A Preliminary Study On The B2m Gene Silencing Mediated By Lentivirus Of The Marmoset

[Background]Immunodeficient animal is a kind of model,which is defective in one or more components of the immune system,caused by congenital genetic defect or artificial means.It plays an important role in the research of human diseases,development of drugs,organ transplantation and so on.The construction of gene modified animal model is an effective tool to explore the molecular mechanism and therapeutic targets of human immunodeficiency diseases.In recent years,many researchers tried to use the emerging genome-editing technique to build a variety of primate disease models,which had made some progress.Lentiviral vector can be used to construct transgenic animals and gene therapy,which can mediate the stable and efficient expression of foreign genes in non dividing cells.Compared to other methods,lentiviral vector has higher infection rate and lower immune response.The combination of characteristics of lentiviral vectors and the effect of RNAi on the inhibition of homologous gene expression can be used to silence target genes in various cells,thereby reducing the expression of specific genes,that to produce a sustained and stable effect of target gene silencing.Beta-2 microglobulin is a low molecular protein that is highly conserved.B2m is a kind of light chain part of the major histocompatibility complex MHC class I molecule,which can maintain the structure stability of MHC class I molecules and promote its expression on the cell surface.It has been proved that the deficiency of B2m gene may lead to the absence of CD4-CD8+ T cells in mice,which may lead to immune deficiency.This study is to investigate whether the expression of B2m gene in primates will have similar molecular mechanism to rodents or not.The marmoset is a kind of small non-human primate animal modelwith small size,close genetic relationship with human beings.Thus it is an ideal animal model forthe tumor research of pathogenesis and treatment.[Objective]To select the sequences which are completely homologous with shRNA target sequences of human B2m gene,we screen and determine the effective silencing targets of B2m gene at the cellular level in marmoset.RNA interference technology is used to silence the marmoset ?-2m gene.We analysed the changes of CD4 and CD8 cells in peripheral blood,aiming to lay a foundation for the construction of immunodeficiency marmoset model.[Methods)(1)First we screened the effective silencing targets of marmoset B2m gene at the cellular level,thensynthesized shRNA interference sequences,and inserted them into the lentiviral vector mediated by RNA interference.The two lentiviral expression plasmids were transfected into 293T cells mediated by polyethylenimine(PEI).The expression of B2m gene in transfected cells was detected by real-time fluorescence quantitative analysis after transfection for 48 hours.(2)Detection of the normal expression level of CD antigen(CD4 and CD8)in marmosets:There are 20 healthy marmosets have never been used for experiments.Each marmoset was drawed 300ml hindlimb venous blood.Wedetected and analysed the general average level of CD4 and CD8 cells of marmosets by flow cytometry.The aim of this study is to provide experimental basis for the following animal experiments.(3)Lentivirus mediated B2m gene silencing of marmosets:The target with interference efficiency greater than 70%was selected to be packaged into lentivirus mediated RNAi.The marmoset injected with RNAi vector were experimental group;The marmoset injected with the no-load viral vecor were negative control group.500ml peripheral venous blood was drawed every week for flow analysis and blood routine test,to analyze the changes of the levels of CD4?CD8 cells and CD4/CD8 ratio.[Results](1)Compared with the shRNA sequence of human B2m gene,two gene-silencing sequences were screened that lied in 290bp?310bp and 665bp?685bp of the marmoset B2m mRNA,which has statistical significance in silencing rate:(46.54±7.91)%(P<0.05)and(83.22±4.37)%(P<0.0001).The second target with silencing efficiency of more than 70%,which is suitable for subsequent animal level experiments.(2)The 20 venous blood samples from marmosets were analyzed by flow cytometry,the results were statistically analyzed in the form of x ± s,The value of CD4%is(32.9 ±7.3)%;The value of CD4%is(24.9±6.6)%;The CD4/CD8 ratio is(1.47±0.45).(3)The blood samples were analyzed by flow cytometry,the results showed that compared with the control group,in the experimental group,the number of CD4?CD8 cells and CD4/CD8 ratio decreased significantly,thus it can affect the normal immune function of normal marmoset.Conclusions Marmoset B2m genes were successfully silenced on cell level;Statistics analysing the average level of CD4 and CD8 cells of the marmoset in our work unit;The lentiviral RNAi expression vector can be safely and effectively infected in marmoset,and led to marmoset CD4?CD8 cells and CD4/CD8 ratio decrease.