Production of a Heterozygous Mutant Cell Line by Homologous Recombination (Single Knockout)

AbstractGene targeting by homologous recombination allows the introduction of specific mutations into any cloned gene. This unit provides a protocol in which the gene of interest is inactivated by interrupting its coding sequence with a positive selectable marker. A negative selectable marker is included in the construct outside the region of target gene homology in order to enrich for clones in which the target gene has undergone homologous recombination. The altered target gene is then expressed in embryonic stem cells. A support protocol describes a method for transient expression of Cre recombinase to remove sequences between lox sites, which can also be used as a selection method.