Study On The Functional Modification Of CD44 Gene In B16F10 Cells Based On CRISPR/Cas9 System

CRISPR/Cas9 system is a new kind of gene editing technology,which appears after ZFNs(zinc finger nucleases)and TALENs(transcription activator like effector nucleases).Comparing with ZFNs and TALENs,CRISPR/Cas9 system is simple and less costly.There are three types of CRISPR/Cas9 system,which are type I,type II and type III.Type II is the easiest one among them,and people change it into a targeted genome modification technology,which refers to CRISPR/Cas9 system.CD44 is a group of widely distributed multi-molecular form of membrane integrated glycoprotein,which can mediate adhesion in cells and participate in cell-specific adhesion processes,including between cells and cells,between cells and the substrate.A large number of studies have shown that CD44 molecules in many malignant tumors,involved in tumorigenesis and regulation,but the current mechanism of CD44 molecules is not clear,so the establishment of CD44 expression decreased mouse melanoma(B16F10)cells which have great significance for studying CD44 protein.Research purpose: To establish a cell line with reduced expression of CD44 in mouse melanoma cells by CRISPR / Cas9 gene gene editing technique,and to study the biological activity of cells with reduced CD44 expression.Research method:(1)According to the CD44 gene designed sgRNA sequence,the sgRNA sequence were ligated with linearized PX459 plasmid to form recombinant plasmid.The sgRNA / Cas9 expression vector was constructed by sequencing.(2)The recombinant plasmid was transfected into B16F10 cells by electroporation,and the effect of recombinant plasmid on B16F10 cells was detected by flow cytometry and Western Blot.(3)The effect of decreased CD44 protein on the migration of B16F10 cells was detected by wound-healing assay.(4)MTT assay was used to detect the effect of decreased CD44 protein on the proliferation of B16F10 cells.(5)Flow cytometry was used to detect the effect of decreased CD44 protein on the apoptosis of B16F10 cells.Experimental results:The recombinant plasmids sgRNA2 and sgRNA3 were successfully constructed for CD44 gene specific target sites.The transfected B16F10 cells were identified by flow cytometry and Western Blot,and the expression of CD44 protein was decreased.B16F10 cells with reduced protein expression was detected by scratch test,and the migration ability of B16F10 cells decreased after CD44 expression was decreased.B16F10 cells with reduced protein expression were confirmed by MTT assay,and the proliferation of B16F10 cells decreased after CD44 expression was decreased.The results of flow cytometry showed that the decrease of CD44 could significantly increase the apoptosis of B16F10 cells.Conclusion:CRSPR/Cas9 technology can reduce the CD44 protein in B16F10 cells,and impact on the migration of B16F10 cells by CD44 protein reduction.Further experiments show that CD44 protein decreased can cause B16F10 cell viability,cell apoptosis and reduce proliferation.The experimental result will help us to further study the CD44 in tumor cells regulatory mechanisms,to provide other malignant tumor cell high expression of CD44 gene potential application value.