Abstract: The aim of this study was to construct a lentiviral overexpression vector containing mouse PKM1 gene,screen out a C₂C₁₂ cell line stably overexpressing PKM1,and preliminarily evaluate the effect of PKM1 on cell glycolysis. Total RNA from the longissimus muscle of mouse was extracted and reversely transcribed into cDNA. The FLAG-tagged PKM1 gene expression vector was constructed by 'Golden Gate’ method and transfected into 293T cells for lentiviral package. Lentiviruses were collected and the viral titer was determined. Then the lentiviruses were transfected into C₂C₁₂ cells with optimized transfect conditions. A stable transgenic cell line carrying the resistance gene puro was screened by puromycin. The expression of target gene was detected from transcriptional level and translation level by qPCR and Western Blot,respectively. The effect of PKM1 on glycolysis was examined by measuring pH,lactic acid and glucose consumption of the cell culture medium. The results indicated that the mouse PKM1 gene lentiviral vector was successfully constructed. The detected lentivirus titer was 3.4×10⁸ TU/mL.The optimal multiplicity of infection was 30,and the optimal concentration of puromycin used for screening stable cell lines was 1.2 μg/mL. As showed by microscopy results,the virus transduction effect was well,and the fluorescence rate was over 80%. The mRNA of FLAG-PKM1 in the overexpression group was 94,000 times higher than that of the empty vector group. The FLAG-PKM1 fusion proteins were successfully expressed in host cells. Compared with wild type group and empty vector group,the lactic acid content and glucose consumption of the overexpressed group increased while the pH value decreased. These results indicated that the cell glycolytic activity of the overexpressed group was intensified. In conclusion,a C₂C₁₂ cell line stably overexpressing mouse PKM1 was screened out. The selected stable cell line could provide a material basis for further study about the mechanis of PKM1 post-translational modification on its enzymatic activity and cellular glycolysis.