Pig gene knockout by rAAV-mediated homologous recombination: comparison of BRCA1 gene knockout efficiency in Yucatan and Göttingen fibroblasts with slightly different target sequences.

In this study, we compared the gene targeting efficiencies of two rAAV-BRCA1 KO targeting constructs in Yucatan and Göttingen minipig fibroblasts. The homology arms of the constructs consisted exclusively of exonic sequences amplified by PCR from Yucatan genomic DNA. The sequences were identical to those of the reference porcine genome of a Duroc sow (Ensembl Susscrofa 9) and the BRCA1 gene of the Landrace breed (NCBI acc. no. AB271921). Surprisingly, we found that the very efficient gene targeting observed for Yucatan fibroblasts (35% targeting efficiency) was completely absent using either of the two constructs in Göttingen fibroblasts. Sequencing of the relevant BRCA1 exon 11 region (~2 kb) in the Göttingen minipig revealed three single nucleotide differences in the sequence targeted by the left homology arm of the construct (0.3% of the bases) and three or seven in the two right homology regions (0.3 or 0.7% of the bases, respectively). Construction of a novel rAAV-BRCA1 targeting vector based on the Göttingen genomic DNA sequence re-established gene targeting although the efficiency was somewhat lower than that observed for Yucatan fibroblasts. These BRCA1 KO Göttingen fibroblast clones have been used as nuclear donor cells for somatic cell nuclear transfer to generate a Göttingen BRCA1 KO pig model as previously done with the Yucatan breed. The present study illustrates that even a few mismatches present in the homology arms of an efficient rAAV-targeting construct can completely abolish gene targeting by homologous recombination emphasizing the importance of using isogenic DNA even for creating targeting constructs consisting of exon sequences only.