Generation of conditional Xpac/− mouse with a floxed genomic/cDNA fusion construct.
A) Schematic representation of the wild-type mouse Xpa locus (exon 2 to 6), the targeting construct, the conditional Xpa allele, and the conditional Xpa allele following Cre-recombinase-mediated inactivation. In addition the knock out allele with a neomycin (NEO) marker cassette, as present in the non-conditional Xpa mouse model is shown . In the conditional construct, exon 4 was fused in frame to the mouse Xpa cDNA (containing the remaining coding sequence and including a synthetic polyA sequence), followed by a PGK promoter-driven hygromycin (HYGRO) selectable marker gene. LoxP sites were introduced in intron 3 and downstream of the hygromycin marker to allow Cre-mediated excision of the cDNA and hygromycin marker gene, yielding an Xpa allele lacking exon 4, known to act as a null allele. A LacZ-GFP fusion marker gene, preceded by a splice acceptor (SA) and an internal ribosomal entry (IRES) was included to allow visualization of inactivation of the conditional Xpa allele. Open and gray boxes represent exon and cDNA sequences, respectively. The splice acceptor-Murfi cassette ensures proper splicing and translational stops in all frames respectively when the Xpa gene is knocked out. B) Southern analysis of wild-type ES cell DNA transfected with the conditional Xpa construct. DNA was digested with EcoRI and hybridized with an intron 5/exon 6 probe, external to the targeting construct. Wild-type and targeted alleles gave fragments of 16 and 12 kb, respectively. C) Southern blot analysis of DNA from E13.5 Xpac/− and Xpac/−/Cag-Cre embryos. DNA was digested with EcoRV and hybridized with a LacZ probe. Intact and Cre-inactivated conditional Xpa alleles are represented by 2.4 and 10 kb fragments, respectively. D) LacZ staining of E13.5 Xpac/− and Xpac/−/Cag-Cre embryos, indicating inactivation of the conditional Xpa allele in Xpac/−/Cag-Cre embryos only. E) Survival of wild-type, Xpac/− and Xpac/−/Cag-Cre MEFs, exposed to increasing doses of UV-C light. F) Photograph of 16-day old Csb−/−/Xpac/− and Csb−/−/Xpac/−/Cag-Cre littermates, the latter showing severely reduced size.