Construction of Zinc Finger Nuclease-induced Targeting Vector of Luchuan Pig (Sus scrofa) Fat1 Gene and Transgenic Study In vitro
HUANG Xing1,2, YAN Ai-Fen2, DENG Ting-Xian2, OUYANG Hong-Jia1, LIU Lian2, FENG Juan2, ZHU Xiang-Xing2, NIE Qing-Hua1, TANG Dong-Sheng2,*, ZHANG Xi-Quan1,*
1 College of Animal Science, South China Agricultural University, Guangzhou 510000, China; 2 Guangdong Gene Editing Engineering Technology Research Center, Foshan University, Foshan 528000, China
Abstract：ω-3 polyunsaturated fatty acid (PUFA) dehydrogenase, the expression product of Fat1 in Caenorhabditis elegans, can convert ω-6 PUFAs to ω-3 PUFAs. Mammals cannot synthesize ω-3 PUFAs due to lack of Fat1 gene. ω -3 PUFAs can only be obtained through the diet to ensure the balance of ω-6/ ω-3 and body's health. In order to obtain the transgenic pig (Sus scrofa) with stable integration and expression of Fat1, experiments on transgenic technology at the cellular level were performed by zinc finger nuclease (ZFN)-mediated multi-site targeting technology. Expression vectors pcDNA3.1-NLS-ZFN-R and pcDNA3.1-NLS-ZFN-L, which were used to target to internal transcribed spacer-1 (ITS1) sequence in Luchuan pig rDNA gene, were constructed. Secondly, primary kidney fibroblast (PKF) cells were successfully isolated from kidney. After 7 d of co-transfection of targeting vector and ZFN vectors into PKF cells, the genomic DNA was extracted and the integration of exogenous genes were detected by PCR. The results showed that Fat1 gene was successfully integrated into the genome of Luchuan pig PKF cells, but not in a site-specific way. This study provides a theoretical basis for further obtaining transgenic pigs.
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