Gene transfer mediated through adeno-associated virus type 2 induces transgenicexpression of bone morphogenetic protein-7 in adipose-derived adult stem cells in vitro
- VernacularTitle:腺相关病毒介导人骨形态发生蛋白7基因体外转染脂肪源性干细胞的研究
- Author:
Yan KANG
;
Weiming LIAO
;
Puyi SHENG
- Publication Type:Journal Article
- Keywords:
Adeno-associated virus;
Adipose-derived adult stem cell(ADAS cells);
Human bone morphogenetic protein7 (HBMP7);
Gene therapy
- From:
Chinese Journal of Orthopaedic Trauma
2004;0(11):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct vectors based on adeno-associated virus type 2 (rAAV2) carrying bone morphogenetic protein-7 (BMP-7) and observe their expression in adipose-derived adult stem cells, which can be served as a new gene therapy method and cell source for bone tissue engineering. Methods The coding sequence (1. 3 kb) of BMP-7 was amplified by PCR from the pcDNAl. 1 ( + ) plasmid containing the human BMP-7 cDNA. After purified, the gene fragments were cloned into a plasmid pUC18 and termed plasmid pUC18-hBMP7. The recombinant pUC18-hBMP7 was digested and further ligated to the pSNAV by T4DNA ligase and termed plasmid pSNAV-hBMP7. BHK-21 cells were transfected with the purified pSNAV-BMP7 plasmid according to a standard calcium phosphate precipitation method. The cells were isolated and the integrity of hBMP7 gene was determined by polymerase chain reaction (PCR) using the above PCR primers. To package the virus, stably transfected BHK-21 cells were subsequently infected with recombinant herpes simplex virus type 1 (rHSV-1). The collected cells were processed by chloroform treatment, PEG8000/NaCl precipitation and chloroform extraction for purification. The tiler was determined using quantitative DNA dot blots and the purity was examined by sodium dodecyl sulphatepolyacrylamide gel electrophoresis. Following infection with rAAV2-BMP7 at multiplicities of infection of 1?105 vector genomes per cell and subsequent culture, adipose-derived adult stem cells were assessed qualitatively for BMP7 production. Results The recombinant plasmid pSNAV-hBMP7 was identified by PCR and digested with restriction enzyme. Transfection showed an efficiency of 90 % in ADAS cells. BMP-7 expression in ADAS cells was identified by Western blot. Conclusions The hBMP-7 recombinant adeno-associated virus vectors can be successfully constructed in vitro and rAAV2-hBMP7 can infect ADAS cells.
