[Screening and identification of mesenchymal stem cell strains to secret mouse interleukin-12 mediated with lenti-viral vector].
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.


CONCLUSIONS
The recombinant mMSC strains lentivirally engineered to secret mIL-12 were obtained.
Xu
Jian-Rong
JR
State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, China.
Li
Hong-Xia
HX
Wang
Guo-Qing
GQ
Du
Xiao-Bo
XB
Wei
Yu-Quan
YQ
Zhao
Ju-Mei
JM
D000818:Animals; D001854:Bone Marrow Cells / Q000166:cytology*; D002460:Cell Line; D005260:Female; D005822:Genetic Vectors; D018664:Interleukin-12 / Q000096:biosynthesis* / Q000235:genetics; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D014162:Transfection
D011994:Recombinant Proteins; D018664:Interleukin-12