Construction and identification recombinant eukaryotic expressive vector of mouse GM-CSF gene

Objective: Construction mouse GM-CSF gene effective eukatyotic expressive vector, selection high GM-CSF expressing mouse leukemia cell line RMA after transfected with the constructed vector, and study the method of treatment leukemia with tumor cells transfected with GM-CSF gene.Methods:770 bp of GM-CSF 3' end cDNA was amplified by PCR and inserted into pcDNAS vector.The constructed vector was transfected into RMA cells by electroporation. After screening by G418 and cloning by limiting dilution, a relative high GM-CSF expressing cell clone was selected by RT-PCR, hematopoietic progenitor cell proliferative assay and hematopoietic progenitor cell colony formation assay. The cells of this clone were inactivated by mitomycin-C and vaccinated mice to induce antitumor immune reaction. Results: The orientation and sequence of the insert was found to be correct, and a GM-CSF high expressing cell line RMA-GM was selected , which can induce mice obtain anti-tumor protective immune ability after inactivated by mitomycin-C.Conclusion:Tumor cells transfected with GM-CSF gene may be used an effective anti-T lymphycoma tumor vaccine.