ZFNs Mediated Liver-specific Exogenous Gene Hsfat-1’s Pig ROSA26 Site-Specfic Integration And Reconstructed Embryo Preparation

As the essential fatty acids of the human body, n-3 PUFAs may reduce the risk of a variety of diseases. Lacking of n-3 or n-6/n-3 ratio imbalance are not only harmful to the developmental stages of the human body but although have a negative impact on metabolism throughout the whole life. However, due to the lack of appropriate enzymes in the human body, products of n-3 PUFAs were limited,we must rely on food intake. Fatty acid desaturase encoded by C. Elegans fat-1 gene, involved in catalize the n-6 fatty acids to the n-3 fatty acids and balancing body n-6/n-3 ratio. Therefore, animals transfected by fat-1 gene will become an important source of essential n-3 fatty acids. In the process of traditional transgenic pigs preparation, target gene usually integrate in a random way, it is hard to control the expression and regulation of the target gene. In this study we used zinc finger nuclease enzymes combined with traditional targeting technology to guide hsfat-1 gene integrate into pig ROSA26 locus, achieved site-specific integration, improved the integration efficiency and reduced the security risks of transgenic animals, researches are as follows:1-In our study, C.elegans fat-1 gene was optimized to hsfat-1 gene to facilitate its expression in mammalian cells, according to the mammalian codon usage preference. Mammalian expression vector pcdna3.1 (+) hsfat-1 was then constructed and transfected to PEF cells, after G418 selection positive clone were then cultured with AA, fat acids were calculated by GC-MS to evaluate catalyze ability of fatty acid desaturase. Statistics show that, fatty acid desaturase encoded by the optimized hsfat-1 gene was capable of convert n-6 fatty acids into n-3 fatty acids, reducing n-6/n-3 ratio at least 2 times when comparing to negative control group.2^ The "friendly loci"-mouse ROSA26 (Gt (ROSA) 26Sor) gene locus was widely used in transgenic animal production, until now nearly 130 knock-in mouse lines had been constructed successfully. In this research, we used mouse ROS A26 locus as template to search the whole pig genome published online and got the pig ROSA26 locus, based on this result zinc finger nucleases were designed and selected to facilitate site-specific cutting. Then site-specific knock-in vector concluding pig liver-specific ApoE promoter、 hsfat-1 gene were builded and were transfected to PEF cell line after linearization. Under G418 selection 125 clones were collected and identified by PCR,16 of were confirmed as positive with correct integration, correct integration rate reached to 12.8%, this provided enough materials for the preparation of cloned pigs.3% Oocytes extracted from healthy sow were matured in vitro, enucleated and fused with positive hsfat-1 cells to prepared reconstructed embryos. Reconstructed embryos were then implanted to 6 estrus sows,cloned pigs are expected to be born in this August.