Site-directed Preparation Of XABT Cell Lines Transfected Into Swine Cep112 Locus

Non-starch polysaccharide and phytic acid are the main anti-nutrition factors in the feed,which can significantly reduce the digestibility of nitrogen and phosphorus in the feed and cause nitrogen and phosphorus pollution.In the previous study,our research group has prepared a transgenic pig with co-expression of glucanase,xylanase and phytase in the saliglands,which has significant grain saving and emission reduction effects,but the pig cannot digest cellulose,and the expression level of the target gene is not ideal.Piggybac transposon mediated random integration is the transgenic method,which is not conducive to the cultivation of new transgenic strains.In order to make up for the above defects,further improve the grain saving and environmental performance of transgenic pigs.On the basis of previous studies,this study aims to construct a specific,highly efficient and coexpressed transgenic vector of cellulase,glucanase,xylanase and phytase in site-specific integration,laying a foundation for the preparation of a new generation of environmentally friendly pigs.In this study,CRISPR-Cas9 mediated site-directed integration transgenic technology was used to knock XABT into the stop codon of pig endogenous PSP gene,and the mouse PSP promoter was used to regulate XABT gene fusion expression to obtain a site-directed transgenic cell line.The main research results are as follows:(1)Comparing the effects of 2A and A3 binding peptides,A3 was significantly superior to 2A,we were obtained XABT series of xylanase,phytase,glucanase and cellulase gene.(2)The results of the sensitization and toxicity analysis of the target gene XABT product protein showed that there was no potential safety hazard in the XABT product protein.(3)Recombinant PSP promoter was cloned by homologous recombination technology and cloned into the porcine Cep112 site to obtain four fixed-point targeting plasmid.(4)Cas9 expression vector sgRNA-Cas9 co-transfected PFFs cell lines with three optimized promoter homologous target plasmids,we obtained Cep112 locus fixed-point XABT-positive cell lines.