The Study On CRISPR/Cas9 Mediated EGFP Gene Targeting At Cattle NANOG Locus

Nanog is a new transcription factors that is expressed in the inner cell mass,primordial germ cell and embryonic stem cells(ESCs).Nanog,a ANTP and a NK family gene,plays an important role in embryonic development and stem cell differentiation.In this study,we designed a gRNA for a NANOG gene sequence and constructed the expression vector CRISPR/Cas9.Based on the cleavage site of CRISPR/Cas9,homologous targeting vector pNlrkt was constructed,with the plasmid for the skeleton vector,which has Neomycin resistance gene(Neor)expression cassettes and reporter gene EGFP.EGFP is the exogenous gene and the gene upstream region contains the 801bp 5'homologous arm,containing 370bp 3'homologous arm at the downstream region of Neomycin resistance gene(Neor)expression cassettes.The results of restrictive enzyme fragment analysis and sequencing analysis indicated that the constructed vector pNlrkt is correct.The vectors of pNlrkt constructed successfully were transfected into MelI cell lines of human embryonic stem cells with lipofectin transfection.After 24 hours in culture,green fluorescence was monitored under fluorescent microscope.The results indicated that pNlrkt can guide EGFP and make it emit fluorescence in human embryonic stem cells.To obtain the cell lines of EGFP targeted integration into NANOG gene locus and detect the integration efficiency of CRISPR/Cas9 mediating exogenous gene,we transferred CRISPR/Cas9:pNlrkt into Qinchuan cattle fetus fibroblasts with electrotransfection.Finally,we obtained 69 cell lines by G418 screening,monoclonal cells sorting with mouth pipette and flow cytometer,monoclonal cells cultivation and amplification,and we obtained 4 cell lines of exogenous gene targeted integration by the PCR detection of 3'homologous arm and 5'homologous arm.In this study,we finally obtained 4 cell lines of NANOG targeted integration.These cell lines play important roles in the following study of Nanog in Qinchuan cattle,improving embryo development rate and promoting the development of animal husbandry.Furthermore,these cell lines lay the foundation for the building of stem cell lines and the induced pluripotent stem cell lines in Qinchuan cattle and provide the materials for the somatic cell nuclear transfer and embryo transfer.