Gene Targeting: A Practical Approach / Edition 2

Gene Targeting: A Practical Approach / Edition 2

by Alexandra L. Joyner, A. L. Joyner
Pub. Date:
Oxford University Press, USA

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Gene Targeting: A Practical Approach / Edition 2

Since the publication of the first edition of Gene Targeting: A Practical Approach in 1993 there have been many advances in gene targeting and this new edition has been thoroughly updated and rewritten to include all the major new techniques. It provides not only tried-and-tested practical protocols but detailed guidance on their use and applications. As with the previous edition Gene Targeting: A Practical Approach 2e concentrates on gene targeting in mouse ES cells, but the techniques described can be easily adapted to applications in tissue culture including those for human cells. The first chapter covers the design of gene targeting vectors for mammalian cells and describes how to distinguish random integrations from homologous recombination. It is followed by a chapter on extending conventional gene targeting manipulations by using site-specific recombination using the Cre-loxP and Flp-FRT systems to produce 'clean' germline mutations and conditionally (in)activating genes. Chapter 3 describes methods for introducing DNA into ES cells for homologous recombination, selection and screening procedures for identifying and recovering targeted cell clones, and a simple method for establishing new ES cell lines. Chapter 4 discusses the pros and cons or aggregation versus blastocyst injection to create chimeras, focusing on the technical aspects of generating aggregation chimeras and then describes some of the uses of chimeras. The next topic covered is gene trap strategies; the structure, components, design, and modification of GT vectors, the various types of GT screens, and the molecular analysis of GT integrations. The final chapter explains the use of classical genetics in gene targeting and phenotype interpretation to create mutations and elucidate gene functions. Gene Targeting: A Practical Approach 2e will therefore be of great value to all researchers studying gene function.

Product Details

ISBN-13: 9780199637928
Publisher: Oxford University Press, USA
Publication date: 03/28/2000
Series: Practical Approach Series , #212
Edition description: New Edition
Pages: 312
Product dimensions: 9.10(w) x 6.10(h) x 1.00(d)

Table of Contents

List of Contributorsxv
1.Gene targeting, principles, and practice in mammalian cells1
Targeting vectors2
2.Replacement vectors2
Design considerations of a replacement vector5
Recombinant alleles generated by replacement vectors7
Replacement vectors: screening for targeted events7
3.Insertion vectors11
Vector design for insertion vectors12
Screening for recombinant alleles generated with insertion vectors14
4.Maximizing the targeting frequency and selection of targeted clones15
Homology to the target locus15
Enrichment schemes for targeted clones in culture17
5.Selection markers21
Promoters and polyadenylation sites used for selection markers21
Effects of selection markers on phenotypes23
6.Generating subtle mutations with gene targeting techniques23
Subtle mutations generated by microinjection24
Non-selectable mutations generated by co-electroporation24
Subtle mutations generated with a hit-and-run vector25
Subtle mutations generated by double replacement28
7.'Knock-in' targeting vectors: simultaneous study of gene function and expression29
2.Site-specific recombination in cells and mice37
2.Site-specific recombinase systems: Cre-loxP and Flp-FRT37
General properties38
Target sites and recombinase action41
Making recombination events irreversible42
3.Cre, Flp, FlpL, Flpe: distinguishing properties suggest specific applications43
4.Extending conventional gene replacement schemes using site-specific recombination46
Removal of selection genes49
Generating larger deletions along with marker gene removal53
Engineering subtle mutations54
Swapping sequences56
Engineering large scale chromosomal rearrangements in vitro57
Tamere: engineering duplication and deletion chromosomes in vivo61
5.Conditional gene targeting63
Conditional gene knock-out versus conditional gene repair63
Engineering target genes for lineage-specific mutagenesis68
Engineering target genes for lineage-specific repair73
Generating recombinase mice75
Generating indicator mice81
Breeding schemes for conditional gene targeting82
6.Switching-on transgenes in the living mouse82
7.Fate-mapping by site-directed recombination84
Tagging cell lineages in the mouse85
Combining recombinase-mediated fate-mapping with mouse mutations to yield high resolution studies of gene function87
8.Targeted integrations: isogenic cell and mouse lines for structure-function studies87
9.Inducible recombination: gaining temporal and spatial control over genome modifications88
10.Tools: Cre-loxP and Flp-FRT vectors and mice91
3.Production of targeted embryonic stem cell clones101
2.Propagation and maintenance of ES cells102
General conditions102
ES cell culture media and solutions103
Production of fibroblast feeder layers104
Culturing ES cells111
Testing serum batches113
Freezing, storage, and thawing of ES cell lines115
3.Electroporation of DNA into ES cells116
Standard electroporation conditions116
4.Screening single colonies for homologous targeting events119
Screening for homologous targeting events by Southern blot analysis119
Screening for homologous targeting events using PCR124
Selecting ES cells homozygous for the targeted allele126
5.Establishing new ES cell lines129
4.Production of chimeras by blastocyst and morula injection of targeted ES cells133
2.The starting material134
ES cells134
Mice: setting up for embryo recovery and transfer137
Recovering embryos144
3.Injection of ES cells into embryos149
Micromanipulation apparatus149
Injection procedures156
4.Embryo transfer159
Detection and quantification of chimerism (see Chapter 5 for additional markers of chimerism)165
Phenotypic effects of chimerism166
6.Maintaining a targeted mutation167
Animal husbandry167
Test breeding168
Breeding schemes to maintain targeted alleles and determine phenotype169
5.Production and analysis of ES cell aggregation chimeras177
Types of aggregation chimeras178
Uses of ES cell [left and right arrow] diploid embryo aggregation179
Uses of ES cell [left and right arrow] tetraploid embryo aggregation182
2.Embryonic stem cells183
3.Production of embryo partners184
Recovery of 8- and 2-cell stage embryos187
Production of tetraploid embryos189
4.Aggregating ES cells with cleavage stage embryos193
Preparation of aggregation plate193
Preparation of diploid or tetraploid embryos for aggregation194
Assembly of aggregations195
Culture and transfer of embryos198
Caesarian section198
5.Characterization of mosaicism in diploid or tetraploid chimeras199
Marker systems199
6.Prospects and limitations205
6.Gene trap strategies in ES cells207
2.General properties and design of gene trap vectors208
Basic gene trap vectors208
Reporter genes210
Modifications of basic gene trap vectors214
3.Establishment of cell lines carrying reporter gene integrations219
Introduction of reporter gene constructs into ES cells219
Identification of lacZ-expressing ES cell clones222
4.Screening strategies225
Analysis of GT activation patterns in chimeric embryos225
Identification of GT integrations activated or repressed during embryoid body formation228
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 factors233
5.[beta]-Galactosidase staining234
Staining of attached cells, embryos, and tissues235
Staining of cryostat sections237
Histological analysis of stained whole-mount embryos and tissues237
In vivo staining of cells to detect lacZ expression241
6.Identification of trapped genes242
Cloning genomic DNA flanking insertion sites by inverse PCR242
Cloning cDNAs containing endogenous sequences fused to lacZ or selector gene transcripts by RACE-PCR246
7.Classical genetics and gene targeting255
2.Genetic considerations in gene targeting255
Implications of genetic heterogeneity among 129 mouse strains255
Molecular and genetic nature of targeted mutations257
3.The significance of genetic background on phenotypes created by gene targeting260
Developing mouse models of human diseases263
Genetic mapping of modifier loci265
4.Targeting multiple genes272
Redundancy and analysis of multiple mutations272
Limitations, controls, and caveats273
5.Application of ENU and radiation mutagenesis in gene targeting experiments274
Generating allelic series274
Deletion complexes in ES cells276
6.Future prospects278
List of suppliers285

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