Double knockouts. Production of mutant cell lines in cardiovascular research.

Abstract

Double knockouts by homologous recombination is a method for producing cell lines with an inactivating mutation in any desired gene. The biochemical analysis of genetically altered cell lines has been important in determining the function of specific proteins. Until recently, mutant cell lines have been produced by random mutagenesis and then selection for a particular phenotypic change. Recent technological advances in gene targeting by homologous recombination now enable the production of mutants in any desired gene. Diploid cells contain two copies or alleles of each gene encoded on an autosome (nonsex) chromosome. In most cases, both alleles must be inactivated to produce a phenotypic change in a mutant cell line, hence the term "double knockout." We and others have described the production of mutationally altered cell lines by inactivating both alleles by the production of two targeting vectors, two separate homologous recombination events, and selection. A simpler procedure, involving considerably less effort and time, has been used to inactivate several alpha-subunits of G proteins and other genes. This method facilitates the inactivation of more than one gene in a single cell line.