Generation Of Transgenic Pigs Which Integrated Glucanase-Xylanase-Phytase Fusion Gene In PSP Site Through Site-Directed Knockin Technology

Feed non-starch polysaccharides and phytate are two major anti-nutritional factors.They have adverse effects on digestion and absorption of nitrogen and phosphorus.To minimize those adverse effects,we have produced transgenic pigs which secrete glucanase,xylanase,and phytase in their salivary glands through piggybac transposon system.However,the exogenous genes integration manner of piggybac transposon system is random,so it poses a great inconvenience for the breeding of homozygous transgenic lines.Rstricted by the promoter length of parotid gland secretory protein(12.6kb),the integtration efficiency of the homologous recombination based transgenic technology is very low.In order to reduce the difficulty of generating environmental-friendly transgenic pigs,we explore the feasibility of using endogenous PSP regulatory elements to control expression of non-starch polysaccharide enzyme and phytase.Ous expesriment will pave a way for the future development of parotid gland reactor and the production of new environmental-friendly transgenic pig model.Based on the CRISPR-Cas9 mediated site-directed transgenic technology,our study integrated BgEgXyAp into 3'-end site of PSP locating right before the termination codon.A P2 A sequence is used for the fusion expression of PSP and BgEgXyAp.The main results are as follows:(1)In order to obtain high efficiency target site,four sgRNAs were designed to target the DNA sequence along the termination codon of pig PSP.T7 EI assay showed that the indel rates of these four sgRNA were 70.0%,69.9%,69.9%,and 0% respectively.Since sgRNA3 is located at the termination codon,it was selected for subsequent experiments.(2)The homologous target vector pMD-BgEgXyAp was successfully constructed using In-fusion technology.(3)The Cas9 expressing plasmid sPSP-Cas9 and the homologous target vector pMD-BgEgXyAp were cotransfected into Duroc boar fetal fibroblast line 6F10 and 7F3 respectively.After continuous screening by G418,three cell lines with high purity green fluorescence were obtained,named 6-1,6-2,and 7-1 respectively.PCR detection assay and sequencing analysis showed that the exogenous gene of all three cell lines have been integrated correctly and the three cell lines are all single allele knock-in.Among them,11 bp deletion of wild-type allele was detected in cell line 7-1,but the wild-type allele of cell line 6-1 and 6-2 did not mutate.(4)Cell screening marker neoGFP was successfully deleted using cre recombinase.Eight live cloned pigs were produced uing SCNT tehnology and embryo transfer technology.PCR detection assay showed that all the cloned pigs are positive transgenic.Sequencing analysis showed that the screening marker has been successfully deleted in all positive transgenic pigs.(5)Two transgenic pigs from cell line 6-1 and 7-1 were used to detect mRNA of the exogenous gene BgEgXyAp by RT-PCR technology.Results showed that no mRNA of BgEgXyAp was detected in parotid gland,submandibular gland,and sublingual gland of these two transgenic pigs.(6)Saliva collected from 2-months-old transgenic pigs was used to detect NSPase and phytase activity.Results showed that no ?-glucanse activity,xylanase activity,and phytase activity were found in saliva of all of the positive transgenic pigs.In summary,we have successfully integrated multi-cistronic BgEgXyAp into the 3'-end of pig PSP and achieved gene fusion of endogenous PSP and exogenous BgEgXyAp through a P2 A sequence.We did not detect any BgEgXyAp expression in mRNA level and protein level in our transgenic pigs.The absence of the BgEgXyAp expression indicated that the endogenous regulatory elements of pig PSP can not initiate BgEgXyAp expression.