Construction Of Human PTPN12 Gene Konckout A549 Stable Strain By CRISPR/Cas9 Technology

PTPN12(Tyrosine protein phosphatase non-receptor type 12)is a gene encoding human PEST protein,located on human autosomal 7q11.23,highly expressed in related cells of hematopoietic system and mainly expressed in cytoplasm.It consists of an NH2-terminal catalytic domain and a long COOH-terminal domain.The Nterminal has a highly conserved specific motif of the tyrosine phosphatase family,while the hydrophilic C-terminal is rich in proline,glutamate,serine and threonine,regulating the interaction between PEST and the substrate protein.PTP-PEST plays a key role in cell migration,cell proliferation,cell differentiation and other physiological processes through dephosphorylation of phosphate groups.Currently,the researchers found that the decrease of PTP-PEST protein level was related to several known cancers,but the research reports on the function of PEST and the occurrence and development of tumor of PEST were still limited,so the influence of PEST on cell function needs to be further discussed.CRISPR/Cas9 is a new gene editing technology,which has the advantages of high efficiency,high specificity,high precision,etc.,as well as simple and fast operation,and has rapidly developed into a hot new method in recent years.The CRISPR/Cas9 system is composed of CRISPR related genes and Cas9 endonuclease.SgRNA homologous to the target gene was used to specifically bind the DNA sequence of the target cell genome to form heterozygous double strand.Subsequently,the cell repaired the broken double chain through non-homologous terminal connection,homologous recombination repair and other mechanisms,inserted abnormal base or made normal base deletion at the fracture,which led to the shift mutation,so that the protein could not be translated normally,and finally realized the knockout of the target gene.In this paper,CRISPR/Cas9 gene editing technology was applied to the knockout of PTPN12 gene in human lung adenocarcinoma cell line A549,and the stable cell line A549 with PTPN12 gene knockout was constructed to study the function of ptppest.First according to the rules of CRISPR/Cas9 targets design,design specific recognition PTPN12 gene exon 6 related sequence of two sgRNA(Single guide RNA),recombinant eukaryotic expression plasmids PX458-PTPN12-sgRNA1 and PX459-PTPN12-sgRNA2 were constructed.After successful construction,two recombinant plasmids were co-transfected into A549 cells,and the PTPN12 gene knockout A549 cells and stable monoclonal cell lines were obtained by continuous culture.Finally,the monoclonal cell lines were identified by PCR,gene sequencing and Western Blot,and the results showed that we successfully constructed the A549 stable cell line with PTPN12 gene knockout.In addition,in order to study the function of PTPN12,we conducted a series of in vitro experiments on A549 stable cell lines with the final PTPN12 gene knockout.We used cck-8 and in situ counting experiments to observe the effect of PTPN12 knockout on the proliferation of A549 cells,and the results showed that the absence of PTPN12 accelerated the proliferation of cells.The cell cycle was then analyzed with giemsa cell staining and flow cytometry,and the results showed that after PEST knockout,the number of cells in the G2 phase increased,which promoted cell division.Finally,we used cell scratch experiment to verify the changes in cell migration ability,and the results showed that the cell migration ability was enhanced after the knockout of PTPN12 gene in A549 cells.In conclusion,we successfully established A549 stable cell line with PTPN12 gene knockout,providing a gene knockout cell model for the future study of PTPPEST function.Meanwhile,a series of in vitro experiments verified the effect of PTPN12 on cell proliferation and migration,and the results showed that PTPN12 significantly enhanced the ability of cell proliferation and migration.