Abstract: Objective To establish a simple and effective method used to isolate and purify bone marrow mesenchymal stem cells (BMSCs) of Tibetan miniature pigs, and further set up the lentivirus-mediated in vitro gene delivery system for BMSCs of Tibetan miniature pigs. Methods ①MSCs of Tibetan miniature pigs were initially isolated by density gradient centrifugation, followed by purifying MSCs using differential attachment method, and further passage 3 BMSCs of Tibetan miniature pigs were characterized by CD45, CD90 and CD105. ②According to the standard protocol from Invitrogen, lentiviruses carrying EGFP gene were produced, and then infected MSCs, followed by EGFP assay under fluorescent stereo microscope and fluorescence activated cell sorting (FACS) for determining the infecting efficiency. Results BMSCs of Tibetan miniature pigs obtained by the combined use of density gradient centrifugation and differential attachment method were shown to enjoy the characteristics of MSCs. Additionally, the BMSCs of Tibetan miniature pigs were successfully and efficiently infected by lentivirus supernatant, and the infecting efficiency was 84.1%. Conclusion High purified BMSCs of Tibetan miniature pigs can be obtained by the combined use of density gradient centrifugation and differential attachment method, and lentivirus-mediated in vitro gene delivery systems for BMSCs of Tibetan miniature pigs were successfully established.

Key words: Tibetan miniature pigs, Bone marrow mesenchymal stem cells (BMSCs), Lentivirus, Enhanced green fluorescent protein (EGFP)