Talen-mediated SP110 Knockin In The PRNP Locus Endows Goat With Increased Resistance To Tuberculosis And Scrapie

Tuberculosis is a class of disease caused by Mycobacterium tuberculosis(MTB).The spread and epidemic of tuberculosis has a serious impact on the development of the world's agricultural economy and public health.The SP110 is a gene that regulates the innate immunity of tuberculosis-also known as intracellular pathogenres(1).A large number of studies have shown that SP110 gene can effectively inhibit the proliferation of Mycobacterium tuberculosis in macrophages,and can control the death of macrophages.Sheep pruritus is one of prion diseases,which is caused by mutated prion protein(PrP),which is contagious and non-curbable.Knockout of animal endogenous prion protein can resist prion infection,so that animals have disease resistance.TALENs targeted knockout(knock-in)technology is a relatively new molecular biology tool that enables efficient and specific modification of the genome of animal and plant cells.The aim of this study was to insert the SP110 gene into PRNP site of goat genome by the TALENs gene targeting technique,and to produce double-transgenic goat with both anti?tuberculosis and anti-rash pruritus.The main contents are as follows:1.Functional validation of SP110 gene in sheep macrophages.The eukaryotic expression vector pGH-SP110,which regulates the expression of SP110 with macrophage-specific promoter,was constructed.The pMSR-SP110?pCDNA3-SP110 and pCDNA3 vector were transfected into sheep alveolar macrophages.Macrophages were then challenged with M.Smegmatis in vitro.The results showed that the number of viable M.Smegmatis released from the alveolar macrophages transfected with pGH-SP110 was significantly lower than that of pCDNA3 control group,indicating that SP110 has enhanced macrophage resistance to the growth and proliferation of Mycobacterium smegmatis.2.Construction and activity identification of TALENs expression vector.Six groups of TALENs targeting PRNP gene locus were designed,constructed,and then transfected into sheep fibroblasts to analyze their cleavage activity.The sequence near the target gene site was amplificated by PCR.The gene mutation effiency of TALENs vector was analyzed by T7E1 endonuclease identification,BamHI digestion and TA cloning and sequencing.Of the six pairs of TALENs developed,TALEN-L4/R1 cleaved the target site more efficiently,the efficiency is about 35%.3.Construction of MSR-SP110 vector targeting goat PRNP locus and production of transgenic cell lines.The MSR-SP110 targeting vector was designed for the PRNP locus in goat chromosome 13.The 5 'homologous arm and the 3' homologous arm sequence on the third exon of the PRNP gene were amplified by PCR,the puromycin gene was used as positive screening gene and DTA as a negative screening gene,to build the target vector pT-SP110.Then,the pT-SP110 and TALEN-L4/R1 were co-transfected into 30-day-old goat primary fetal fibroblasts,102 resistant cell clones were isolated by drug screening.The genotypesof those cells were identified by two rounds of PCR.The results showed that five cell clones were undergo homologous recombination,the targeting efficiency was 4.9%.4.The production of transgenic cloned goat by somatic cell nuclear transferand the preliminary analysisof cloned goat.The recombinant cells(#78)were designated as nuclear donors for nuclear transfer,andfinally two pregnant goats were obtained.One cloned fetus at day 65 of gestation wasisolated by surgery for analysis.Genotype detection showed that the PRNP gene locus was homologously recombined(targeting the SP110 gene),RT-PCR detection showed that the mRNA of protein gene was down-regulated,Western blot analysis confirmed that the expression of endogenous prion protein in the cloned goat was decreased significantly,and the susceptibility to goat scrapie will be reduced.Meanwhile,the inserted MSR-SP110 will initiate macrophage-specific SP110 gene expression when the goat grows up,thus enhancemacrophage antibacterial functions.Coclusion:In this study,MSR-SP110 expression cassette was inserted into the PRNP gene locus by homologous recombination in transgenic goats.The inserted MSR-SP110 expression cassettedestoryedone PRNP allelefunctionally,also will express spllO in macrophage in later.This study will result in both anti-tuberculosis and anti-pruritus transgenic goats,which are not only important for the defense and control of tuberculosis and prion diseases,but also provide new ideas for animal disease breeding and disease model development.