Mouse Genetics and Transgenics

Transcription

1 Mouse Genetics and Transgenics A Practical Approach IAN J. JACKSON, CATHERINE M. ABBOTT

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3 List of Contributors Abbreviations xx i xxiii 1. Mouse care and husbandry 1 Colin M. Hetherington, Brendan Doe and Donald Hay 1. Introduction 1 2. Housing 1 Types of facilities 1 Conventional accommodation 1 Barrier accommodation or specified pathogen free 1 Germ-free (axenic) and gnotobiotic accommodation 2 Maintenance of pathogen-free animals 2 Breeding performance and production 2 Interpretation of data 2 Collaborative experiments 2 Health screening 3 Quarantine 3 Flexible film isolators 4 Individually ventilated cage (IVC) racks 4 Fumigation 5 3. Husbandry 8 Caging 8 Bedding 9 Diet 9 Water 1 0 Environmental control 1 0 Environmental enhancement Animal identification 1 1 Ear punch 1 1 Toe clip 1 1 Ear tags 1 1 Tail and paw tattooing 1 1 Electronic tags Record keeping Breeding systems 1 6 Timed mating 1 7 Pseudopregnancy and vasectomy 1 8 Superovulation 19

4 7. Rederivation Euthanasia Transportation Legislation 24 Acknowledgements 25 References 25 Further reading Cryopreservation and rederivation of embryos and gametes 2 7 Peter H. Glenister and William F. Rail 1. Introduction Proliferation of mouse models 27 Long-term maintenance of mouse models 28 Cryopreservation and banking of mouse germplasm : resources for future research Principles of cryopreservation 29 Controlled freezing 30 Vitrification Skills required for embryo and gamete cryopreservation Special equipment required for cryopreservation Cryopreservation and recovery of mouse embryos 34 Source of embryos and preferred developmental stages 3 5 Freezing embryos using propylene glycol as cryoprotectant 3 6 Recovery of embryos and live mice from embryos frozen according to Protocol Freezing embryos using glycerol as cryoprotectant 4 0 Vitrification of embryos in vitrification solution VS3a 42 Recovery of embryos and live mice from embryos cryopreserve d according to Protocols 4 and Cryopreservation and recovery of mouse gametes 4 6 Cryopreservation of mouse metaphase II oocytes 4 7 Recovery of oocytes frozen using Protocol? 4 8 Cryopreservation of mouse sperm 4 9 In vitro fertilization of mouse oocytes Hints and tips 5 3 Embryo cryopreservation 53 Sperm cryopreservation and in vitro fertilization 54

5 9. Long-term storage of embryos and gametes 5 5 Expected overall survival of cryopreserved embryos and gametes Genetic resource banks 5 7 Acknowledgements 58 References Spatial analysis of gene expression 6 1 Stefan G. Nonchev and Mark K. Maconochie 1. Introduction Dissection of post-implantation embryos Whole-mount non-radioactive in situ hybridization 63 Precautions for RNA work 63 Theory of in situ hybridization 64 Generation of riboprobe 65 Fixation and pre-treatment of embryos 67 Hybridization and removal of non-specific hybrids 68 Immunodetection of signals 69 Troubleshooting Reporter transgenes 72 General considerations 72 Introduction and uses of reporter genes 72 Detection of 3-galactosidase activity 73 Detection of alkaline phosphatase activity 7 4 Use of the green fluorescent protein (GFP) in transgenesis Multiple and combined detection systems 77 Whole-mount staining of early ( dpc) embryos 77 Multiple detection on cryostat and paraffin sections Whole-mount skeletal analysis 83 Acknowledgements 85 References Mapping phenotypic trait loci 8 7 Benjamin A. Taylor 1. Introduction Rationale for mapping 87 Genetic definition of trait locus 8 7 Identification of candidate genes 8 7 Genetic manipulation 87

6 3. Background information for mapping phenotypic trait loc i of the mouse 88 Definitions 8 8 Mouse genome 89 Mapping basics A backcross mapping experiment : a model for simpl e mapping experiments 8 9 Identifying the phenotypic variant 89 Choosing a tester strain 90 Mating mutant and tester stock to produce Fl progeny 92 Producing backcross progeny 92 Classifying progeny for the target locus 93 Extracting a DNA sample 93 A linkage testing plan 93 Genotyping a subset of backcross progeny for selected markers 95 Recording data 96 Scanning data for linkage 96 Finding markers that flank the target 97 Finding closer markers 97 Analysing the complete data set to determine gene order and distance 97 Reporting linkage data 98 Size of cross 99 Markers for fine mapping Incomplete penetrance, phenocopies and reduced viability Mapping by intercross QTL mapping, including the use of derivitive strains 102 General nature of QTL mapping 102 QTL mapping using strain crosses 103 Strain selection 10 3 Backcross versus intercross 10 3 Number of progeny 104 Marker spacing 10 4 Selective genotyping 10 5 Significance thresholds 106 Confidence intervals 10 7 Fine mapping QTLs 10 7 Selective phenotyping 10 7 Progeny testing 108 Congenic strain construction 10 8 Advanced intercross populations 108 Derivative inbred strains for QTL mapping 109 Recombinant inbred strains 109 Congenic strains 11 1 Recombinant congenic strains Consomic strains

7 8. Markers DNA pooling 114 Acknowledgements 11 8 References Mapping genomes 12 1 Paul Denny and Stephen D. M. Brow n 1. Introduction Applications of genetic and physical mapping Genetic mapping 124 Types of genetic marker 124 Genotyping using silver-staining of SSCP gels 124 Genotyping using fluorescently-labelled dctp incorporation int o PCR products from SSLP markers, for analysis on AB I sequencers 126 Linking different maps together 12 8 Generating new genetic markers in specific genomic regions 129 Criteria for making the decision to construct physical maps Physical mapping 13 1 Introduction to physical mapping 13 1 Primary contig generation 132 Clone identification using hybridization assay 134 Chromosome walking/gap filling 136 Isolating insert end sequences 136 Validation of clone contigs 138 Acknowledgements 140 References Mouse cytogenetics and FISH Introduction 143 6A. Analysing mouse chromosomal rearrangements with G-banded chromosomes 14,4 Ellen C. Akeson and Muriel T. Davisson 1. Introduction Metaphase chromosomes 144

8 3. Stimulation of peripheral mouse lymphyocytes b y phytohaemagglutinin and lipopolysaccharide 14 8 Phytohaemagglutinin 14 8 Lipopolysaccharide G-bands Identifying and karyotyping mouse chromosomes Recognizing aberrant chromosomes 152 Acknowledgement 153 References B. Fluorescent in situ hybridization (FISH) to mous e chromosomes 15 4 Margaret Fox and Sue Povey 1. Introduction FISH and chromosome identification 154 Repeat DNA 154 Mouse chromosome paints 155 P1 probes Obtaining mouse chromosomes Probes and labelling 158 DNA 15 8 Labelling by nick translation 15 8 Chromosome paints and direct labelling FISH 160 Probe preparation 160 Hybridization Analysis and microscopy 167 References Electronic tools for accessing the mous e genome 171 Janan T. Eppig 1. Introduction Databases of genomic information for the mouse 171 The Mouse Genome Database (MGD) 17 1 The MRC Mammalian Genetics Unit 17 5

9 3. Specific data sets for genetic, radiation hybrid and physical mapping 17 5 Genetic data 175 The Whitehead Institute/MIT map 175 EUCIB 175 Jackson Laboratory backcross 176 Copeland-Jenkins interspecific backcross 176 M. Seldin interspecific backcross 176 Radiation hybrid data 176 The Jackson Laboratory mouse radiation hybrid database 177 The EBI radiation hybrid database 17 7 Physical mapping data 177 Genome-wide physical map 177 X-Chromosome physical map Gene expression data 17 8 The Gene Expression Database (GXD) 17 8 The mouse 3D atlas Databases of transgenics, knock-outs and other induce d mutations 17 9 The transgenic/targeted mutation database (TBASE) 17 9 Mouse Knockout and Mutation Database (MKMD) 17 9 Induced mutant resource (IMR) Animal resources lists 18 0 International mouse strain resource (IMSR) 18 0 The Jackson Laboratory: JAX mice IMR, MMR, DNA resource 18 0 European Mouse Mutant Archive (EMMA) mgi-list, an electronic bulletin board for the mous e community Summary 18 1 References Mutagenesis of the mouse germline 18 5 Monica J. Justice 1. Introduction Mouse mutagenesis 185 Spontaneous mutations 18 5 Systems for assessing mutation rate 186 Reporter genes 186 Specific locus test 187 Dominant phenotype assays 188 Other assays 188 Developing a mouse mutant resource 188 Determining functional complexity of genomic regions 189

10 Human disease models 18 9 Allelic series 19 0 Unravelling biochemical or developmental pathways High-efficiency mutagenesis with N-ethyl-N-nitrosourea 191 Mode of action 191 ENU: chemical properties, stability and half-life 19 1 Types of DNA lesion caused by ENU 19 1 Effects on protein products 192 Induction of mutations in the mouse germline 19 2 Doses and treatment protocols 19 2 Genetic screens to isolate mutations 19 6 Dominant mutations 196 Single-locus screens for recessive mutations 197 Three-generation breeding scheme for recessive mutations 197 Two-generation breeding scheme using deletions 199 Modifiers and sensitized pathways Practical considerations for ENU mutagenesis 201 Breeding considerations 20 1 Male rotations 20 1 Gamete sampling for spermatogonial stem-cell mutagenesis 203 Strain background effects 206 Inbred strains to use for mutagenesis 20 6 Fl hybrids 207 Other observations 20 8 Rate of recovery to fertility 208 Mutation rates for different loci 208 Cancer susceptibility and lifespan Other mutagens 209 Radiation mutagenesis 209 X-rays: treatment and mutations recovered 20 9 Other types of radiation 210 Chlorambucil Future prospects 21 1 DNA repair 21 1 Sequence-based screening for lesions 21 1 The future of mutagenesis 21 1 References Generation of transgenic mice from plasmids, BACs and YACs 217 Annette Hammes and Andreas Schedl 1. Introduction 21 7 Principles and general considerations 217

11 Difficulties and limitations 21 8 Construct design 21 9 Perspectives 220 Factors influencing the efficiency of transgenesis 220 Choice of mouse strains DNA isolation 221 Plasmid DNA 221 YAC DNA 22 3 BAC DNA 22 6 Testing the DNA concentration and quality The microinjection set-up 22 8 Location and design of the injection table 22 9 Microscope 23 0 Micromanipulators 230 The holding pipette 23 1 Microinjection needles The microinjection experiment 232 Superovulation and isolation of fertilized oocytes 232 Microinjections 234 Timing of injections 234 Injections 234 Oviduct transfer 236 Pseudopregnant females 236 Preparation of the transfer pipette 237 Oviduct transfer Analysis of transgenic founders 240 Tail tipping and ear punching 243 Isolation of genomic DNA from tail biopsies 24 3 Southern blot analysis 245 References Directed mutagenesis in embryonic stem cells 24 7 Antonius Plagge, Gavin Kelsey and Nicholas D. Alle n 1. Introduction Basic elements of construct design 249 Replacement versus insertion 249 Regions of homology 250 The mutation 25 1 Components of the targeting cassette 25 1 Enrichment for targeted events 252 Screening strategies 253 Alternative approaches for construct design 254

12 3. The use of site-specific recombinases in targeting 254 Excision of heterologous DNA from a targeted locus 255 Excision from ES-cell clones with recombinase expressio n plasmids 256 Excision in mice using transgenic mice expressing recombinase 257 Oocyte microinjection of recombinase 257 Use of recombinases to generate conditional somatic knock-outs 257 Spatially restricted knock-outs using tissue specific promoters to drive recombinase expression 258 Temporally regulated knock-outs using inducible recombinases 259 Knocking-in: recombinase mediated integration int o chromosomally positioned target sites Subtle mutations without site-specific recombinases 263 Double replacement 264 `Hit-and-run' Chromosome engineering in ES cells 265 Deletions, inversions and duplications 265 Translocations Generating, analysing and maintaining knock-out mice 268 ES-cell culture and pluripotency 268 Analysis of ES-cell potency in chimeras with tetraploid embryos 26 9 Equipment for injection of ES cells into embryos 269 Host embryos 271 Blastocyst injection 272 Morula injection 275 Morula aggregation 277 Transfer of embryos to the uterus 27 8 Genetic background 279 Maintenance of mutations as co-isogenic and congenic lines an d mutant analysis in a hybrid background 280 Chimera analysis 281 Acknowledgements 282 References 282 List of suppliers 285 Index 290