A Preliminary Study On The Technical System Of Knockout Of Cynomolgus Monkey APOBEC3G Gene

Acquired immunodeficiency syndrome(AIDS)is a disease of the fatal lethal immune system which causes by immunodeficiency virus(HIV).Although a large number of exploratory studies have been conducted on the disease at home and abroad,one of the main reasons for the fact that the disease is still ineffective is the lack of a related animal disease model that is resistant to HIV pathogenesis.Many studies have shown that APOBEC3 G is an important limiting factor for the inhibition of retroviral infection.A polipoprote in B mRNA editing enzyme catalyzes peptide-like protein with cytosine deaminase activity.In the reverse transcription reaction,APOBEC3 G catalyzes the cytosine deaminasil in the negative chain cDNA of the virus,triggering a large number of base mutations in the genome and play an antiviral effect.The HIV-1 encoded VIF protein can antagonize the antiviral activity of APOBEC3 G by ubiquitin-proteasome degradation pathway.CRISPR / Cas9 has been modified to become a genomic editing tool for efficient and specific recognition and cutting.When the exogenous body invades the body,the body will recognize the exogenous fragments at the same time CRISPR sequence will remember RNA,when the phage and other invasion of the second time the machine will automatically identify the CRISPR system of the enzyme in the identification of the genome at the recognition,so as to achieve editing.And this technique is used to modify the target after the target gene for cutting,play a role in the modification of gene expression changes.This paper hopes to knock out the genes that express CD domains on APOBEC3 G,making it impossible to synthesize CD domains,resulting in APOBEC3 G unable to bind to the virus and thus fail to play antidepressant transcription.To this end,this paper designed three consecutive experiments,the specific experimental design,research content andexperimental results are as follows:Experiment 1: Build effective sgRNA on APOBEC3 G.First,two batches of wild-type cynomolgus monkey embryonic fibroblasts were cultured.The primers were designed to obtain the sequences of their exon 6,and the sixth exon SNP was obtained by repeated comparison.And then compared with NCBI,and finally the sixth exon exact sequence,the use of CHOPCHOP and other software design of multiple sgRNA,in vitro validation of the design target site activity.Found that sgRNA1 and sgRNA3 in vitro target site activity is strong,the synthesis of the corresponding plasmid.Experiment 2: Verify knockout on cynomolgus monkey embryonic fibroblasts.Cells were transfected with synthetic plasmids and lopi3000 transfected cells,and the number of proliferating cells after resistance screening was obtained.All of the cells were transfected into the cells.The amplified fragments were amplified and sequenced.It was found that the transfected cells had fluorescent and digested fragments,and the fragments were found to be knocked out in the vicinity of the designed target sites sgRNA1 and sgRNA3.Experiment 3: Verify gene knockout on cynomolgus monkeys.In combination with the site activity and cell knockout,sgRNA3 was selected as an eight-point sequence validated on the embryo.After in vitro synthesis of sgRNA3 and Cas9 mRNA mixed,the use of embryonic injection of 17 embryos were injected,after training to detect the case of knockout.Found that sgRNA3 in the same embryonic knockout.Based on the above experimental results,the sgRNA1 and sgRNA with strong activity were obtained and the corresponding plasmid vector was constructed.The vector was tested on cynomolgus monkey embryonic fibroblasts and cynomolgus monkeys.Points are knocked out.In this paper,we established the corresponding technical system for establishing the APOBEC3 G gene of cynomolgus monkey,which provided the idea for establishing the animal model of AIDS susceptible disease.For the early get the AIDS susceptibility model took a solid step.