[Transduction of recombinant marrow stem cells using lentiviral vector and adenoviral vector: a comparative study].
OBJECTIVE
To compare the efficiency of transfection of marrow stem cells (MSCs) by using lentiviral vector and adenoviral vector.


METHODS
MSCs were obtained from the femur bone marrow of rat and cultured. Recombinant plasmid pHIV-CS-CG-PRE containing green fluorescent protein (GFP) gene, and package plasmids pRSV-Rev, pMDLg/pPRE, and pMD.G were infected into the human embryonic kidney cells of the line 293T. The rat MSCs were transfected with the DFP recombinant lentiviral particles, and flow cytometry was used to detect the expression of GFP 1, 3, and 5 weeks later. Full-length human bone morphogenetic protein = 2 (hBMP2) gene was searched out from the GenBank and isolated from human liver cDNA from a sample of liver tissue resected from a patient with liver rupture and then cloned. Thus the lentiviral vector with BMP-2 gene, pHIV-CS-CDF-CG-PRE-BMP-2 was constructed and transfected into 293 cells. Indirect immunofluorescence assay, ELISA, and Western blotting were used to detect the expression of hBMP2 gene.


RESULTS
After infected by the viral vectors the GFP expression was significantly better in the lentiviral vector-infected rMSCs than in the adenoviral vector-infected ones. FCM showed that the GFP expression rates of the lentiviral vector-infected rMSCs 1, 3, and 5 weeks after the infection were 90%, 83.2%, and 79% respectively, and he GFP expression rates of the adenoviral vector-infected rMSCs 1, 3, and 5 weeks after the infection were 71%, 0.13%, and 0.05% respectively, all significantly lower than on the former group. Indirect immunofluorescence assay, ELISA, and Western blotting showed that the recombinant lentivirus successfully expressed the target protein in the transfected 293T cells.


CONCLUSIONS
Lentiviral vector with hBMP2 gene can be constructed successfully. The transfection efficiency of BMP2 gene by lentivirus is significantly higher than that of adenovirus.
Ma
Xiao-sheng
XS
Department of Orthopedic Surgery, Huashan Hospital, Fudan University, Shanghai 200040, China. maxiaosheng@hotmail.com
Jiang
Jian-yuan
JY
Lü
Fei-zhou
FZ
Ma
Xin
X
Li
Xiao-kang
XK
Huang
Huang-yuan
HY
D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D015153:Blotting, Western; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D019084:Fluorescent Antibody Technique, Indirect; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D023461:Myeloid Progenitor Cells / Q000166:cytology / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000235:genetics / Q000378:metabolism
C525718:BMP2 protein, human; C525720:Bmp2 protein, rat; D055396:Bone Morphogenetic Protein 2; D019485:Bone Morphogenetic Proteins; D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta; D049452:Green Fluorescent Proteins