Study On Silencing Of P53 And P21 Gene In Marmoset Associated With Adeno - Associated Virus

Background P21 and P53 are two important tumor-suppressor genes, involved in cell cycle regulation, promote apoptosis, gene repair, correlations between tumorigenesis and tumor progression.There are lots of researchs and studies in rodents, but because the rodents are different with human in genetics and physiology, its applications were seriously restricted.Nonhuman primates have highly homology with human on the tissues structure,immune, physiological and metabolic, the application in biomedical research is not very wide in case of the data not comprehensive because of economic condition and the experimental conditions, so the P21 and P53 genes research have an important meaning in marmoset. It can be used to gene function and mechanism of tumor formation research, new drugs development, etc.Objective We used RNA interference to knockdown marmoset P21 and P53 gene expression in cell and animal level, in order to establish a strategy of gene regulation in non-human primates.Methods (1) At cellular level,firstly design shRNA inserted into the adeno-associated virus vector after bioinformatics analysis, then P53mRNA and P21 mRNA inhibiting effect were detected by qPCR after transfected vector into cos-7, p53 and p21 protein change were detected by Western blot. (2) In animal level, optimized the P53 gene interference sequence to be packaged adeno-associated virus; The adeno-associated virus infection effectively and strength were detected in vivo fluorescence imaging after intravenous injection, take a little of liver tissue by surgery and HE staining test cell morphology, p53 protein were detected by Western blot and IHC.Results (1) At cellular level,all of the P53 and P21 gene interference sequence lies in the ORF origin. We screened three effective interference sequences for P21 gene. The expression of P21 mRNA was suppressed (65.2±8.3)%, (66.8 ±8.6)%,(71.4±7.9)% (P<0.05) respectively (P<0.05), and the expression of p21 protein was decreased (64.7 +6.5)%,(65.3±5.9)%,(68.1±4.8)% respectively (P<0.05). We screened two effective interference sequences for P53 gene. The expression of P53 mRNA was suppressed (82.7±8.1)% and (80.7±7.5)%, respectively (P<0.05), and the expression of p53 protein was decreased (77.3±11.5)% and (73.7±10.7)%, respectively (P<0.05).(2) In the overall level of marmoset, the 8 adeno-associated virus tilters reaches above 1×1012vg/ml, Two experimental marmoset can see visible distribution in the liver,testes,neck in vivo fluorescence imaging after virus infection;Four weeks later, take a little of liver tissue. The p53 protein was detected no obvious changes in the liver by Western blot, immunohistochemistry.Conclusions We knockdown the marmoset P53 gene and P21 gene expression at cellular level; However, the p53 protein is not significantly changes in the liver of marmoset; The 8 adeno-associated virus can be safe and effective infection a part of organs in marmoset; We should optimize the way of infection, virus dose, protein detection method in the future.