Production of a Homozygous Mutant Embryonic Stem Cell Line (Double Knockout)

Abstract

Homozygous mutant embryonic stem (ES) cell lines have proven very useful in studying gene and protein function, and there are several reasons why this system might be preferred. Because many genes express their phenotype at the level of individual cells, using these cell lines eliminates the time and expense of producing a mutant animal. They can be isolated directly from a heterozygous mutant cell line without rederiving a cell line or using primary tissues from a mutant animal. In some cases, analysis of the phenotype of homozygous mutant cells can be performed more easily in culture. In addition, homozygous mutant ES cell lines may be produced even when a homozygous mutation is lethal to the animal. Finally, microinjection of homozygous mutant ES cells into blastocysts may allow investigation of the developmental potential of cells with such lethal mutations. This unit was moved from Chapter 9 to reflect a more appropriate placement, and has been updated and revised.