The Effect Of Screening Markers On Transgene Expression In CHO Cells And Molecular Mechanism

Background Mammalian cell expression systems are capable of providing complex post-translational modification functions and are widely used for the production of recombinant therapy proteins.About 70% of recombinant therapy proteins were produced by the Chinese hamster ovary cells(CHO)expression system.There are many factors that could affect the expression of recombinant proteins in CHO cells,of which the expression vector is one of the most important factors.However,due to the random integration of the target gene into the cell,the transgene is often silenced and the expression level is unstable,which greatly affect the expression of the target gene in CHO cells.Therefore,the optimization of the vector has become one of the key research directions for research.Objective To analyze the effects and molecular mechanism of weakened selection markers,different cis-acting elements and screening markers on transgene expression levels in CHO cells.Methods1)The G418 marker gene was mutated,and CMV and SV40 were ligated to construct new vectors.The resulted vectors were transfected into CHO cells by liposome transfection method.After 48 h,e GFP fluorescence was observed by inverted fluorescence microscopy.G418 was used to screen stable cell clones.And the expression of e GFP was detected by flow cytometry.The expression of e GFP was detected by flow cytometry(FCM).2)An cis-acting element-woodchuck hepatitis post-transcriptional regulatory element(WPRE)was cloned into the expression vector driven by CMV promoter.Then,they were transfected into CHO cells,and the expression level of e GFP was detected by flow cytometry.Transgenic copy number and m RNA expression levels were also be detected.The stably cells were cultured for two months and detected the transgene expression3)Four kinds of common mammalian cell selection markers were selected,including blasticidin,bleomycin,puromycin and hygromycin for CHO cell drug sensitivity experiments;G418 resistance gene was replaced by the drug resistance gene which had the shorter screening time and constructed a new vector,followed by transfecting into cells,and the expression level of e GFP was analyzed by flow cytometry.4)We cloned the e GFP reporter gene upstream of the G418 resistance gene and added Erythropoietin(EPO)to construct the vector containing the EPO-EGFP-G418 chimeric gene,and then transfected into CHO cell;The polyclonal cell pool was construced;We screened 11 monoclones;The intensity of the green fluorescence signal were observed by the fluorescence inverted microscope.;The e GFP expression level of the eleven clones were detected by flow cytometry,and marked with high,middle and low;We also detected the protein expression levels of the cell line marked high,middle and low by Western blot;At last,we analysed whether there was a positive correlation between the protein expression levels and e GFP expression levels.Results1)Two new vectors with different promoters(CMV and SV40)were successfully constructed using the weakened G418 resistance gene.The transient expression results showed that the G418 weakened gene could improve the expression of e GPF under two different promoters.The results showed that CMV+G418mut and SV40+G418mut could improve the expression of transient transgene.Especially driven by the CMV promoter,the expression level of e GFP was significantly increased by 2.62 times(p<0.05).2)The transgenic expression level of CMV+G418mut+WPRE was lower than that of CMV+G418 after inserting the WPRE element fragment into the vector which contained CMV promoter.The e GFP expression level of CMV+G418+WPRE was the highest among all vectors,which was 3.02 times higher compared with the control(p<0.05).The copy number of CMV+G418mut was as high as 10.76.The expression level of m RNA of CMV+G418mut was the highest,was 9.61 versus the control.After two months of stable culture,the results showed that G418 mut had the most significant reduction in transgenic expression,while the expression level of the vector with WPRE was relatively stable,which still showed a high trend.3)Blasticidin(4-5d)and hygromycin B(10-12d)showed a strong advantage in screening marker selection experiments and could completely kill the untransfected CHO cells in a short period of time.So they were selected to carry out the following experiments.After 48 h,the fluorescence signal showed that the vector containing blasticidin resistance gene was successfully transfected into the cells.The transfection efficiency was lower than that of the G418 resistance gene,but the e GFP expression level was higher than that of the G418 resistance gene under stably state.4)The eleven monoclonal clones of EPO-EGFP-G418 chimeric gene could be divided into three groups according to their fluorescence intensity.The results of flow cytometry were consistent with those of fluorescence intensity.The results of Western blot analysis showed that the EPO expression of clones with e GFP expression level was also relatively higher.Conclusions1)The G418 mutation can increase the expression level of transgene in CHO cells mediated by different promoters.2)WPRE cis-acting expression elements can effectively improve the transgene expression level of the vector in CHO cells.3)Blasticidin showed a better advantage.4)Selection marker with EGFP reporter gene method can be uesd to predict high-yield protein.