Knockout Of HGPRT In Rabbit Cell By CRISPR/Cas9 Technique

Objective:In this work we have used CRISPR/Cas9 to design target sgRNA against the rabbit HGPRT gene,which is engaged to perform HGPRT gene knock-out,and then we have selected the HGPRT gene-deficient rabbit myeloma line by monoclonal techniques.HGPRT gene-deficient cells are widely used in the cell-fusion hybrid parents,this experiment used CRISPR technology knock out rabbit cells HGPRT gene,which can be used for rabbit anti-monoclonal cell hybridization.Methods:The exon region of HGPRT genome are find out from GenBank.We have imported the first and longest exon sequences into the online website as the target sequences,designed the sgRNA,selected and screened the highest scoring results.A recombinant CRISPER/Cas9 eukaryotic plasmid harbored the working fragments was constructed,and a lentiviral packaging technique was used to obtain a virus strain harboring the fragment for the knockout of rabbit HGPRT gene by293T cell.The lentiviruses obtained by packaging the recombinant plasmids were used to infect rabbit myeloma cells and screened by Puromycin,6-TG and HAT selection media.The limiting-dilution method was used to obtain the target positive cell clones,which was HGPRT gene-defective rabbit myeloma cells.Results:1.We are successfully designed the corresponding sgRNA,and verify the construction of CRISPR/Cas9 recombinant plasmid with the HGPRT fragments with knockout ability of the target gene.2.After the HEK293T cells were co-transfected with the plasmids psPAX2,pMD2.G,and the recombinant plasmid lenti CRISPR v2,a lentivirus strain with knockout efficiency was packaged.3.The rabbit myeloma cells with well growing condition were infected with concentrated recombinant lentivirus and were respectively treated with 0.8?g/mL Puromycin and gradient-concentration 6-TG(6-thioguanine guanine).After screening,they are verified by growing in HAT selection medium.Target-positive monoclonal cells were isolated by limiting-dilution monoclonal methods.4.The cells in the experimental group were expanded and cultured,DNA was extracted and verified by T7E1?digestion,and two methods of direct sequencing were used for identification.HGPRT gene-defective monoclonal cells were successfully screened.Conclusion:We have successfully constructed a lentiCRISPR v2 recombinant plasmid targeting knockout rabbit HGPRT gene by CRISPER/Cas9 technique,packaged and concentrated a Lentiviral strain for the knockout HGPRT gene,A series of screening and verification methods are used to verify the defective cells after infection.A rabbit myeloma HGPRT gene-deficient cell have been established.The genetically engineered defective cell lays the foundation for subsequent rabbit anti-monoclonal cell hybridization experiments.