Laboratory Protocols for Conditional Gene Targeting

About this book

Conditional gene targeting is an emerging technique for introducing mutations into the genes of laboratory mice. This collection of protocols comes from one of the world's premier laboratories for gene targeting The Institute of Genetics in Cologne and is intended for newcomers to the field as well as more experienced researchers. Topics covered include: the Cre/loxP recombination system and gene targeting in ES cells; gene targeting strategies; Cre/loxP recombination system; Gene targeting vectors consideration and use of loxP; nonselectable modifications and removal of selection marker genes; large deletions; gene replacement; chromosomal translocations; targeted integrations; conditional gene modification; LoxP-containing transgenes; conditional gene targeting; working with ES cells; ES cell transfection overview; embryonic feeder cells; ES cells handling and use; ES cell culture and transfection; ES clone picking; identification of homologous recombinants; freezing ES clones; thawing ES clones; genomic DNA preparation; cre-mediated neomycin deletion in ES cells; generation of mice preparation; mouse breeding for ES cell injection or aggregation; injection needles and holders; blastocyst preparation/injection; embryo aggregation; blastocyst transfer;.

Contents

Section 1: The Cre-loxP recombination system and gene targeting in ES cells; 1. Gene targeting strategies; 2. Cre-loxP recombination system; 3. Gene targeting vectors - consideration and use of loxP; 4. Nonselectablae modifications and removal of selection marker genes; 5. Large deletions; 6. Gene replacement; 7. Targeted integrations; 9. Conditional gene modification; 10. LoxP-containing transgenes; 11. Conditional gene targeting; Section 2: Working with ES cells; 12. ES cell transfection overview; 13. Embryonic feeder cells; 14. ES cells - handling and use; 15. ES cell culture and transfection; 16. ES clone picking; 17. Identification of homologous recombinants; 18. Freezing ES clones; 19. Thawing ES clones; 20. Genomic DNA preparation; 21. Cre-mediated neomycin deletion in ES cells; 22. Generation of mice - preparation; 23. Mouse breeding for ES cell injection or aggregation; 24. Injection needles and holders; 25. Blastocyst preparation/injection; 26. Embryo aggregation; 27. Blastocyst transfer; Appendices; References; Selected references for further reading

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