title: Generation of knockout alleles by RFLP based BAC targeting of polymorphic embryonic stem cells.
abstract: The isolation of germ line competent mouse Embryonic Stem (ES) cells and the ability to modify the genome by homologous recombination has revolutionized life science research. Since its initial discovery, several approaches have been introduced to increase the efficiency of homologous recombination, including the use of isogenic DNA for the generation of targeting constructs, and the use of Bacterial Artificial Chromosomes (BACs). BACs have the advantage of combining long stretches of homologous DNA, thereby increasing targeting efficiencies, with the possibilities delivered by BAC recombineering approaches, which provide the researcher with almost unlimited possibilities to efficiently edit the genome in a controlled fashion. Despite these advantages of BAC targeting approaches, a widespread use has been hampered, mainly because of the difficulties in identifying BAC-targeted knockout alleles by conventional methods like Southern Blotting. Recently, we introduced a novel BAC targeting strategy, in which Restriction Fragment Length Polymorphisms (RFLPs) are targeted in polymorphic mouse ES cells, enabling an efficient and easy PCR-based readout to identify properly targeted alleles. Here we provide a detailed protocol for the generation of targeting constructs, targeting of ES cells, and convenient PCR-based analysis of targeted clones, which enable the user to generate knockout ES cells of almost every gene in the mouse genome within a 2-month period.
authors.affiliation: Department of Reproduction and Development, Erasmus MC, University Medical Center, Room Ee 09-71, PO Box 2040, 3000 CA, Rotterdam, The Netherlands, t.barakat@erasmusmc.nl.
authors.forename: Tahsin Stefan
authors.initials: TS
authors.lastname: Barakat
authors.forename: Joost
authors.initials: J
authors.lastname: Gribnau
mesh_terms: D000483:Alleles; D000818:Animals; D022202:Chromosomes, Artificial, Bacterial / Q000737:chemistry / Q000378:metabolism*; D018274:Electroporation; D004622:Embryo, Mammalian; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism*; D016678:Genome; D015698:Genomic Library; D059765:Homologous Recombination; D051379:Mice; D018345:Mice, Knockout; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length