Figure 1.

Strategy for pre-emptive complementation of Lig3-null mouse embryonic stem (ES) cells. DNA ligase transgenes (Tg) are stably introduced into Lig3 conditional cells to determine which allow the survival of Lig3^KO/KO cells. In the parental Lig3^KO/cKOneo+ cells, the Lig3^KO allele is null due to deletion of exons 6–14 which encode the Lig3 catalytic core and the Lig3^cKOneo+ allele is conditional, with LoxP sites flanking exons 6–14 and an intronic neomycin resistance gene (neo). Cre recombinase is transiently expressed and colonies are grown in normal (drug-free) media. Colonies are replica plated into media containing G418 to determine which had successfully undergone Cre-mediated recombination to delete the neo gene and hence had become Lig3^KO/KO at the endogenous Lig3 locus; colonies that had retained the neo gene continue to grow in G418 and hence maintain the parental genotype (Lig3^KO/cKOneo+). (A) A Lig3 transgene which expresses both nuclear and mitochondrial Lig3 allows the survival of Lig3^KO/KO colonies. After Cre expression, both G418^S colonies (with Cre deletion; Lig3^KO/KO) and G418^R colonies (parental, without Cre deletion; Lig3^KO/cKOneo+) are recovered. (B) Expression of nuclear Lig3 from a transgene (NucLig3) does not allow the survival of Lig3^KO/KO colonies, indicating that mitochondrial localization of Lig3 is critical for cell survival. Only G418^R colonies (Lig3^KO/cKOneo+) are recovered after Cre expression.