To generate mice with a loxP-containing HNF6 allele (HNF6flox), we constructed a loxP-FRT HNF6flox-neo targeting vector using BAC recombineering. To avoid disrupting potential regulatory regions within the HNF6 locus, mouse and human HNF6 gene homology was compared (using http://genome.ucsc.edu) and loxP sites introduced flanking the cut-domain in regions of less than 50% sequence conservation. A clone containing HNF6 genomic DNA was obtained from a mouse RPCI-22 BAC library (BACPAC Resources, Children's Hospital Oakland, CA). The HNF6 targeting construct also contained a neomycin resistance cassette (neoR) located 5' to the second loxP site and flanked by FRT sites to facilitate deletion by Flp recombinase; and a herpes simplex virus-thymidine kinase (TK) cDNA located outside of the HNF6 gene homology region. Deletion of the cut domain renders the allele a null.

Schematic diagram of HNF6^flox-neo targeting vector and Cre-mediated HNF6 inactivation. (A) Representation of the mouse HNF6 locus showing exon one (the cut DNA binding domain). Also shown: HNF6^flox-targeting construct, HNF6^flox-targeted allele, and recombined HNF6 allele following Cre exposure. The 5' and 3' Southern blot hybridization probes are indicated as horizontal lines below the floxed allele. Restriction sites shown in italics indicate introduced sites to allow for genotyping. (B) Southern blot of SpeI/EcoRV digested genomic DNA from HN6^flox-neo targeted ES cells using the 5’ external probe, detected bands specific to wild type (WT; 13.3kb) and HNF6^flox (fl; 11.6kb) alleles. Using 3’ the external probe, Southern blot of HpaI/SacII digested genomic DNA from HN6^fl targeted ES cells detected bands specific to wild type (WT; 7.6kb) and HNF6^fl (fl; 9.6kb) alleles. (C) PCR analysis of mouse tail genomic DNA using primers flanking the 5’-most loxP site resulted in amplification of a larger product (590bp) from the targeted versus wild type allele (550bp).

PMID: 19766716