Cloning And Expression Of Murine M-CSF In Mammalian Cells

Objective: To clone and express the encoding region of murine macrophage colony-stimulating factor (M-CSF) gene in mammalian cells. Methods: Extract the total RNA from the L929 cells. The cDNA encoding M-CSF was amplified by RT-PCR method. The PCR product was cloned into vector pGEM-T Easy and sequenced. Further, the M-CSF encoding region gene was inserted into mammalian expression vector pEGFP-N3. Finally, the constructed recombinant plasmid pEGFP-N3-M-CSF was transfected into CHO cell line. The expression of EGFP and M-CSF gene was determined by fluorescent microscope, RT-PCR and real time PCR. Results: The size of amplified M-CSF was 1690bp. The correct recombinant plasmid pGEM-T Easy-M-CSFa was isolated and confirmed by restriction analysis. Recombinant plasmid EH,EB,SB,SH,SG was constructed by subcloning for sequencing. The positive subclone was sequenced, compared with the standard sequences, we found an G base was lacked in 9 site, the A base in 1093 site was replaced by the G base. These mutations caused sense mutation. In order to correct the mutation gene, we cloned recombinant plasmid X and D. The correct recombinant plasmid pGEM-T Easy-M-CSF was constructed by subcloning. Recombinant plasmid pEGFP-N3-M-CSF was constructed successfully and transfected into CHO cell line. Green fluorescence was semitled from transfected cells under fluorescent microscope. The expressing product was detected by RT-PCR and real time PCR. Conclusion: The pEGFP-N3-M-CSF eukaryotice expression plasmid was constructed successfully and M-CSF gene can be expressed in CHO cells. These results enable for further studies in fuction of M-CSF.