FIG. 1.
Gene targeting of the CDK4 gene in ES cells and generation of CDK4-null mice. (a) Targeting replacement vector. A genomic fragment containing the six exons encoding CDK4 was isolated from a 129/svj mouse genomic library, and a 6.0-kb EcoRI-NotI fragment was subcloned into the pBluescript plasmid vector. The homologous recombination was constructed by replacing a 2.2-kb MscI fragment of this plasmid with a neomycin-phosphotransferase gene under the control of the thymidine kinase promoter for the positive selection. An HSV thymidine kinase gene under the control of the polyoma enhancer was then inserted at the 3′ side of the 1.2-kb homologous flanking region for the negative selection. A 1.2-kb BamHI-EcoRI genomic fragment upstream of the 2.6-kb 5′ homologous region was used as a probe to distinguish the wild-type (wt) and mutant (mut) alleles. (b) Germ line transmission of the CDK4 mutation. The gene targeting event was identified by Southern blotting with BamHI-digested genomic DNAs and the probe shown in panel a. Germ line transmission was confirmed by the Southern blot with genomic DNAs of the F1 offspring from crossbreeding of chimeras with wild-type C57BL/6 mice. An analysis of a representative F1 litter is shown. (c) PCR genotyping of day 12.5 embryos obtained from intercross between CDK4-heterozyous mice. Wild-type (+/+), homozygous (−/−), and heterozygous (+/−) mice were identified by the presence of PCR products specific for either the wt or mut allele. (d) Immunoblotting for CDK4 with CDK4 monoclonal antibody (DCS-31) with extracts of fibroblasts obtained from (+/+), (−/−), and (+/−) embryos.