Production Of Transgenic Goat By Site-specific Integration Of Natural Resistance-Associated Macrophage Protein 1 (Nrarymp1) Gene

Tuberculosis is a chronic,malignant,infectious zoonotic disease that threatens global health,there is currently no effective means to prevente and cure it.Mycobacterium tuberculosis entering the body through the respiratory systemand forms a phagocytic body by binding to the receptor of membrane surface of the pulmonary macrophages.Intracellular parasitic Mycobacterium tuberculosis obtains iron and other metal ions to produce a large number of enzymes,which can resist the cytotoxic effect of macrophages.So,intracellular parasitic Mycobacterium tuberculosis is difficult to be removed by the host.Through enhancing the expression of goat Nrampl gene to cultivating the disease-resistant population becomes a new point of control of goat tuberculosis.The Cre/loxP recombination system consists of Cre recombinase and loxp site.Cre recombinase can recognize the specific loxP site and deleted or reorganized the gene between loxP sites.This method has the following advantages:(1)The foreign gene can be accurately integrated into the specific locus of the host chromosome.(2)Reducing the copy number of the foreign gene in the genetically modified organism,and the majority of the foreign gene is single copy.In this study,the Nramp1 transgenic goat was prepared by integrating the Nrampl gene into the genome of human lysozyme(hLYZ)transgenic goat cells through the Cre/loxp recombinase system.hLYZ transgenic fibroblasts were isolated from the ears of human lysozyme transgenic goats.The hLYZ cell genome includes the expression framework of box exchange system loxpl-Neo-Tk-loxp.loxp1 means the reverse repeat sequence "ATAAC" of the loxp site was mutated to "CACCT" The expression profile of the Nramp1 gene was synthesised.Its amino acid at position 132 is mutated from isoleucine(I)to threonine(T).This mutein is specifically expressed in macrophages and has a stronger resistance to intracellular bacteria,which is particularly useful for the prevention and treatment of infections caused by intracellular bacteria.The Nrampl expression vector plasmid pTM-Nrampl,that is the framework of loxp2-polyA-Nrampl-Purocoda-loxp.was obtained.The pBS185 plasmid and pTM-Nramp1 plasmid were cotransfected into hLYZ fibroblast which named 3-376?.After screened by puromycin and identified by PCR and sequencing,22 monoclonal cells were obtained.Among these cells,3 monoclonal cells were verified that Nrampl had integrated into the predetermined site.The targeting integration rate is 13.6%(3/22).No.11 monoclonal cell was selected as the nuclear donor because of its good growth.The 50 donor goats were superovulated and 13 transplant recipients were induced oestrous synchronization.The total number of oocytes was 155,the number of eggs was 111,and 105 eggs were obtained after fusion.The fusion rate was 94.59%(105/111).The reconstructed embryos were transplanted into the fallopian tubes of 13 recipient goats.After the somatic cell nuclear transfer,B-ultrasonography was performed on 13 recipient goats at 35 days.There were 6 recipient goats pregnant.The pregnancy rate was 46%(6/13).One of them was taken out by caesarean section at 47 days.Now one lamb was born.The fetus and the newborn lamb were all Nrampl targeted rams by PCR detection and sequencing identification.The new lamb is healthy till now.This study laid a solid foundation for the expansion of the Nrampl transgenic goat.