Production Of Cloned Transgenic Pigs And Preliminary Study On ZFNs Mediated Gene Knock-out In Porcine Genome

Transgenic somatic cell nuclear transfer in pigs has wide applications in basicresearch areas, human medicine and agricultural production. And the new geneticengineering tools, Znic finger nucleases, have largely improved the efficiency oftransgenic pig production. Compared to traditional gene targeting method, zinc fingernuclease is a very powerful gene targeting tool. The application of zinc fingernuclease technology to produce transgenic animals, especially transgenic farmanimals will be the main direction for future development. In this study, we developeda technical platform for transgenic pig production and produced7green fluorescenceprotein transgenic pigs, and we also primarily explored the application of Zinc fingernuclease in gene knock-out pig production.First of all, we optimized three fibrolytic enzyme genes: bgl4, xynB and egxA intwo different codon optimization methods and compared the expression level in fourdifferent mammalian cell lines. We found that “one codon-one amino acid” optimizedsequences had higher expression level than the original ones and the “Humanized”ones. We established two PK15cell lines, which can stable express BGL4and XYNB separately, and the enzyme activity was measuredFive embryo fibroblast cell lines from Lantang pig, ten embryo fibroblast celllines from Landrace pig and one skin fibroblast cell line from a30days old Landracepiglet were established. Embryo fibroblast cell lines from Lantang and Landrace pigwere transfected with pEGFP-N1and pEGFP-BGL4, pEGFP-XYNB plasmids DNAby NeonTMtransfection system, and six transgenic cell lines were gained. We didnuclear transfer experiments using the transgenic cell lines as donor cells and thereconstructed embryos were transplanted into female pigs. At last, we obtained7liveGFP transgenic piglets.In order to design ZFNs which targeting the IGF2and Kit gene of pig, weamplified and sequenced the genomic fragment of Kit gene from intron8to intron11,we also found that this sequence has a10bp insertion differs from the largewhite Kitgene in NCBI. We also sequenced partial fragment of IGF2intron3, verified the QTNtype of IGF2gene intron3of the Landrace and Langtang pig. Landrace is A type andLantang pig is G type. Based on these sequencing results, we custom made zinc fingernucleases (ZFNs) from Sigma Corporation. By T-A cloning sequencing method andCEL-I method, we measured the cleavage activity of ZFNs in PK15cells and PEFcells. Results shows that in Langtang Pig PEF cells, IGF2ZFN set3has the highestactivity,8.91%cells were gene knock-out. And the highest sctivity of Kit ZFNs isfrom set1, which is7.14%. At the same time, by comparing ZFN activity in the PK15and PEF cells, we found that ZFN activities are not consistent in the two types of cells,indicated that cell type has impact on ZFN activity. Through this study, we have amore in-depth understanding of ZFN activity, and most importantly, laid thefoundation for the production of IGF2and the Kit gene knockout pigs.