Pig gene knockout by rAAV-mediated homologous recombination: comparison of BRCA1 gene knockout efficiency in Yucatan and Göttingen fibroblasts with slightly different target sequences
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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.
KeywordsrAAV-mediated gene targeting BRCA1 Homologous recombination Isogenic DNA Yucatan minipig Göttingen minipig
We would like to thank Bert Vogelstein & Kenneth W. Kinzler, John Hopkins University, Baltimore, US, for kindly providing the pNeDaKO plasmid vector and Dr. R. Jude Samulski and the UNC Vector Core Facility, Chapel Hill, North Carolina, US for the rAAV packaging. We thank Ellegaard Göttingen Minipigs A/S for authorizing us to use the Göttingen fibroblasts for cloning. We also would like to thank Trine Skov Petersen for skilled technical assistance. The project was supported by a grant from the ‘‘Pigs and Health Platform’’ of the Danish National Advanced Technology Foundation (Højteknologifonden), the Danish Agency for Science, Technology and Innovation (grant no. 274-05-0535), the DAnish Genetically Modified Animal Resource (DAGMAR), and the ‘‘Sino-Danish Breast Cancer Research Centre’’ under the auspices of the Danish National Research Foundation (Grundforskningsfonden) and the National Natural Science Foundation of China.
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