Foxp3 Gene Targeted Editing In Mouse Lymphocytes Based On CRISPR/Cas9 System

The CRISPR/Cas system is an adaptive immune defense mechanism for bacteria and archaea in the evolving process that protects its own genome of interference and destruction by exogenous nucleic acids such as phages and viruses.CRISPR/Cas system as a set of adaptive immune system,used CRISPR RNA(crRNA)in the form of complementary bases to guide Cas protein to identify the invasion of foreign genomes,and cleave DNA.The CRISPR/Cas system is divided into Type I,Type II and Type III according to the sequence and structure of the Cas protein.Which type I and type III systems require multiple copies of the original protein,and type II only requires the Cas9 protein.The CRISPR/Cas system(CRISPR/Cas9 system)has evolved into an ideal set of procedural editing tools.The main purpose of this paper is to design the target site gRNA for Foxp3 gene,and to construct the target modified vector system by CRISPR/Cas9 system,and to edit the Foxp3 gene locus.Then the biological function of Foxp3 was explored.The Foxp3 gene was knocked out by CRISPR,and Foxp3 knockout cells were utilized to study the biological characteristics.We are applied this technique to tumor mouse models to reduce the expression of Foxp3 gene in tumor cells,prevent the production of Treg cells,and inhibit the growth of tumor cells in mice.The main methods and results used in this article:1.Using the UCSC website to determine the mouse Foxp3 gene exon,utilizing CRISPR Design to retrieve the potential target site,by BLAST to determine the final targeting sequence.2.The synthesized target sequence primers were connected with the carrier plasmid PX458 digested by endonuclease Bbs I,and the target plasmids were successfully constructed by enzyme digestion,electrophoresis and universal primers sequencing.3.The mouse spleen cells were successfully transfected by electroporation transfection technique and the expression of GFP tags carried by plasmid plasmids was detected by fluorescence microscopy.4.Using RT-PCR technique and flow cytometry to detect Foxp3 cell staining,the transfection of Foxp3 gene targeted knockout plasmid was identified at mRNA and protein levels,and the expression of Foxp3 in spleen cells of mice was reduced.5.The target knockout vector plasmid was successfully transferred into tumor mice by intravenous injection and in vivo gene introduction technique,and the transfection of target knockout vector plasmid was effectively determined by anatomically to reduce the number of tumors in tumor-bearing mice And tumor rate.6.Analysis of spleen cell count,blood cells and flow cytometric detection of cytokines in lymphocytes by targeting plasmid transfected mice,to determine the expression of Foxp3 in mice reduced effect on spleen cell number,lymphocytes and cytokines.The main conclusions of this paper:1.In the first part of the experiment,we constructed and screened the CRISPR/Cas9 vector targeting the Foxp3 gene in mice.2.In the second part of the experiment,the plasmid was expressed in mouse spleen cells,and it was shown that the target vector we constructed in the previous part played a role in the cell and reduced the overall expression of Foxp3.3.In the third part of the experiment,targeted knockout vectors can function in mice and prevent the production of Treg cells.Detection of targeted knockout vector in vivo transfection of mouse blood lymphocyte percentage increased obviously,while the content of serum IL-2,IFN-? rebounded significantly,the proportion of T helper cells increased growth and inhibition of tumor development in mice.