| Bovine tuberculosis is a kind of zoonotic infectious disease,and its main pathogen is Mycobacterium bovis(M.bovis).Due to the lack of effective vaccine,bovine tuberculosis has not been effectively controlled in developing countries without sufficient financial support.Natural resistance-associated macrophage protein 1(NRAMP1)gene has pleiotropic effect on macrophage activation pathway and can inhibit the infection of many intracellular parasites.Its expression level in the body also affects the body’s resistance to M.bovis.Therefore,improving the expression of NRAMP1 gene in cattle by transgenic method will provide a new way for the prevention and control of bovine tuberculosis.In the early stage of the laboratory,the Clustered Regularly Interspaced Short Palindromic Repeats and CPISPR-associated protein 9(CRISPR/Cas9)gene editing system combined with somatic cell nuclear transfer(SCNT)was used for the first time to breed the cloned cattle with the NRAMP1 gene integrated at the ROSA26 locus.In this study,3 NRAMP1 transgenic cloned cattle were used as experimental materials,and molecular identification and antituberculosis function tests were carried out to prove that the transgenic NRAMP1 gene cloned cattle can be used for further genetically modified organism safety evaluation and improved breeding.The main contents of this study are as follows:1.Precise target detection of NRAMP1 transgenic cattle.The ear tissues of transgenic Holstein calves were collected,and calf fibroblasts were isolated and cultured by the tissue block adherence method;after the calf fibroblast genome was extracted,three NRAMP1 cloned cattle were detected by Junction PCR and Sanger sequencing.A precise insertion was performed at the target site of the ROSA26 locus,and then the directional integration of the NRAMP1 gene was further verified by Southern Blot experiments,and it was a heterozygous single-copy insertion in transgenic cloned cattle;Cas-OFFinder software was used to predict potential off-target sites,the 8 potential off-target sites with the highest offtarget risk were detected.Sanger sequencing showed that there were no typical indels and site mutations in all off-target sites analyzed.2.NRAMP1 transgenic cattle anti-tuberculosis function test.Real-time fluorescent quantitative PCR was used to detect the expression levels of the adjacent genes(SETD5,THUMPD3 and LHFPL4)of the ROSA26 locus.The results showed that the foreign insertion of NRAMP1 gene in the transgenic cattle genome did not affect the transcription of adjacent genes;Heart,liver,spleen,lung,kidney,skin and muscle tissues were tested,and it was found that the NRAMP1 gene was only expressed in the spleen rich in macrophages,indicating that the expression of the inserted gene was tissue-specific;isolated by Western blot The NRAMP1 gene expression in transgenic bovine macrophages was significantly increased after challenge and the expression level was higher than that of wild-type cattle.The CFU analysis was performed on bovine peripheral blood macrophages after challenge.It shows that NRAMP1 transgenic cloned cattle can inhibit the proliferation of M.bovis in macrophages and show stronger anti-tuberculosis ability than wild-type Holstein cows.In summary,this study proved the precise insertion of the NRAMP1 gene in the ROSA26 locus in the transgenic cloned cattle,and the cloned bovine macrophages had stronger anti-tuberculosis infection ability,which provided an important research basis for biosafety evaluation and improved breeding of transgenic anti-tuberculosis cattle. |
