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Gene Knockout Protocols

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M E T H O D SINM O L E C U L A RBIOLOGYGene KnockoutProtocolsEdited byMartin J. TymmsNational Vision Research Institute of Australia,Carlton, Victoria, AustraliaandIsmail KolaCentre for Functional Genomics and Human Disease,Institute of Reproduction and Development, Monash University,Clayton, Victoria, AustraliaHumana Press m Totowa, New Jersey1

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PrefaceAs the major task of sequencing the human genome is near completionand full complement of human genes are catalogued, attention will be focusedon the ultimate goal: to understand the normal biological functions of thesegenes, and how alterations lead to disease states. In this task there is a severelimitation in working with human material, but the mouse has been adopted asthe favored animal model because of the available genetic resources and thehighly conserved gene conservation linkage organization.In just of ten years since the first gene-targeting experiments were preformed in embryonic stem (ES) cells and mutations transmitted through themouse germline, more than a thousand mouse strains have been created. Theseachievements have been made possible by pioneering work that showed thatES cells derived from preimplantation mouse embryos could be cultured forprolonged periods without differentiation in culture, and that homologous recombination between targeting constructs and endogenous DNA occurred at a frequency sufficient for recombinants to be isolated. In the next few years themouse genome will be systematically altered, and the techniques for achieving manipulations are constantly being streamlined and improved.Recently new technologies have developed for inducible gene expression in transgenic mice that in combination with conventional gene targetingcan give temporal and tissue-specific expression. These advances have beenspurred on by the desire to study the function of genes that show an embryonic-lethal phenotype when deleted or "knocked out". Gene targeting in miceinitially concentrated on making gene knockouts or null mutations, but increasingly the technology is being used to create subtle point mutations to simulatehuman disease states.Gene Knockout Protocols brings together distinguished contributors withextensive experience in the gene targeting and mouse genetics fields. In linewith the successful format of Methods in Molecular Biology, the volume contains step-by-step protocols for the design of targeting constructs to protocolsfor the analysis of the mouse phenotype. Emphasis has been paid to the inclusion of other techniques used in mouse genetics that are relevant to researchers performing gene targeting. These include embryo transplantation, In vitro

viPrefaceES cell differentiation, creation of aggregation chimeras, mouse pathology, embryocryopreservation, and transplantation. Issues such as the use of existing mousemutation resources and the influence of genetic background and epigeneticeffects upon phenotype are also covered. We hope that Gene Knockout Protocols will be an invaluable source of proven protocols for those just enteringthe field of gene targeting, but also a valuable reference for researchers in theprocess of describing the phenotype of mutant mice.Martin J. TymmsIsmail Kola

ContentsPrefacevContributorsix1 Overview: Gene KnockoutsPaul J. Hertzog and Ismail Kola2 Isolation and Maintenance of Primate Embryonic Stem CellsVivienne S. Marshall, Michelle A. Waknitz,and James A. Thomson3 Gene Targeting in ES CellsThomas M, DeChiara4 Manipulating Mouse Embryonic Stem CellsLino Tessarollo5 Gene Targeting in a Centralized FacilityLouise D. Barnett and Frank Kontgen1111947,.656 The LoxP/CRE System and Genome ModificationTrevor J. Wilson and Ismail Kola837 Creation and Use of a Cre Recombinase Transgenic DatabaseAndras Nagy and Lynn Mar958 Choice of Mouse Strains for Gene TargetingMireille H. Lahoud, Trevor J. Wilson, and Paul J. Hertzog9 Isolation, Microinjection, and Transfer of Mouse BlastocystsAngelika Bonin, Susan W. Reid, and Lino Tessarollo10 Aggregation Chimeras: Combining ES Cells, Diploidand Tetraploid EmbryosMika Tanaka, Anna-Katerina Hadjantonakis,and Andras Nagy11 How to Study Pathologic Phenotypes of Knockout MiceRoderick T. Bronson12 Analysis of Hematopoietic Phenotypesin Knockout Mouse ModelsIvan Bertoncello and Brenda Williams,vii107121135155181