OBJECTIVE: To screen the stable expression cell strains of mouse interleukin-12 (mIL-12) from mouse Mesenchymal Stem Cells (mMSCs) transfected with lenti-mIL-12 virus. METHODS: The mIL-12 cDNA was amplified from plasmid pORF-mIL-12 (Invivogen) by PCR. The cDNA was subcloned into pENTR 11 to generate recombinant plasmid pENTR-mIL-12. Then, pENTR-mIL-12 was homologously recombinated with pLenti6/V5-Dest. The recombinant was named as pLenti6/V5-mIL-12 and confirmed by PCR and DNA sequencing. The Lenti6/V5-mIL-12 virus was packaged using 293FT cells. The Lenti-mIL-12-MSC monoclone was picked from the mMSCs infected by the Lenti6/V5-mIL-12 virus using Blasticidin and verified by RT-PCR and ELISA. RESULTS: The recombinant pLenti6/V5-mIL-12 was constructed. The sequence of amplified mIL-12 gene was consistent with that reported in GenBank. By RT-PCR and ELISA, it was confirmed that the mIL-12 protein could be expressed and secreted into the supernatant of MSC strain culture. CONCLUSION: The recombinant mMSC strains lentivirally engineered to secret mIL-12 were obtained.
OBJECTIVE: To screen the stable expression cell strains of mouse interleukin-12 (mIL-12) from mouse Mesenchymal Stem Cells (mMSCs) transfected with lenti-mIL-12 virus. METHODS: The mIL-12 cDNA was amplified from plasmid pORF-mIL-12 (Invivogen) by PCR. The cDNA was subcloned into pENTR 11 to generate recombinant plasmid pENTR-mIL-12. Then, pENTR-mIL-12 was homologously recombinated with pLenti6/V5-Dest. The recombinant was named as pLenti6/V5-mIL-12 and confirmed by PCR and DNA sequencing. The Lenti6/V5-mIL-12 virus was packaged using 293FT cells. The Lenti-mIL-12-MSC monoclone was picked from the mMSCs infected by the Lenti6/V5-mIL-12 virus using Blasticidin and verified by RT-PCR and ELISA. RESULTS: The recombinant pLenti6/V5-mIL-12 was constructed. The sequence of amplified mIL-12 gene was consistent with that reported in GenBank. By RT-PCR and ELISA, it was confirmed that the mIL-12 protein could be expressed and secreted into the supernatant of MSC strain culture. CONCLUSION: The recombinant mMSC strains lentivirally engineered to secret mIL-12 were obtained.