[Screening and identification of mesenchymal stem cell strains to secret mouse interleukin-12 mediated with
Jian-Rong Xu1, Hong-Xia Li, Guo-Qing Wang
1State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
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.
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