR screen, the restriction pattern for all clones was consistent with the homology arm- flanked Neo cassette. Sequencing of these plasmids further verified that each clone contained all three elements in the desired orientation.
5.3.4 Minitargeting of Introns 14 and 15
With the successful construction of the minitargeting plasmids, the subcloned
BAC in pL253 was ready for modification via recombineering. The first step was the
targeting of intron 14 with the homology arm-flanked loxP-Neo-loxP cassette (Figure
5.9A). This was accomplished by induction of the λ-prophage recombination system in empty DY380 cells, followed by co-electroporation of the NotI/SalI-excised intron 14 minitargeting cassette and the retrieval plasmid containing the subcloned BAC DNA.
Recombineered clones were selected on Kanamycin (Kan) plates. Hundreds of KanR colonies were obtained. Approximately 50 colonies were obtained from the negative
105 control transformation, in which the same DNA elements were co-electroporated into un-
induced DY380 cells. Seven isolates from the induced condition were screened by
digestion with NotI/NdeI, and the expected 13 kb and 5.2 kb bands were present in all
seven clones (Figure 5.9B). However, this digest indicated a mixed population of
untargeted and targeted plasmids. To obtain a pure population of targeted plasmids, a
small amount of DNA (500 pg) was retransformed into electrocompetent DH10B cells.
One transformant was re-screened by NotI/NdeI digestion, which indicated that a pure population of targeted plasmid had been isolated.
In order to leave a single loxP site in intron 14, it was necessary to excise the Neo cassette with Cre recombinase. Ten nanograms of the intron 14-targeted plasmid was transformed into electrocompetent EL350 cells in which Cre had been transiently induced in arabinose-containing LB medium. Transformants were selected on LB-Amp plates and four isolates were screened by digestion with NotI/NdeI. Three of the four clones exhibited a shift in the high molecular weight fragment from 13 kb to 11.2 kb, indicating excision of the DNA between the two loxP sites in intron 14. Sequencing of these plasmids verified the presence of a single NdeI-marked loxP site in intron 14 (Figure
5.10).
The final step in generating the Hydin conditional targeting construct was the
targeting of intron 15 with the homology arm-flanked frt-Neo-frt-loxP cassette (Figure
5.11A). This procedure was performed in a manner similar to the targeting of intron 14.
Induced DY380 cells were co-electroporated with NotI/SalI-excised intron 15
minitargeting cassette and the intron 14-targeted BAC subclone. Several KanR isolates
106 were screened by digestion with NotI/NdeI, and the majority of these clones exhibited the
9.4, 5.2 and 3.7 kb fragments expected from the desired targeting of intron 15 (Figure
5.11B). As before, a mixed population of targeted and untargeted plasmids was obtained.
Retransformation of these plasmids resulted in the isolation of pure intron 15-targeted clones. One clone, designated Hydin CKO, was used for gene targeting in ES cells.
5.3.5 ES Cell Targeting and Screening
After linearization with NotI, the Hydin CKO plasmid was used for gene targeting
in ES cells. ES cells were targeted and cultured in the laboratory of Dr. Michael
Weinstein. After electroporation of the targeting construct, ES cells were cultured in ES cell medium supplemented with G418 and gancyclovir. Approximately 150 ES cell colonies were picked, 105 of which grew well enough for screening by southern analysis.
Our primary southern strategy for screening ES cells was to utilize the targeted allele-specific NdeI sites flanking exon 15. While NdeI is a suitable enzyme for this purpose, we decided to use BglII, a very efficient enzyme for digestion of crude DNA samples, as an initial screen for homologous recombination at the 3’ end. Hybridization of the 3’ Southern probe to BglII-digested DNA would reveal a shift in size from 5 kb to
6.9 kb in the wild-type and targeted alleles, respectively (Figure 5.3). Two of the 105 ES cell clones, clones 11 and 24, exhibited the 5 kb/6.9 kb doublet expected if the desired targeting event occurred. Clones 11 and 24 were subsequently digested with NdeI and screened using the 5’ and 3’ probes (Figure 5.12). Both clones exhibited the wild-type
13.8 kb fragment in addition to the smaller targeted fragment (7.1 kb and 4.9 kb for the 5’ and 3’ probes, respectively). These results indicated that clones 11 and 24 carried the
107 desired targeted allele for Hydin exon 15.
5.4 Discussion
Here, we have reported the successful generation of a conditional targeting construct for Hydin exon 15 using a recombineering-based approach. In addition, this construct was suitable for the targeting of Hydin exon 15 in ES cells. Currently, mice carrying the floxed exon 15 allele have not been generated. Therefore, it is unknown whether the targeting of exon 15 is sufficient to create a Hydin null allele. Our targeting strategy, while carefully considered, can only be validated after breeding mice homozygous for the floxed allele to Cre-expressing lines.
For reasons unknown, the ES cell targeting efficiency was sub-optimal (1.9%).
One limitation to the recombineering approach reported here is that the length of the homology arms for ES cell targeting is restricted by the amount of BAC DNA that can be retrieved into the pBluescript-based pL253 plasmid. The construct used to target exon 15 contained 5.2 kb and 2.2 kb of homology at the 5’ and 3’ ends, respectively. While these homology arms were capable of generating targeted ES cells, it is likely that additional homology at both ends of the construct would have resulted in a higher targeting efficiency (105).
A potential improvement to the approach reported here would be to make the desired modifications (minitargeting of introns with the desired loxP-containing elements) in the intact BAC clone. This would allow the entire BAC, with homology arms on the order of 50 kb or greater, to be used as the ES cell targeting construct. A recent report, in which entire BAC clones were used as targeting constructs,
108 demonstrated a dramatic improvement in ES cell targeting efficiency (105). Using this
method with our floxed allele, selection for targeted ES cells would rely solely on G418
resistance in the absence of an additional recombineering step in which a TK negative
selection cassette is inserted into the BAC vector.
The rate-limiting step of the described recombineering-based approach was the
cloning of the intron 14 and intron 15 minitargeting plasmids. The ligation reactions
required to generate these plasmids were cumbersome, as they involved four distinct
DNA elements (two homology arms, the Neo cassette and the vector). A potential
improvement to this method would be to use PCR-amplified Neo cassettes as the
minitargeting constructs. In this case, the primers used for amplification of the Neo
cassette would include 50 bp overhangs at their 5’ ends as the homology arms for minitargeting. The 3’ ends of these primers would be specific to the sequences at the boundaries of the Neo cassettes in plasmids pL452 and pL451. Our laboratory has previously demonstrated that 50 bp arms are sufficient for recombinogenic cloning. As these PCR-amplified Neo cassettes could be generated in several hours, their use could dramatically decrease the time it takes to make the desired modifications in the BAC
DNA.
To investigate the function of Hydin in vivo, we plan to use several different Cre-
expressing lines. First, to determine the functional role of Hydin during spermatogenesis
and in mature sperm, we plan to cross Hydinflox/flox mice to mice carrying the PrP-Cre28.8
transgene (106). PrP-Cre28.8 mice express an estrogen receptor-Cre fusion protein
specifically in spermatagonia and spermatocytes. The fusion protein is confined to the
109 cytosol in the absence of the estrogen analog tamoxifen. When PrP-Cre28.8 mice are
supplemented with tamoxifen, the fusion protein translocates to the nucleus where the
Cre recombinase efficiently excises loxP-flanked sequences. These mice will be a
valuable tool for answering questions regarding spermatocyte development, sperm
motility and fertility status in Hydin homozygous mutants.
It is currently unknown whether hydrocephalus in hy3 and OVE459 mice is
developmental in origin. The absence of Hydin function prior to the onset of
hydrocephalus (during the establishment of the ependymal cell lineage, for example) may
initiate a cascade of events that leads to compromised CSF homeostasis postnatally. It is
equally plausible that Hydin is not required early in development, but rather serves an essential role in CSF homeostasis that must be maintained throughout the life of the
animal. A second Cre line of interest, the ubiquitous tamoxefin-inducible Cre line
CAGGCre-ER (107), will allow the role of Hydin in the adult animal to be determined.
The CAGGCre-ER mice express a tamoxefin-inducible Cre fusion protein similar to that
described for the PrP-Cre28.8 transgene. The CAGGCre-ER transgene is expressed in the
vast majority of cellular compartments, including those of the brain and respiratory
system. It will be important to determine whether the inducible inactivation of Hydin in the adult animal results in the onset and progression of hydrocephalus. Such a result
would indicate that Hydin function in the mature ependyma is essential and must be
maintained throughout the life of the animal. Inducible inactivation will also be useful in assessing Hydin function in other ciliated cell-types in which Hydin is normally expressed.
110 A
(Adapted from Copeland et. al., 2001) B
(Adapted from Lee et. al., 2001)
Figure 5.1 The λ-prophage recombination system and DH10B-based recombineering strains (5.1A) The genetic organization of the λ-recombination system includes the genes gam, bet and exo. Gam inhibits RecBCD activity of the host cell. Exo degrades linear double-stranded DNA in a 5’ to 3’ manner, leaving single stranded 3’ ends. Beta binds to the 3’ ends generated by Exo and anneals them to homologous strands in the target molecule. (5.1B) The recombineering strains DY380, EL250 and EL350 (red border) contain the λ-recombination system (blue border) and tetracycline-resistance, arabinose- inducible Flpe and arabinose-inducible Cre cassettes (green border), respectively.
111 Figure 5.2 The completed Hydin exon 15 targeting plasmid An 11 kb region encompassing exon 15 was retrieved from a BAC clone into plasmid pL253, which contains the MC1 TK negative selection cassette. A single loxP site was inserted into intron 14 via recombineering of a loxP-Neo-loxP cassette and subsequent excision of Neo by Cre recombinase. An frt-Neo-frt- loxP cassette was inserted into intron 15 via recombineering, completing the construction of the targeting plasmid.
112 Figure 5.3 Southern strategy for ES cell screening The relevant exons in the wild-type allele (top) are encompassed by a 13.8 kb NdeI site. In correctly targeted ES cells (bottom), the 5’ and 3’ NdeI fragments decrease in size to 7.1 and 4.9 kb, respectively. The relevant BglII sites for screening of the 3’ end are also indicated. Addition of the frt-Neo-frt-loxP cassette in intron 15 shifts the size of the BglII fragment from 5 kb to 6.9 kb.
113 A 5’ Probe Exon 15 Probe 3’ Probe
B 228P8 193L17 21B7 C t I u t c o n N u 500 bp
400 bp
13 15 18 13 15 18 13 15 18 194 kb
7 7 1 1 145 kb L B L B D 3 3 V 9 V 9 F 1 F 1
13.8 kb
5’ probe 3’ probe
Figure 5.4. Identification and characterization of BAC 193L17. (A) Screening of RPCI-22(129S6/SvEvTac) BAC clones by hybridization to 5’-, exon15- and 3’-specific probes. Red boxes indicate BAC 193L17. (B) PCR screening of BAC 193L17 to determine whether this BAC contains the entire region to be subcloned by gap repair. PCR products specific to exons 13, 15 and 18 are indicated by the arrows, and are 406, 408 and 423 bp, respectively. BACs 228P8 and 21B7 serve as negative and positive controls, respectively. (C) Pulse-field gel electrophoresis of NotI-digested and uncut BAC 193L17, indicating an approximate insert size of 180 kb. (D) Southern blot of NdeI-digested genomic (FVB) and BAC 193L17 DNA probed with the 5’ (left panel) and 3’ (right panel) probes to be used for ES cell screening. The signal corresponding to 13.8 kb represents the wild-type NdeI fragment encompassing the targeting locus, confirming the presence of this entire fragment on BAC 193L17.
114 BAC Clone 5' Probe Ex15 Probe 3' Probe 74O8 193L17 228P8 261I9 282F16 340N16 350E13 381G4 397H19 404L14 536B19 538I10 541G15 549B20 549O7 566H10 569F24 569P17
Table 5.1. Summary of 129 BAC Library Screening Eighteen RPCI-22(129S6/SvEvTac) BAC clones are listed in the left column in numerical order from top to bottom. Positive hybridization to the 5 prime, exon 15 or 3 prime probe is indicated by the grey shading.
115 Figure 5.5 Retrieval of the targeting region from BAC 193L17 into plasmid pL253 The 5’ and 3’ homology arms for retrieval were PCR amplified using primers containing the indicated restriction sites. After restriction digestion of the PCR products, both arms were ligated into NotI/SpeI-digested pL253, generating the retrieval plasmid. To retrieve the relevant portion of genomic DNA, the retrieval plasmid was digested with HindIII and transformed into induced EL350 cells containing BAC193L17.
116 Figure 5.6 Confirmation of clones harboring the retrieved targeting locus Four AmpR clones were screened by digestion with NotI and SpeI (blue) to determine whether the 11 kb portion of BAC 193L17 had been retrieved into plasmid pL253. All four clones exhibited the predicted restriction digest pattern. The 202 bp fragment is produced by the two closely situated SpeI sites upstream of exon 15.
117 Figure 5.7 Minitargeting Plasmid Construction 5’ (purple) and 3’ (blue) intron 14-specific homology arms were PCR amplified and digested with NotI/EcoRI and BamHI/SalI, respectively. The homology arms were ligated simultaneously to the EcoRI/BamHI-excised Neo cassette and NotI/SalI- linearized pBluescriptII (pBSII) vector. Construction of the intron 15 minitargeting plasmid was performed in the same manner.
118 Figure 5.8 Minitargeting Plasmid Screen (Right) A map of the intron 14 minitargeting plasmid, which is also representative of the intron 15 minitargeting plasmid. (Left) Two minitargeting clones for intron 14 and intron 15 were double-digested with NdeI/SalI or NdeI/BamHI. The NdeI/SalI digest results in two bands of 3.2 kb and 2.2 kb. The NdeI/BamHI digest results in two bands of 3.5 and 1.9 kb. Both clones for each intron exhibited fragments of the expected sizes, indicating that the orientation of the three elements ligated into pBSII was correct.
119 A
B
Figure 5.9 Intron 14 Minitargeting (A) The floxed Neo cassette is targeted to intron 14 via homology arm-mediated recombineering. (B) Confirmation of intron 14-targeted clones by digestion with NotI and NdeI (red text). The diagnostic 5.2 kb fragment was present in all seven clones analyzed. The 13 kb fragment specific to the targeted DNA lies just beneath the NotI- linearized contaminating untargeted plasmid (16.4 kb). Retransformation of this mixed population yielded a pure population of targeted plasmid.
120 Figure 5.10 Confirmation of Cre-mediated excision of Neo in intron 14 by sequence analysis Sequencing of a putative intron 14-targeted clone using a genomic primer upstream of the 5’ homology arm revealed the presence of a single NdeI-marked loxP site (red) in intron 14 (yellow). The region immediately flanking the loxP site (black) is residual sequence from the loxP-Neo-loxP cassette following Cre- mediated recombination.
121 A
B
Figure 5.11 Intron 15 Minitargeting and completion of the Hydin CKO plasmid (A) The frt-Neo-frt-loxP cassette is targeted to intron 15 via homology arm- mediated recombineering. (B) Confirmation of intron 15-targeted clones by digestion with NotI and NdeI (red). The diagnostic 3.7 kb NdeI restriction fragment was present in the two clones analyzed (lanes 3 and 4). The intron 14 targeted clone after Cre treatment (lane 1) served as the negative control.
122 Figure 5.12 Confirmation of targeted ES cell clones by southern hybridization (Right Panel) Hybridization of the 5’ probe revealed the presence of the targeted allele in ES cell clones 11 and 24 digested with NdeI (see Figure 5.3 for the restriction map). Clone 25 serves as a negative control, as it was determined to be untargeted in the first southern screen. (Left Panel) Hybridization of the 3’ probe revealed the presence of the targeted allele in ES cell clones 11 and 24 digested with NdeI and BglII.
123 CHAPTER 6
CONCLUSIONS
6.1. Hydin encompasses the OVE459 transgene insertion site
The initial objective of this project was to identify expressed sequences near the
OVE459 transgene insertion site, as we hypothesized that genes nearest the transgene are
most likely to be causative for hydrocephalus in OVE459 homozygotes. We were successful in this endeavor. Direct cDNA selection identified exons from two novel genes, Hydin and Vac14. The cDNA selection experiment advanced the project beyond a positional cloning phase to a candidate gene approach to identify the hydrocephalus- inducing mutation.
It was plausible that the expression of Vac14, beginning just 12.7 kb distal to the transgene array, was disrupted by the insertion event. The transgene is located in a region where putative Vac14 regulatory elements may be situated. Such elements have yet to be
124 characterized. However, quantification of Vac14 mRNA by northern analysis revealed no differences in transcription levels between wild-type and either homozygous mutant line.
The presence of Vac14 ESTs from nearly every tissue suggests that this gene is ubiquitously expressed. For this reason, along with its notable conservation in yeast, we predict that null mutations in Vac14 will result in a broad spectrum of phenotypic abnormalities (and likely embryonic lethality) not observed in OVE459 or hy3 homozygous mice.
Our characterization of Hydin began with the identification of selected cDNA 17 and ended more than three years later when the 5 prime end of this gene was determined by RACE. Hydin is extraordinarily large, consisting of 87 exons and covering more than
340 kb in the mouse genome. The full-length Hydin transcript is 15.9 kb and encodes a putative 5099 amino acid protein. Despite its size, the primary amino acid sequence of
Hydin offers few clues concerning its function. The lack of conserved domains, with the exception of modest homology to caldesmon near the middle of the protein, indicates that
Hydin has a unique evolutionary origin. It is notable that a Hydin homologue is present in
the biflagellated single-celled alga Chlamydomonas reinhardtii. Such evolutionary conservation indicates that Hydin is an ancient gene. We hypothesize that the appearance of Hydin is coincident with the appearance of organisms possessing 9+2 cilia. Hydin homologues are not present in sequenced organisms that lack 9+2 cilia, such as
Drosophila melanogaster and Caenorhabditis elegans.
Hydin became our primary candidate gene once we discovered that exons from
this gene are situated within 2.5 kb of the OVE459 transgene array. We subsequently
125 determined that the transgene inserted within Hydin, causing a complex chromosomal rearrangement that disrupts the order of Hydin exons in the transgenic allele. The complexity of the genomic rearrangement is evident from overlapping RT-PCR amplification of the entire Hydin transcript from homozygous OVE459 mice. Despite
this, the utility of the transgene as a molecular tag for the gene most likely disrupted in
these mice was invaluable.
The order and orientation of genes surrounding Hydin on mouse chromosome 8 is
conserved on human chromosome 16q22. Interestingly, a human fibroblast cell line
isolated from a hydrocephalic patient was shown to carry a (4;16)(q35;q22.1) translocation (108). In an attempt to define the translocation breakpoint, Sakuragawa and
Yokoyama detected a rearranged 1.2 Mb NotI fragment using a calretinin (CALB2)-
specific probe (109). Based on this information, these authors suggested that the human
homologue of the gene disrupted by the hy3 mutation would lie within 1.2 Mb of
calretinin. In agreement with this prediction, the current public mouse and human
genome assemblies place Hydin 99 kb and 160 kb from calretinin in the mouse and
human genomes, respectively (Figure 2.17). A relationship between HYDIN and human
hydrocephalus remains to be established.
6.2 Hydin is expressed specifically in compartments responsible for CSF homeostasis
and cell types that contain 9+2 cilia or flagella
Direct cDNA selection provided the first evidence for Hydin expression in the
brain. Selected cDNAs derived from neonatal head RNA indicated that Hydin is
expressed in the head at a time coincident with the onset of hydrocephalus in OVE459
126 and hy3 homozygotes. P0 brain RNA was subsequently used to amplify all portions of the Hydin transcript. However, prior to expression pattern analysis by in situ hybridization, the specific compartments within the head that express Hydin were not known.
Our results showing Hydin expression in the choroid plexus and ependymal layer lining the cerebral ventricles strengthened our hypothesis that Hydin is the relevant hydrocephalus-inducing gene. The choroid plexus is responsible for producing approximately 80% of the total volume of CSF in the brain, while the barrier function of the ependymal layer limits the rate of CSF entering the ventricular lumen from the brain parenchyma. Hydin expression in the brain is exclusive to these compartments. Multiple in situ hybridizations were carried out in which the layers between the brain and skull were examined for Hydin expression, as it was suggested in earlier papers that defects in
CSF reabsorption in the subarachnoid space causes hydrocephalus in hy3 homozygotes
(63). We did not detect Hydin messages in this compartment, calling into question the hypothesis concerning defective reabsorption in these mice. Our findings are consistent with targeted mutations in E2F5. E2F5 is highly expressed in the choroid plexus and ependymal layer, and loss of E2F5 results in lethal communicating hydrocephalus. The pathogenic mechanism in the E2F5 mice, as with Hydin mutants, remains to be elucidated. However, both mutations result in communicating hydrocephalus that does not likely involve obstruction between the ventricles or reabsorption defects. Rather, CSF overproduction by the choroid plexus or hindered CSF flow (possibly due to ependymal failure) appear to be the primary culprits. Additionally, the barrier function of the
127 ependyma may be compromised in these mice, resulting in an excess volume of fluid entering the ventricular lumen from the brain parenchyma.
The expression pattern of Hydin strongly suggests that this gene plays a fundamental role in the function or maintenance of 9+2 cilia and flagella. In the central nervous system and beyond, Hydin is expressed exclusively in cell types that possess these structures. This finding is consistent with other mouse mutations where cilia dysfunction and hydrocephalus are clearly linked (37-41). In light of these other mouse mutations, the cilia- and flagella-specific expression pattern of Hydin further pointed towards this gene as the underlying cause of hydrocephalus in OVE459 and hy3 mice.
6.3 Hydin mRNA levels are reduced in OVE459 and hy3 mice, and the hy3 allele of
Hydin contains a frameshift mutation in exon 15
The transgene-induced genomic rearrangement and expression pattern of Hydin provided strong circumstantial evidence that its disruption is causative for hydrocephalus in OVE459 and hy3 homozygotes. However, the definitive evidence came from quantitative analysis of Hydin mRNA levels in wild-type and mutant mice, as well as sequencing the hy3 allele of Hydin. Northern analysis indicated that Hydin mRNA transcribed from either the OVE459 or hy3 mutant allele is unstable, resulting in markedly lower levels compared to wild-type littermates. The stability of the message, rather than a block in transcription, causes this reduction because every portion of the full-length Hydin transcript is detected in homozygous mutants by standard RT-PCR.
The disrupted order of Hydin exons in the OVE459 allele is likely to blame for destabilizing the transcript. Due to its apparent complexity, and the availability of the hy3
128 mice, we chose not to fully characterize the OVE459 rearrangement. Rather, we chose to
sequence each exon from hy3 homozygous genomic DNA to search for mutations that disrupt Hydin expression. This search was fruitful, as we identified a CG base-pair deletion in exon 15 that shifts the reading frame and causes a premature termination signal two codons downstream of the mutation. The stop codon created by this deletion is presumed to have a devastating effect on the Hydin gene product, as 89% of the open reading frame lies downstream of this codon.
Northern analysis on wild-type testis RNA using a cocktail of two Hydin-specific probes indicated that alternative splicing of Hydin mRNA occurs (Figure 2.12). The exon composition of each splice variant has not been determined. If exon 15 is present in each
Hydin isoform, we are confident that the hy3 allele is a null allele. The importance of exon 15 will certainly be established in our mice carrying a conditional exon 15 allele.
6.4 ES cells harboring a conditional Hydin allele have been generated
Despite having definitive proof that mutations in Hydin are responsible for hydrocephalus in OVE459 and hy3 mice, the function of Hydin is currently unknown. We
have circumstantial evidence that Hydin plays a role in 9+2 cilia/flagella function or
maintenance. Hydin is expressed in a variety of ciliated (and flagellated) compartments
outside of the brain, some of which are more amenable for functional analysis than the
ependyma. However, the lethality of OVE459 and hy3 homozygotes prior to weaning age
makes it difficult to assess the phenotypic consequences of Hydin disruptions in these
other tissues. Such phenotypes may only be apparent later in life. For instance, the
OVE459 and hy3 mutations may lead to male infertility. Currently, we cannot address the
129 fertility status of Hydin mutants because they do not reach sexual maturity. In addition,
cultured tracheal epithelium constitutes an excellent system for studying cilia motility
and signaling. Attempts by experienced investigators with this system to culture hy3
tracheal epithelia have failed due to the immaturity of the mice from which the cells were
derived (Scott Randell, personal communication). For these and other reasons, we chose
to generate a conditional allele of Hydin to permit loss of Hydin function in specific
ciliated/flagellated compartments.
A recombineering-based approach was successfully employed to generate the
conditional targeting construct. The efficacy of this method was two-fold. First, it
enabled us to precisely choose the intronic regions flanking exon 15 where the loxP sites
and Neo cassette were situated. This method obviates the requirement for appropriate restriction enzyme sites in the introns to be targeted. Second, this approach allows the construct to be generated in less time than traditional cut and paste methods.
Southern analysis of 105 ES cell clones indicated that two clones, 11 and 24, were correctly targeted at Hydin exon 15. Both clones have been injected into blastocysts.
Clone 24 has failed to yield high percentage chimeras. Injection of clone 11, however, has resulted in several males exhibiting greater than 70% chimerism. We are hopeful that these mice will pass the conditional allele through the germline so that our planned functional studies of Hydin can proceed.
6.5 Future investigations concerning Hydin function
Hydin’s expression pattern suggests a specific functional role in cell types that
possess 9+2 cilia and flagella. The precise cellular function of the Hydin protein remains
130 to be elucidated. Several lines of investigation have been chosen to characterize Hydin
function. As previously discussed in Chapter 5, the breeding of mice carrying a
conditional Hydin allele to various Cre lines will allow Hydin loss-of-function
phenotypes in specific cellular compartments to be assessed.
A critical piece of information regarding Hydin’s function will be its location in
the cell. We predict that Hydin functions in cilia/flagella formation, motility or
maintenance. If this prediction is true, Hydin will likely reside within the ciliary axoneme
or at the base of the cilium near the apical surface of the epithelium. Apical localization
of basal bodies, from which axonemal microtubule filaments of cilia are nucleated, is
accomplished via anchoring to the apical actin cytoskeleton. In Foxj1 null mice, where
cilia are completely absent in epithelia that is normally ciliated, basal bodies are
randomly distributed throughout the cell (110). The loss of apical basal body localization
in Foxj1 null epithelia is the likely cause for the absence of cilia in these mice.
A recent report by Gomperts et al. reveals a mechanism by which Foxj1 regulates
the anchoring of basal bodies to the apical cytoskeleton in ciliated pulmonary epithelia
cells (111). The expression of calpastatin, an inhibitor of the protease calpain, is down- regulated in Foxj1 homozygous mutants. Ezrin, a target for proteolysis by calpain, was found to localize to the basal bodies of cilia in wild-type epithelial cells. In the absence of
Foxj1, calpastatin mRNA and protein is reduced, calpain levels remain unchanged, ezrin protein levels are diminished and apical localization of basal bodies is disrupted. When cultured Foxj1 null epithelial cells were treated with a calpain inhibitor, ezrin levels
131 returned to near wild-type levels and apical localization of basal bodies was restored.
Cilia reappeared on Foxj1 null pulmonary epithelium treated with the calpain inhibitor.
Ezrin is one of the founding members of the ezrin/radixin/moesin (ERM) family
of proteins. ERM proteins act as linkers between the F-actin cytoskeleton and apical membrane proteins (112). Interestingly, a 262 amino acid region (residues 2284-2545) of the predicted Hydin protein exhibits significant sequence similarity (>66%) to the canonical FERM domain, which resides at the N-terminus of most ERM proteins (112).
The FERM domain is the site at which ERM proteins interact with apical membrane-
associated proteins. A second founding member of the ERM family, radixin, was recently
found to localize to the base of stereocilia in the inner ear (113). A specific role for
radixin in anchoring actin filaments of the stereocilium to the apical membrane has been
proposed.
As discussed in chapter 2, Hydin also exhibits amino acid similarity to the actin-
binding protein caldesmon. The sequence similarity between Hydin and ERM
proteins/caldesmon suggests that Hydin interacts with the cytoskeleton. The recent
findings that ezrin and radixin are associated with apical localization/anchoring of cilia
and stereocilia, respectively, leads to the current hypothesis that Hydin plays a role in
anchoring the basal body to the apical cytoskeleton. It is unlikely that a single protein is
sufficient for basal body anchoring, as more than 250 proteins are implicated in the
formation, function and maintenance of cilia (114). Localization of Hydin protein to the
basal body would lend support to the hypothesis concerning Hydin and apical anchoring
of the basal body. Congenital hydrocephalus in hyh and orpk mice results from mutations
132 in the genes encoding αSNAP and Polaris, respectively (30, 31, 115). αSNAP and
Polaris are required for apical localization of proteins (30, 39), and Polaris localizes exclusively to basal bodies in ciliated epithelium. These examples indicate a clear precedence for epithelial apical-basolateral polarity defects in hydrocephalus. It is the author’s prediction that Hydin will function at a similar step in the differentiation of ciliated epithelium.
The ependymal cells of hy3 homozygous mutants possess cilia (69), and we have observed cilia on the tracheal epithelium of OVE459 homozygotes. Therefore, mutations in Hydin are not sufficient to completely abolish ciliogenesis. Loss of Hydin function may result in more subtle defects, such as diminished structural integrity and/or function of cilia.
In order to perform localization experiments, an anti-Hydin antibody suitable for immunocytochemistry must be generated. Currently, cDNAs encoding three peptides specific to the mouse Hydin protein have been cloned into expression vectors. Purified
Hydin peptides will be used to raise antibodies against mouse Hydin in rabbits. Cultured tracheal or ependymal epithelium will be stained with a suitable anti-Hydin antibody to determine its location in these cells by immunofluorescence. Higher resolution co- localization studies may be performed by immunoelectron microscopy to determine whether Hydin localizes specifically to the basal bodies of cilia. In addition, inducible
Cre lines may be used to disrupt Hydin in adult mice homozygous for the conditional
Hydin allele. Following Cre treatment, tracheal or ependymal epithelium can be analyzed
133 by electron microscopy to determine whether the basal bodies exhibit apical or random localization.
Another approach to visualize Hydin localization will utilize the single-celled,
biflagellated green alga Chlamydomonas reinhardtii. As discussed in chapter 2, we have identified a Chlamydomonas Hydin homologue. Interestingly, northern analysis revealed an increase in Chlamy Hydin expression following deflagellation (data no shown). This result suggests a role for Hydin in flagellar assembly or function. A FLAG epitope tag has been inserted via recombineering between the first and second codons of the Chlamy
Hydin gene present on BAC clone 29D10. Transformation of this modified BAC clone and subsequent anti-FLAG immuofluorescence will allow localization of the Chlamy
Hydin protein to be determined. In addition, loss-of-function studies utilizing siRNA against Chlamy Hydin transcripts should clarify the role of Hydin in this organism.
Once the cellular location of Hydin has been determined, putative interactions
between Hydin and proteins that co-localize with Hydin will be assessed. If Hydin localizes to the basal body, co-immunoprecipitation (co-IP) assays of Hydin and other proteins that localize to basal bodies, such as ezrin (111) and Polaris (39), will be performed. The region of the Hydin protein critical for protein interactions could subsequently be determined in a yeast-two hybrid system in which successive portions of
the Hydin protein are expressed along with the full-length protein previously shown to
interact with Hydin. Alternatively, plasmids expressing successive portions of the Hydin
protein fused to an epitope tag could be co-transfected into cultured cells with plasmids
expressing putative interacting proteins, followed by co-immunoprecipitation.
134
APPENDIX
Table A.1. Mouse Hydin RT-PCR primers.
product primer set size (bp) TA Sequence ACA AAT AAA AGG CTA GAG GAG TC 5 Prime-F1/R1 536 54.2 CAG GTC AAC ATG TGG GCG TAA TC CAC GGA TGG TAA AAG TTG TTG AA 5 Prime-F2/R2 544 55.7 GTT GGC CAG AGA TAT GTA GGT TTT CCT GCA TCA CCG AAA GAG AAA AGT 5 Prime-F3/R3 515 56.2 GAG TTG GGC CAG ACA TCA CCT CAA AGA CTC CAT GTT CCA CCT C 5 Prime-F4/R4 534 53.6 CTT TGC CAA TGT CCA GTA AT TGT CTG GCC CAA CTC ATC 5 Prime-F5/R5 400 53.1 GAC ATC AAT CAG GAA CTC TT CTT GGG GGC CTG CTT TGT CTT C 5 Prime-F6/R6 406 57.1 TTT GGG TTT TGT GGT AGG CAT TTC TGG CGA AGG CAT GAG TAG CA 5 Prime-F7/R7 541 56.5 CCC AAA GAC GGA GAT GTA AAC AGC CCC TCC CCC TGT GG 5 Prime-F8/R8 575 57.9 ACT CGG CGT CCT TTG TTG GTC TGA CAT CCC ACT GAC ACT GAC TGC 5 Prime-F9/R9 412 58.6 TCC CGG GTA CAA CTC CAT CCT GAC 5 Prime- CCA GTA GCC GTC GCC AGT CCA 518 55.8 F10/R10 TGC CAC CCA GTT GTT CAG 5 Prime- TCC CTA CTG CCT GCA ACC CCT AAG 572 56.7 F11/R11 CCC GCG GAG CTG TAT TTT 5 prime 1a/1b 141 58.9 ACG CTT GCT TGG CTC CTT TAG TGG 135
GTG GAT GCC GCG GGC TTT TTG ATT AGC CCT ACA GCG CCT TCT TG new Ex2-1a/1b 466 58 GAC GCC TCC CCC TTC AGT AGT GGC CCT TCC GAG AGT GT 5 prime 2a/2b 517 55.9 CTT TGG CCC GGG CGA TGA TGT AAT CAA AAA GCC CGC GAC ATC CAC 5 prime 3a/3b 558 56 TCA GGT ATT CCA GGC AAG TAG TAA GTA CCA CTT ATC CTG CCT CTG T 5 prime 4a/4b 405 53.8 GAT GCT TCT CTG GTG TTC C ATA TGA GGT GAT GGC TGA GGA ACT 5 prime 5a/5b 559 58 CAC CAT GCA CTG CCC ACA CTG AAT GTG GGG AAC AAA GAA GTC ATT 5 prime 6a/6b 443 56 TGG GCC AGA AGT GTC AGC CCG GCG AGA GGG CTA ATG TGC 5 prime 7a/7b 496 59.7 TTT GTG CTG GTG GTT CCT TGG TCA TGC GAC AGC TGA AGG GCT ACG A hy2b-1a/1b 495 56.5 TCC CGT GGA CGA TAA TGG CAG GGG AGA AGC TGC CAC CAG hy2b-2a/2b 624 57.9 CTG ACT CCT CTG CTT CCC TCA T TGA AGA AAT TGA AAG AGG AGC ACA T hy2b-3a/3b 487 54.3 AGG GCT TCT AAG ACG ATG GAG TCA A AGC CTG CCT GAA CAT TGA CTC hy2b-5a/6b 428 57.4 GGT CTC CCC GCC AAT ACT G TCT AAT AAC CCA GGA ATA CAA AGT hy2b-8a/8b 422 58.5 TGA CGA CAT AAT CCT GAG ACA T hy7-1a/hy2b- GGG GAG ACC GGG CTG ATG AG 674 56.5 5b ATG GCT TTC TTC TTC CTG GTG GTT ATG TCT CAG GAT TAT GTC GTC hy7-3a/3b 560 53.9 GCC TGG CGC TGG TCG TC GGA CGA GTA TGA CGC CCT AAC hy2b-9a/9b 555 56.3 ATC TCG TCC GTG CCT GTA TGA AAG AAG AAA GCC ATG ACC GAA TAC hy2b10a/10b 599 57.9 TAA GAG ATG ATG GAG AAC AAG ACG hy7-2a/2b 472 58.5 GAC GTG GAA GGG GAA GGG GAA GA 136
TGC TGG AGT AAG TGC GTC GTT TTT
CCG CAA GCA AGG GAT GAT GGT A C2-1a/1b new 454 60.8 TTC CCG CAC TTG CTC TGG TTT GTC AGA CCA GGC TCA AGA GGA ACA AC C2-2a/2b 545 56.5 TGC CAG GGA ACA AGG ATG ACT CAC CTA CCC ATA CAT CTG C C2-3a/3b 503 52.9 TTA CTA TCT TGG TTT CTT TCC TAT GGA GCC CAG GCA GTT ACA TTT C2-4a/4b 430 56.5 CTC ATA TTT TCC ACG GTT TTT CAG GAA AAT CTT GTT GGT GTC GT C2-5a/5b 452 54.3 GGG CTG ATG GTG AAT TTG GAG TAT TGC GCT GCC AGA TTA TTG A C2-6a/6b 530 56.5 GGC AGG ATT TTC TCT AGG GTC TCG GCA AAC CAG GCC CGC TTC A C2-7a/7b 565 59.7 GGT AGG TCT GCA TGG TCT GTG GTG AGC CGC TCA CAT GCC TTC GTC C2-8a/8b 448 55.6 CAT CAA ATT CAG CCG TGT C ACT GCC TCT CAT CCT TAA G C2-9a/9b 480 52.8 GTG AAG CTC ACA TTG TAG CTG TGA CAG GCA CCA TCC ACT C2-10a/10b 583 54.9 GGC TCA GGG TCC ATC TCT GAT GCT CCC CGA AAT ACA C2-11a/11b 586 54.4 TCA GGT CAA AGT GGC AGA A GAG AAC AAG GTC CTA TTT TG C2-12a/12b 385 52.3 AGT CCA GAA CTC TTC CGT G CTC TCG AAC CTA ATA CCC GTG TGA C2-13a/13b 479 55.6 AAT GAG GCT GTT GTT GAA TCC AGA CCA CAG ACC CCC TTA TC C2-14a/14b 566 54.8 ACC CAC GTC TAC GCT ACT GT TCT ACG AGG TGG AGT TGA AT 3 prime 1a/1b 431 53.6 GTG TGG GGT GTT GGT GTA GCC TCC ATA TAA GCA AAT CC 3 prime 2a/2b 491 54.8 TGG GGG CAA AGG TAA TC 3 prime 3a/3b 445 54.2 GAG CCC TTC CAC ACG AT
137
TGA GGC ACA GAG GAT TTC
ATA AAA ACA TTC TCT GCT TCA TCC 3 prime 4a/4b 510 56.4 GGT CTG TCT TCT CTG GTT TCA ATA GCA ACT GGC TGA ACA AG 3 prime 5a/5b 561 53.6 GAA CGG TCT GGA AGT GGA AAG CCC AGT CCG CCA GAG TGT AT 3 prime 6a/6b 549 58.7 GGG GGC AGA GCA ATT CCA AAG A TTA TTA ACG CAG CCC CAG GAG 3 prime 7a/7b 480 57.2 GAA GGC CGC CAG TTG ACA CAG T
138 Table A.2. Human Hydin RT-PCR Primers.
Primer Sequence TA Size human RT-F1 GGGCGGCGCATGGAGAGTG 57.8 468 human RT-R1 CTGATGGAAAGGGCTGGAATAATG human RT-F2 TGACCACCGAGCAGAGACT 54.1 512 human RT-R2 GCCAATGTTTCGTACCAGCAGA human RT-F3 CGCCCATACGTTGACCTGTGTTAC 55.2 526 human RT-R3 CTATATTTTTCTCTGTCCTC human RT-F4 GGAGTTTGAGCCACAGAGT 54.1 577 human RT-R4 GATTCGGAGGGGCAGACGGATTT human RT-F5' GTGCATTACTGATCCTTTACTC 53.9 586 human RT-R5' TCCAATGACACAGCCTCTA human RT-F6 AAGAGTTCCTGGTCAATGTCAAT 55.9 511 human RT-R6 TGCCCAAAGTCCACCTCTGT human RT-F7 TATTGTGAGCAGCATGTGGACTAC 57.8 416 human RT-R7 CTGGGGGTGGGGCTGGAAAAC human RT-F8 GTGACATTATGCTCCAAC 55.2 594 human RT-R8 CTTCATTGGGTTCAATCGTCC human RT-F9 GACTCTTCCAGGATTCTCAACC 56.6 600 human RT-R9 CACCAGCTTCTCTTTCACTAT human RT-F10 CCAGGCCAGGCAATTGATGTGATA 57.9 598 human RT-R10 ACGGACTGCAGTTGGTGATGGTAA human RT-F11 ATTTTGGCTGCATCCTGAACG 55.7 527 human RT-R11 GGTGAACGATATTTGGTGGCTACT human RT-F12 GTGTTTGATATTTTGCCCCTGTTT 56.8 577 human RT-R12 TGTCTGTGTTTTTCCTGGCTTGAT human RT-F13 CGATCTCCCCAGGAACCTCACAG 54.8 493 human RT-R13 TGGGTCAAATCTCACTTCAAAT human RT-F14 ACATCCTGGACTTTGGCTACATCA 56.8 431 human RT-R14 AGGCTTTTGGCTCTGGACGAACC human RT-F15 GGAGGGCCAACAGTTCACATCT 57.9 505 human RT-R15 TCACTATCACCTCGGCCTCGTCTC human RT-F16 GAAATTCTACAGCCAAACC 53.4 425 human RT-R16 TCTGATGAGGTTATATCTTCCTCT human RT-F17 CAAAAGAGGAGCAGATGAG 53.9 444 human RT-R17 AGTACTTGGCCACGCTAACG human RT-F18' CCACCTGGGCATTGACATTTC 59.2 543 human RT-R18' CCGACACTGGGACTAACACTGAG human RT-F19 AAGAGTGTTCGTGGGAGCGTGGTG 60 535 human RT-R19 TCCCCCGATCGAATGTGAGTTT human RT-F20 CCGAGGAGTGGTGTTTGAT 55.7 593
139 human RT-R20 CGCCCATGTTCCTTTTCTTCTTAT human RT-F21' GTTCCTCCGCTCACCAAAGT 58.5 486 human RT-R21' CCAGTCGCTCCGCTCCTCCAG human RT-F22 CCTGGAGGAGCGGAGCGACTG 55.7 441 human RT-R22 ATCTTTTCTTTCATTTTCTGTTTG human RT-F23 GGCCACCACAAACACTACAAAG 54.8 433 human RT-R23 CCGCTGAGGAAATACCACT human RT-F24 GCCGAGGCATCTGCACTTACCC 55.7 515 human RT-R24 GAAGAACTACCCTGGATTCCTGT human RT-F25 TGCATCAATGACAACCCAGAG 55.7 542 human RT-R25 GTTGAAATCCCTACAGAGTCC human RT-F26 AAGGGGGACTGGATTTTGGGATTG 57.9 513 human RT-R26 CTTGCATGTCTGACGTGGCTGGCT human RT-F27 TTGATCTGTGGCACTCGTAAAA 54.8 426 human RT-R27 TTCAGCTAGCAAAGTGTAAAGA human RT-F28 CTGACGCCATGGGAAAGTGTGAGG 59.2 500 human RT-R28 GGGCAAGCCATCCAAGGTAGC human RT-F29 CCCGCATCGTCGACATTTTTGAA 56.7 442 human RT-R29 CTTTTACATGTGGTTTATTTTCC human RT-F30 CTGCATGTTGACCTGCAGGA 57.5 484 human RT-R30 CTTGGCTGTGATTTGTGACTGTGA human RT-F31 CCCCCACCAGCCAGGAAGACATAA 56.7 527 human RT-R31 CACATTGGCACTGATTTGAAGC human RT-F32 AACATGCCTGGGACTTTCACTACA 58.2 558 human RT-R32 GCTCCGCCCTTTTGCTACCA human RT-F33 CCCCGTTGGACATTGGAGAC 57.5 520 human RT-R33 GAGATGAGAGGTTCGGTAGTTTTG human RT-F34 TAATTCCCGAGCACAACATCACA 55 576 human RT-R34 ATTCACACTGGACATTCATTTA human RT-F35 ACCAAAGCAGGAAGGAGATGTG 53.3 552 human RT-R35 GTAATTACCAGGGTTTGTTTG human RT-F36 ACAACTTTGGGACCTGCTTTATCT 55 450 human RT-R36 CTTGGGTTCCTGGAGTTCTGG human RT-F37 CCAGGGCAGGTTGTGAAA 56.7 498 human RT-R37 CTCGGTGGGATGGTAGGTC human RT-F38' AGCCTCATTTCTCCATTAG 54.9 535 human RT-R38' AGCTCAGTGTAGGGGGTCTT human RT-F39 ATGGGACCGGCTGGCTGTATG 57.5 420 human RT-R39 TGATGGAGGCTGACGCAACTT human RT-F40 TCTTCCGAAACGAGGTGACAAATG 59.2 556 human RT-R40 AGGTGGCTGGGCTCGAATAAGAC
140 Table A.3. Mouse Hydin exon genomic primers.
exon product exon start size primer set size TA Sequence CCA AGA GGC AGG TGT TCG 1 99609 41 99609-F/R 418 56.5 GGC CAT CTC ATC AGT CCC CAT AAG GAG ATG GCC CCT ACT GTT CAC C 2 99897 172 99897-F/R 472 57.3 ACT CGG GCG CTT GTA ATG TTC C AGG GGA TCC AAG GCT CTG T 3 130798 158 130798-F/R 538 54.5 GAC GGA AAC TCT TGC TGT AAT G AGT GCA TGG GAT TTC TCT TGT 4 132648 126 132648-F/R 493 54.5 TTA ATT TCG TGC ATA TAG GGT AGG TTT TGA GTG GCC CTT GTG TTT TT 5 141801 120 141801-F/R 449 55.1 GGG AGC TTG CCA TCT GTC G GCC CTT ACT GTG CTC TAC TCT GA 6 144874 135 144874-F/R 458 55.6 TTC TGC CAC AAT AAC AAA ACC AC TGC CTG CCA TAG CCC CTG TTT CT 7 159075 179 159075-F/R 405 60.2 CAC CCC CAC CCA CCG ACT CC ATC GGC AGG AAA ATC ATA CCA CAC 8 167431 109 167431-F/R 544 57.1 ACA TTC ACA ACC TCT GCC CAT CAT TCC TCT TCC CCA GCA CCA AAA CTC 9 187680 202 187680-F/R 493 56 GCG GGA CTA GAA GGG GTA ATG TGA CAG GGC AGG TGC TAA ATA AT 10 198110 184 198110-F/R 561 60.3 ACC GGG GGC TCG ATC GTG AAA AC CAC AGG GAC CAT TTC ACT ATC 11 213291 100 213291-F/R 403 49.7 TAT ATC CTT CTA TTG GTA TCA T ACT GTA GTT CCC TCG GTA TCG 12 225291 119 225291-F/R 524 54.1 AGA AGT TAG AAT TGA GGC AGA AAA CAA GAG TTT CCC GTG GTC ATA A 13 230956 224 230956-F/R 406 55.1 CTA GAA GGC AGA AGG GAA AAC TC CTT CTT AGA CTC TGC CAT TGA CCT 14 237298 68 237298-F/R 576 53 ACA GAT TGC CAG AGT TAG TTT C TGT TGA TCA AGG GAG GCT ACT GG 15 243454 236 243454-F/R 408 57.3 ACG GCA TTC CCT ATC ACT GTC CTT 141 TAA CTC AGC ATA CGA AAG CAT CCA 16 246224 101 246224-F/R 406 56.9 GAA TCC AGT ACT CAG GTT TAG CAG GGA TGC CCC CTC TTA TTG TTA T 17 248715 136 248715-F/R 442 52.2 CCT GCA GCT GCT CGG TCA C AGG CTT CTC TAA ATG ATG A 18 251379 165 251379-F/R 423 53.9 ATA AAG CCT TGT AAC TGT AAA AAC AAC CAG CGG GAA GGG ATG TG 19 252735 153 252735-F/R 509 57.5 CTT GGA GCT AAT GTC TTC AGT G GTG CTG GGA TTA AAG GCG TGT GC 20 279365 239 279365-F/R 484 56.9 AGA GGG ATG GAG GGG GCT TAG TTC AGC CCT TGA CTC CCT GAA TG 21 283231 277 283231-F/R 508 54.8 GGG ACC TGC CTA AGA AAC ACA TGT GTC CCT TTT CTC ATT ATT TTG 22 284969 144 284969-F/R 555 50.5 CCC TTT CCC CTT GGT CGT AG TGT GTT GAG GGT TTT GTT 23 289569 144 289569-F/R 429 55.9 CTA CTG GGC TGG AAG GT TTC AAT TCC CAG CAA CCA CAT 24 295506 314 295506-F/R 521 57.6 GGG AGA GGG AGA GGG GGA GAA AG CAA CCG GGA ATG AGA AGG AAA 25(2) 318439 138 EXON2-F/R 400 59.4 ATG TGA ACG GAC AGG GAG GGA TGG AAA GGG TGG CCA CTG GGA GAC 26(3) 319392 73 EXON3-F/R 446 61.6 TG TGG GGA GAA GCG ACC TGG ACA C GGC TCC ATT CTT ACC ATC ATA GTC 27(4) 322850 111 EXON4-F/R 404 55.3 ATA GTG TCT GGG TTT GGT GGT TGT GGA GGG AAG GGT ATA GGG AAA 28(5) 323658 210 EXON5-F/R 454 57.3 TGT AGG GAG CTG AAT GTG GAT GAA TG
NEW EX6- ATG TGG CTT AAT CGG TAT CTG A 29(6) 327360 138 536 55.3 F/R ACC GAG TAT TCC TGA GTC TAT CTA TGA ACC CGG GGC ACA CCA TC 30(7) 333710 178 EXON7-F/R 453 59 GGC AAC TGA GGC ACA ACA TAG AAT NEW EX8- CTG AGC TGA GAG TCG AGG TTT GA 31(8) 335995 128 480 55.5 F/R ACT TAA TTA TGA CGT AGC ACT TCC 32(9) 338238 131 EXON9-F/R 483 52.7 TTA AAA CTA GGA AAA AGA ATC A 142
CCT ACT GGC TGT GGT GT TGC ACC CAG AAG AGA ACA TAC NEW EX10- ACT 33(10) 338812 137 409 57.7 F/R GAG CAA GGA AAA CAG CAC ATA GAA TGT CCT TCC ACG CAG AGA 34(11) 339163 128 EXON11-F/R 465 55.4 GCC CGG CTT TTG ATA CC NEW EX12- CTA CAT GCC CAG CCA GAG A 35(12) 339497 192 422 55.1 F/R AAG GGA AGT CAG ACA TTT TAC C CTA TGC GTG GGG AAT GGA AGG 36(13) 339950 150 EXON13-F/R 403 56.1 GAA AGG TGG TGT TAC TCA GGT CTA TTG TCC CTT TTG TGG CTC TCA GTT 37(14) 341953 240 EXON14-F/R 459 56.9 CAA GTG GGG AAA TGC TAT GGA AAT CTC ATC CTC GGG TTT TT 38(15) 343905 163 EXON15-F/R 443 55.4 CAG GAC TTG AGT AGG GGT TTC TCT CCC CAT TTC CTG TTT TCT TTT 39(16) 346061 174 EXON16-F/R 452 56.9 GCA TGT GGT AGG TGG GAG GTC A CGG TGC CTG TGT CCC TAT GTA 40(17) 347183 174 EXON17-F/R 420 59.7 AAT CCC CCG CCC CCA CTC CTG TGC CCA GCT CAT CTT CAC AG 41(18) 347899 174 EXON18-F/R 447 56.3 ACT CAC ACT CAC ACG CAC ATA CAC GTC CCC CAC TGT CTC TTC CTT CAT 42(19) 352150 221 EXON19-F/R 450 58.7 ATC CAG TGC GGT GCC AGT AAT AGA ATT TAA GGA GCC TGC AAG AGT 43(20) 356141 138 EXON20-F/R 486 56.8 TTT TCA TGG GCG AGA GCG ACA A TTT GGT TTC CTG GTG TTT ATT TTG 44(21) 358513 184 EXON21-F/R 431 57.6 CAT TTG AAG CCA TTG GAT TGT TAC CAA ACT TGG CAC TAT AGA GAT NEW EX22- GAA 45(22) 360976 142 518 56.6 F/R AGG GAG CAG AGG ACA ACA GAA GAC CCC CTT TGT TTC ATT CAT TCA TTT 46(23) 364118 169 EXON23-F/R 432 58.1 TGC CCC GGG ACA GTA ACC AGC TCC CCA CAT TAC ATA CAA EXON245'- 47(24) 365745 618 496 54.7 AGT F/R GCC TGG CGC TGG TCG TCA EXON243'- CCG CAA GCA AGG GAT GAT GGT A 47(24) 365745 618 455 56.4 F/R AAA GGG CTT GTG GAT TCT 143 AGT CAG AGG AGG GCA GGG AAG ATT 48(25) 368234 222 EXON25-F/R 450 59.1 GGG CAG GAG GAC CAG AGA AAC AA CTG GCC CTA CCC TTT CCT ACG A 49(26) 371374 209 EXON26-F/R 420 56.3 CAC TAT AAC CCT GCA CTT CTC TGG GCA GGA ACA AGG GAG AGG GAG 50(27) 371788 183 EXON27-F/R 522 57.2 GAT GCA TAC GCA CAC TTG GAG GAA T GGA GAT CCC CAC ATT AGC 51(28) 372788 120 EXON28-F/R 447 52.2 GAG GGA AAG GGG AAG CCA AGT T TCA GTC AAC CTT CCA CCA CCT NEW EX29- 52(29) 374590 160 432 55.5 TAG F/R CTC CTT GCC CCT GAT AGA AT GCA TGA CAT AAA AGC AAG CAC CTA 53(30) 374865 173 EXON30-F/R 468 56.3 AGA CAA CAG CAA GCC CAG AAA ATC AAG GCA AAT TCA AGT GGA TAA 54(31) 382658 199 EXON31-F/R 473 54.9 AGA ATG GGA TGG AGA TAG GAA ACA GA AGC CCC TTT TCT TCA CC 55(32) 384342 100 EXON32-F/R 434 52.5 CCC CAC CAA CCC CAC CAT TAA TCT ATA GAG GGG AGC CAG TGC 56(33) 388601 100 EXON33-F/R 433 56.2 AGA GGG GTG TGA TTG TTG AGG AAG TAG TGG GTT TTG CTT GGT ATG A 57(34) 390065 169 EXON34-F/R 418 55.5 TTG GGA CTG AAC TCT GGT GTA G GGG GAA GCA GGC AAG AAG 58(35) 390698 236 EXON35-F/R 408 51.6 CTG GGT GAA TTT AGT TAG GTA AGA AGA AAG GAC CTC AAG CAG 59(36) 394092 118 EXON36-F/R 424 54.9 TGT GTA GCC AGC CTC AAT CAA ATG TT ACC GTC TCC CCA TTT CAT CAT CTC 60(37) 396865 205 EXON37-F/R 409 57.9 ACC CCT CTG TGT CTG CTG GAA ATC CCA AAT TAG CAT GAC AGG AAA CC 61(38) 398036 236 EXON38-F/R 430 54.3 CTA ATA AAG CCC GAG AGC ACT GGC TTG GTT TGT CTA TTT GAG 62(39) 399668 152 EXON39-F/R 401 57.9 AGG AGG AGG AGG AGG AGG AGG TG NEW EX40- CAC CTT GCT TGC TCT TCC 63(40) 399970 190 453 55.5 F/R CAG GGT AGT GCC GCC ATA A 144 ATT GAG GAA GGC ATA CTG TC 64(41) 402394 103 EXON41-F/R 439 52.9 GCT TTG GCT TCT GGT GA TCA GTA GGC AAT GGA GAA C 65(42) 402681 288 EXON42-F/R 535 53.6 GTG TGA CTG AGC CTG GTG T GAT GGG TGG ATG GAT GGA TGC 66(43) 404880 143 EXON43-F/R 463 56.7 TGG TTG AGG GGA GGT TTC TTA GTT TGC TCC TCC ATG AAA CAC TG 67(44) 407910 219 EXON44-F/R 419 54.3 ATG GCG GGC TGG ATG AAG CTC TCC GTT AAT TAC CTG CTA NEW EX45- 68(45) 411220 161 537 51.6 CCC F/R AAC TAC CAT TCC TGA CTG AGA GCC TAG CTG TCC ACT GTC AA 69(46) 412618 98 EXON46-F/R 419 54 AGG CCC ATG CTC TGC TGT TC NEW EX47- GAG CCA AAA GGA AAT CAA TC 70(47) 413740 196 554 55.4 F/R GGC GGG GCT TAG ACT ACA TCC TC AAA GGG ACA ACG GGA TAG AAA 71(48) 415029 213 EXON48-F/R 457 56.7 AAT GTC AAA AGC AAC GCA AAC AGA AC CTC CAT AGC TGC ACC ATT CAT 72(49) 415748 139 EXON49-F/R 402 55.8 TAC TTC TCC CTT CAC ACT CTT GCT ACA GGT GCA TGG CCC TAC TCC 73(50) 416971 166 EXON50-F/R 437 56.1 TGC CCC ACT CCT GAC TGC T GGG GGA GGG GAT GCT GAG AT 74(51) 419092 148 EXON51-F/R 436 55.5 TGC TGG GAA TTG AAC ACA GAG TGC TGG TTT TGG TGT CTT T 75(52) 420103 208 EXON52-F/R 468 53.5 CTC TCC CAT TCT CAT CAT AAC TAC CAC CCA GCT TAT TAA TGT CAC TCT 76(53) 420705 222 EXON53-F/R 415 54.7 TGG GGC TGC TGC AAC TAT CC CAA ACC TAC TGA AAC CGT CCT T 77(54) 422518 170 EXON54-F/R 419 55.1 CCA ACC TCA TCC CCC TGC TAA GCT CTT GGG CTG ACT CTG C 78(55) 425678 199 EXON55-F/R 404 56.6 TGA AGC CCC GCC TAC TGT TGG NEW EX56- GCC GCC TCC CCT CAA GTC ACA 79(56) 426736 159 550 58.1 F/R AAG GGG AGC GAG GAG TTT TC NEW EX57- AAG GGG ATG GTT TGA GC 80(57) 428287 278 502 54.2 F/R ATA TGA GGT GAG GAG ATG C
145 GTG TTG GTT GGT TTG GGT TTT G 81(58) 430856 220 EXON58-F/R 406 56.3 TGG CAG GGA GGA GCA GGA C CAC TAT GGG AAA CAC TTG AAA CCT 82(59) 432028 213 EXON59-F/R 461 56.6 TCC CAT GAT CTA GAT ACC CAC TCC TAC ACA GGT GCA CTC ATC TAC 83(60) 433311 164 EXON60-F/R 424 54.7 ATA TTC TGA CTC CCC CTA AAT CTG AC
NEW EX61- TCC CAT AAA GCA GAG GTG 84(61) 434307 151 563 53.5 F/R GAT TAG GCA ACA TAG AAC ATA GTG TGC CAC ACC CTA CCT TAC ATC A 85(62) 436231 231 EXON62-F/R 489 57.7 CTT GCC ATT CTT CTT GCT TTC TAC AAT TCT CCG GCT CAG GGT CAG 86(63) 440754 225 EXON63-F/R 458 57.9 GCA AGG GAA GGG CCA CTA ACT EXON64_5'- GGT TCC CCG ATG GTC TCT TTA GG 87(64) 442644 679 569 58 F/R AGG CCG CCA GTT GAC ACA GTT G EXON64_3'- ACC GTG CGG CCC AAG AAG AT 87(64) 442644 679 569 58 F/R CTG CGT GAA GGT CCC GAG TGG
146
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