Gene targeting: a practical approach
Gespeichert in:
| Format: | Buch |
|---|---|
| Sprache: | English |
| Veröffentlicht: |
Oxford [u.a.]
Oxford Univ. Press
2005
|
| Ausgabe: | 2. ed., reprinted |
| Schriftenreihe: | The practical approach series
212 |
| Schlagworte: | |
| Online-Zugang: | Klappentext Inhaltsverzeichnis |
| Beschreibung: | XVIII, 293 S. Ill., graph. Darst. |
| ISBN: | 0199637938 019963792X |
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Datensatz im Suchindex
| _version_ | 1806141764028858368 |
|---|---|
| adam_text |
Over the past ten years it has become possible to make essentially any mutation
in the germline of mice by utilizing homologous recombination and embryonic
stem (ES) cells. As the era of gene discovery comes to an end, in vivo approaches
for studying gene function will be utilized to an even greater extent. This new
edition of Gene Targeting: A Practica/ Approach contains a remarkable collection of
state-of-the-art protocols as well as thoughtful discussion of how to design a new
mouse mutation and analyse the resulting phenotype to maximise the
information gained. The techniques described for gene targeting in mouse ES
cells, are equally applicable for gene targeting in other tissue culture cells,
including human cells. The second edition describes additional genetic approaches
for studying gene function in mice, including chimera analysis, gene trap (GT)
approaches and powerful ways to combine gene targeting with classical genetics.
The book starts by describing the design of gene targeting vectors for mammalian
cells and how to distinguish random integrations from homologous
recombination. Topics then covered include: extending conventional gene
targeting manipulations by site-specific recombination using the Cre-loxP and Flp-
FRT systems to produce germline mutations that involve changing only a few base
pairs or conditional (in)activation of genes; methods for introducing mutations
into ES cells via homologous recombination including selection and screening
procedures for identifying and recovering targeted cell clones; the establishment
of new ES cell lines; aggregation versus blastocyst injection to create mouse
chimeras and study mutant phenotypes; GT strategies including the components,
design, and modification of GT vectors and screens; the molecular analysis of GT
integrations; the use of classical genetics in studying targeted mutations and
phenotype analysis.
Gene Targeting: A Practical Approach (second edition) is an indispensable practical
tool and information resource for scientists working on fundamental or applied
aspects of gene targeting, ES cell manipulation, and analysis of mouse mutants. It
will be invaluable to all researchers who wish to use the approaches for studies of
gene function in cells or mice.
UNIVERSITY PRESS
Contents
List of Contributors
Abbreviations
ft
XV
xvii
1. Gene targeting, principles, and practice in
mammalian cells 1
Paul Hasty, Alejandro Abuin, and Allan Bradley
1. Introduction 1
Targeting vectors 2
2. Replacement vectors 2
Design considerations of a replacement vector 5
Recombinant alleles generated by replacement vectors 7
Replacement vectors: screening for targeted events 7
3. Insertion vectors 11
Vector design for insertion vectors 12
Screening for recombinant alleles generated with insertion vectors 14
4. Maximizing the targeting frequency and selection of targeted
clones 15
Homology to the target locus 15
Enrichment schemes for targeted clones in culture 17
5. Selection markers 21
Promoters and polyadenylation sites used for selection markers 21
Effects of selection markers on phenotypes 23
6. Generating subtle mutations with gene targeting techniques 23
Subtle mutations generated by microinjection 24
Non-selectable mutations generated by co-electroporation 24
Subtle mutations generated with a hit-and-run vector 25
Subtle mutations generated by double replacement 28
7. ‘Knock-in’ targeting vectors: simultaneous study of gene
function and expression 29
8. Summary 33
Acknowledgements 34
References 34
Contents
2. Site-specific recombination in cells and mice 37
Susan M. Dymecki
1. Introduction 37
2. Site-specific recombinase systems: Gre-loxP and Flp-FRT 37
General properties 38
Target sites and recombinase action 41
Making recombination events irreversible 42
3. Cre, Flp, FlpL, Flpe: distinguishing properties suggest
specific applications 43
4. Extending conventional gene replacement schemes using
site-specific recombination 46
Removal of selection genes 49
Generating larger deletions along with marker gene removal 53
Engineering subtle mutations 54
Swapping sequences 56
Engineering large scale chromosomal rearrangements in vitro 57
TAMERE: engineering duplication and deletion chromosomes
in vivo 61
5. Conditional gene targeting 63
Conditional gene knock-out versus conditional gene repair 63
Engineering target genes for lineage-specific mutagenesis 68
Engineering target genes for lineage-specific repair 73
Generating recombinase mice 75
Generating indicator mice 81
Breeding schemes for conditional gene targeting 82
6. Switching-on transgenes in the living mouse 82
7. Fate-mapping by site-directed recombination 84
Tagging cell lineages in the mouse 85
Combining recombinase-mediated fate-mapping with mouse
mutations to yield high resolution studies of gene function 87
8. Targeted integrations: isogenic cell and mouse lines for
structure—function studies 87
9. Inducible recombination: gaining temporal and spatial
control over genome modifications 88
10. Tools: Cve-loxP and Flp-FRT vectors and mice 91
Acknowledgements 96
References 96
X
Contents
3. Production of targeted embryonic stem cell
clones 101
Michael P. Matise, Wojtek Auerbach and Alexandra L. Joyner
1. Introduction 101
2. Propagation and maintenance of ES cells 102
General conditions 102
ES cell culture media and solutions 103
Production of fibroblast feeder layers 104
Culturing ES cells 111
Testing serum batches 113
Freezing, storage, and thawing of ES cell lines 115
3. Electroporation of DNA into ES cells 116
Standard electroporation conditions 116
4. Screening single colonies for homologous targeting events 119
Screening for homologous targeting events by Southern blot analysis 119
Screening for homologous targeting events using PCR 124
Selecting ES cells homozygous for the targeted allele 126
5. Establishing new ES cell lines 129
Acknowledgements 131
References 131
4. Production of chimeras by blastocyst and
morula injection of targeted ES cells 133
Virginia Papaioannou and Randall Johnson
1. Introduction 133
2. The starting material 134
ES cells 134
Mice: setting up for embryo recovery and transfer 137
Recovering embryos 144
3. Injection of ES cells into embryos 149
Micromanipulation apparatus 149
Microinstruments 153
Injection procedures 156
4. Embryo transfer 159
5. Chimerism 165
Detection and quantification of chimerism (see Chapter 5 for
additional markers of chimerism) 165
Phenotypic effects of chimerism 166
xi
Contents
6. Maintaining a targeted mutation
Animal husbandry
Test breeding
Breeding schemes to maintain targeted alleles and determine
phenotype
Acknowledgements
References
Appendix
167
167
168
169
172
172
174
5. Production and analysis of ES cell aggregation
chimeras 177
Andras Nagy and Janet Rossant
1. Introduction 177
Types of aggregation chimeras 178
Uses of ES cell diploid embryo aggregation 179
Uses of ES cell tetraploid embryo aggregation 182
2. Embryonic stem cells 183
3. Production of embryo partners 184
Recovery of 8֊ and 2-cell stage embryos 187
Production of tetraploid embryos 189
4. Aggregating ES cells with cleavage stage embryos 193
Preparation of aggregation plate 193
Preparation of diploid or tetraploid embryos for aggregation 194
Assembly of aggregations 195
Culture and transfer of embryos 198
Caesarian section 198
5. Characterization of mosaicism in diploid or tetraploid
chimeras 199
Marker systems 199
6. Prospects and limitations 205
Acknowledgements 205
References 206
6. Gene trap strategies in ES cells 207
Wolfgang Wurst and Achim Gossler
1, Introduction 207
2. General properties and design of gene trap vectors 208
Basic gene trap vectors 208
* ·
XU
Contents
Reporter genes 210
Modifications of basic gene trap vectors 214
3. Establishment of cell lines carrying reporter gene integrations 219
Introduction of reporter gene constructs into ES cells 219
Identification of /acZ-expressing ES cell clones 222
4. Screening strategies 225
Analysis of GT activation patterns in chimeric embryos 225
Identification of GT integrations activated or repressed during
embryoid body formation 228
Identification of GT integrations into genes active in specific cell
lineages and/or downstream of specific factors by ‘induction
trapping’ 232
Identification of target genes of homeobox-containing transcription
factors 233
5. ß-Galactosidase staining 234
Staining of attached cells, embryos, and tissues 235
Staining of cryostat sections 237
Histological analysis of stained whole-mount embryos and tissues 237
In vivo staining of cells to detect lacZ expression 241
6. Identification of trapped genes 242
Cloning genomic DNA flanking insertion sites by inverse PCR 242
Cloning cDNAs containing endogenous sequences fused to lacZ or
selector gene transcripts by RACE-PCR 246
Acknowledgements 250
References 251
7. Classical genetics and gene targeting 255
Scott Bultman and Terry Magnuson
1. Introduction 255
2. Genetic considerations in gene targeting 255
Implications of genetic heterogeneity among 129 mouse strains 255
Molecular and genetic nature of targeted mutations 257
3. The significance of genetic background on phenotypes
created by gene targeting 260
Developing mouse models of human diseases 263
Genetic mapping of modifier loci 265
4. Targeting multiple genes 272
Redundancy and analysis of multiple mutations 272
Limitations, controls, and caveats 273
xiii
Contents
5. Application of ENU and radiation mutagenesis in gene
targeting experiments 274
Generating allelic series 274
Deletion complexes in ES cells 276
6. Future prospects 278
Acknowledgements 278
References 278
List of suppliers 285
Index 289
XIV |
| adam_txt |
Over the past ten years it has become possible to make essentially any mutation
in the germline of mice by utilizing homologous recombination and embryonic
stem (ES) cells. As the era of gene discovery comes to an end, in vivo approaches
for studying gene function will be utilized to an even greater extent. This new
edition of Gene Targeting: A Practica/ Approach contains a remarkable collection of
state-of-the-art protocols as well as thoughtful discussion of how to design a new
mouse mutation and analyse the resulting phenotype to maximise the
information gained. The techniques described for gene targeting in mouse ES
cells, are equally applicable for gene targeting in other tissue culture cells,
including human cells. The second edition describes additional genetic approaches
for studying gene function in mice, including chimera analysis, gene trap (GT)
approaches and powerful ways to combine gene targeting with classical genetics.
The book starts by describing the design of gene targeting vectors for mammalian
cells and how to distinguish random integrations from homologous
recombination. Topics then covered include: extending conventional gene
targeting manipulations by site-specific recombination using the Cre-loxP and Flp-
FRT systems to produce germline mutations that involve changing only a few base
pairs or conditional (in)activation of genes; methods for introducing mutations
into ES cells via homologous recombination including selection and screening
procedures for identifying and recovering targeted cell clones; the establishment
of new ES cell lines; aggregation versus blastocyst injection to create mouse
chimeras and study mutant phenotypes; GT strategies including the components,
design, and modification of GT vectors and screens; the molecular analysis of GT
integrations; the use of classical genetics in studying targeted mutations and
phenotype analysis.
Gene Targeting: A Practical Approach (second edition) is an indispensable practical
tool and information resource for scientists working on fundamental or applied
aspects of gene targeting, ES cell manipulation, and analysis of mouse mutants. It
will be invaluable to all researchers who wish to use the approaches for studies of
gene function in cells or mice.
UNIVERSITY PRESS
Contents
List of Contributors
Abbreviations
ft
XV
xvii
1. Gene targeting, principles, and practice in
mammalian cells 1
Paul Hasty, Alejandro Abuin, and Allan Bradley
1. Introduction 1
Targeting vectors 2
2. Replacement vectors 2
Design considerations of a replacement vector 5
Recombinant alleles generated by replacement vectors 7
Replacement vectors: screening for targeted events 7
3. Insertion vectors 11
Vector design for insertion vectors 12
Screening for recombinant alleles generated with insertion vectors 14
4. Maximizing the targeting frequency and selection of targeted
clones 15
Homology to the target locus 15
Enrichment schemes for targeted clones in culture 17
5. Selection markers 21
Promoters and polyadenylation sites used for selection markers 21
Effects of selection markers on phenotypes 23
6. Generating subtle mutations with gene targeting techniques 23
Subtle mutations generated by microinjection 24
Non-selectable mutations generated by co-electroporation 24
Subtle mutations generated with a hit-and-run vector 25
Subtle mutations generated by double replacement 28
7. ‘Knock-in’ targeting vectors: simultaneous study of gene
function and expression 29
8. Summary 33
Acknowledgements 34
References 34
Contents
2. Site-specific recombination in cells and mice 37
Susan M. Dymecki
1. Introduction 37
2. Site-specific recombinase systems: Gre-loxP and Flp-FRT 37
General properties 38
Target sites and recombinase action 41
Making recombination events irreversible 42
3. Cre, Flp, FlpL, Flpe: distinguishing properties suggest
specific applications 43
4. Extending conventional gene replacement schemes using
site-specific recombination 46
Removal of selection genes 49
Generating larger deletions along with marker gene removal 53
Engineering subtle mutations 54
Swapping sequences 56
Engineering large scale chromosomal rearrangements in vitro 57
TAMERE: engineering duplication and deletion chromosomes
in vivo 61
5. Conditional gene targeting 63
Conditional gene knock-out versus conditional gene repair 63
Engineering target genes for lineage-specific mutagenesis 68
Engineering target genes for lineage-specific repair 73
Generating recombinase mice 75
Generating indicator mice 81
Breeding schemes for conditional gene targeting 82
6. Switching-on transgenes in the living mouse 82
7. Fate-mapping by site-directed recombination 84
Tagging cell lineages in the mouse 85
Combining recombinase-mediated fate-mapping with mouse
mutations to yield high resolution studies of gene function 87
8. Targeted integrations: isogenic cell and mouse lines for
structure—function studies 87
9. Inducible recombination: gaining temporal and spatial
control over genome modifications 88
10. Tools: Cve-loxP and Flp-FRT vectors and mice 91
Acknowledgements 96
References 96
X
Contents
3. Production of targeted embryonic stem cell
clones 101
Michael P. Matise, Wojtek Auerbach and Alexandra L. Joyner
1. Introduction 101
2. Propagation and maintenance of ES cells 102
General conditions 102
ES cell culture media and solutions 103
Production of fibroblast feeder layers 104
Culturing ES cells 111
Testing serum batches 113
Freezing, storage, and thawing of ES cell lines 115
3. Electroporation of DNA into ES cells 116
Standard electroporation conditions 116
4. Screening single colonies for homologous targeting events 119
Screening for homologous targeting events by Southern blot analysis 119
Screening for homologous targeting events using PCR 124
Selecting ES cells homozygous for the targeted allele 126
5. Establishing new ES cell lines 129
Acknowledgements 131
References 131
4. Production of chimeras by blastocyst and
morula injection of targeted ES cells 133
Virginia Papaioannou and Randall Johnson
1. Introduction 133
2. The starting material 134
ES cells 134
Mice: setting up for embryo recovery and transfer 137
Recovering embryos 144
3. Injection of ES cells into embryos 149
Micromanipulation apparatus 149
Microinstruments 153
Injection procedures 156
4. Embryo transfer 159
5. Chimerism 165
Detection and quantification of chimerism (see Chapter 5 for
additional markers of chimerism) 165
Phenotypic effects of chimerism 166
xi
Contents
6. Maintaining a targeted mutation
Animal husbandry
Test breeding
Breeding schemes to maintain targeted alleles and determine
phenotype
Acknowledgements
References
Appendix
167
167
168
169
172
172
174
5. Production and analysis of ES cell aggregation
chimeras 177
Andras Nagy and Janet Rossant
1. Introduction 177
Types of aggregation chimeras 178
Uses of ES cell diploid embryo aggregation 179
Uses of ES cell tetraploid embryo aggregation 182
2. Embryonic stem cells 183
3. Production of embryo partners 184
Recovery of 8֊ and 2-cell stage embryos 187
Production of tetraploid embryos 189
4. Aggregating ES cells with cleavage stage embryos 193
Preparation of aggregation plate 193
Preparation of diploid or tetraploid embryos for aggregation 194
Assembly of aggregations 195
Culture and transfer of embryos 198
Caesarian section 198
5. Characterization of mosaicism in diploid or tetraploid
chimeras 199
Marker systems 199
6. Prospects and limitations 205
Acknowledgements 205
References 206
6. Gene trap strategies in ES cells 207
Wolfgang Wurst and Achim Gossler
1, Introduction 207
2. General properties and design of gene trap vectors 208
Basic gene trap vectors 208
* ·
XU
Contents
Reporter genes 210
Modifications of basic gene trap vectors 214
3. Establishment of cell lines carrying reporter gene integrations 219
Introduction of reporter gene constructs into ES cells 219
Identification of /acZ-expressing ES cell clones 222
4. Screening strategies 225
Analysis of GT activation patterns in chimeric embryos 225
Identification of GT integrations activated or repressed during
embryoid body formation 228
Identification of GT integrations into genes active in specific cell
lineages and/or downstream of specific factors by ‘induction
trapping’ 232
Identification of target genes of homeobox-containing transcription
factors 233
5. ß-Galactosidase staining 234
Staining of attached cells, embryos, and tissues 235
Staining of cryostat sections 237
Histological analysis of stained whole-mount embryos and tissues 237
In vivo staining of cells to detect lacZ expression 241
6. Identification of trapped genes 242
Cloning genomic DNA flanking insertion sites by inverse PCR 242
Cloning cDNAs containing endogenous sequences fused to lacZ or
selector gene transcripts by RACE-PCR 246
Acknowledgements 250
References 251
7. Classical genetics and gene targeting 255
Scott Bultman and Terry Magnuson
1. Introduction 255
2. Genetic considerations in gene targeting 255
Implications of genetic heterogeneity among 129 mouse strains 255
Molecular and genetic nature of targeted mutations 257
3. The significance of genetic background on phenotypes
created by gene targeting 260
Developing mouse models of human diseases 263
Genetic mapping of modifier loci 265
4. Targeting multiple genes 272
Redundancy and analysis of multiple mutations 272
Limitations, controls, and caveats 273
xiii
Contents
5. Application of ENU and radiation mutagenesis in gene
targeting experiments 274
Generating allelic series 274
Deletion complexes in ES cells 276
6. Future prospects 278
Acknowledgements 278
References 278
List of suppliers 285
Index 289
XIV |
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| id | DE-604.BV023179592 |
| illustrated | Illustrated |
| index_date | 2024-07-02T20:01:02Z |
| indexdate | 2024-08-01T00:10:11Z |
| institution | BVB |
| isbn | 0199637938 019963792X |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-016366143 |
| oclc_num | 180041398 |
| open_access_boolean | |
| owner | DE-20 DE-703 |
| owner_facet | DE-20 DE-703 |
| physical | XVIII, 293 S. Ill., graph. Darst. |
| publishDate | 2005 |
| publishDateSearch | 2005 |
| publishDateSort | 2005 |
| publisher | Oxford Univ. Press |
| record_format | marc |
| series | The practical approach series |
| series2 | The practical approach series |
| spelling | Gene targeting a practical approach ed. by Alexandra L. Joyner 2. ed., reprinted Oxford [u.a.] Oxford Univ. Press 2005 XVIII, 293 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier The practical approach series 212 Embryotransfer (DE-588)4113430-8 gnd rswk-swf Methode (DE-588)4038971-6 gnd rswk-swf Maus (DE-588)4169148-9 gnd rswk-swf Gentechnologie (DE-588)4071722-7 gnd rswk-swf Blutstammzelle (DE-588)4146093-5 gnd rswk-swf Gentransfer (DE-588)4194102-0 gnd rswk-swf Transgene Tiere (DE-588)4279021-9 gnd rswk-swf Gen-Targeting (DE-588)4294998-1 gnd rswk-swf Blutstammzelle (DE-588)4146093-5 s Gentechnologie (DE-588)4071722-7 s DE-604 Maus (DE-588)4169148-9 s Gen-Targeting (DE-588)4294998-1 s Methode (DE-588)4038971-6 s Transgene Tiere (DE-588)4279021-9 s Embryotransfer (DE-588)4113430-8 s 1\p DE-604 2\p DE-604 Gentransfer (DE-588)4194102-0 s 3\p DE-604 Joyner, Alexandra L. Sonstige oth The practical approach series 212 (DE-604)BV007921321 212 Digitalisierung UB Bayreuth - ADAM Catalogue Enrichment application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016366143&sequence=000003&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Klappentext Digitalisierung UB Bayreuth - ADAM Catalogue Enrichment application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016366143&sequence=000004&line_number=0002&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 2\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 3\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk |
| spellingShingle | Gene targeting a practical approach The practical approach series Embryotransfer (DE-588)4113430-8 gnd Methode (DE-588)4038971-6 gnd Maus (DE-588)4169148-9 gnd Gentechnologie (DE-588)4071722-7 gnd Blutstammzelle (DE-588)4146093-5 gnd Gentransfer (DE-588)4194102-0 gnd Transgene Tiere (DE-588)4279021-9 gnd Gen-Targeting (DE-588)4294998-1 gnd |
| subject_GND | (DE-588)4113430-8 (DE-588)4038971-6 (DE-588)4169148-9 (DE-588)4071722-7 (DE-588)4146093-5 (DE-588)4194102-0 (DE-588)4279021-9 (DE-588)4294998-1 |
| title | Gene targeting a practical approach |
| title_auth | Gene targeting a practical approach |
| title_exact_search | Gene targeting a practical approach |
| title_exact_search_txtP | Gene targeting a practical approach |
| title_full | Gene targeting a practical approach ed. by Alexandra L. Joyner |
| title_fullStr | Gene targeting a practical approach ed. by Alexandra L. Joyner |
| title_full_unstemmed | Gene targeting a practical approach ed. by Alexandra L. Joyner |
| title_short | Gene targeting |
| title_sort | gene targeting a practical approach |
| title_sub | a practical approach |
| topic | Embryotransfer (DE-588)4113430-8 gnd Methode (DE-588)4038971-6 gnd Maus (DE-588)4169148-9 gnd Gentechnologie (DE-588)4071722-7 gnd Blutstammzelle (DE-588)4146093-5 gnd Gentransfer (DE-588)4194102-0 gnd Transgene Tiere (DE-588)4279021-9 gnd Gen-Targeting (DE-588)4294998-1 gnd |
| topic_facet | Embryotransfer Methode Maus Gentechnologie Blutstammzelle Gentransfer Transgene Tiere Gen-Targeting |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016366143&sequence=000003&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=016366143&sequence=000004&line_number=0002&func_code=DB_RECORDS&service_type=MEDIA |
| volume_link | (DE-604)BV007921321 |
| work_keys_str_mv | AT joyneralexandral genetargetingapracticalapproach |