Construction of recombinant adenoviral vector Ad-CMV-hTGFbeta1 for reversion of intervertebral disc degeneration by gene transfer.
OBJECTIVE
To provide a highly efficient adenoviral vector Ad-CMV-hTGFbeta1 for the study of gene therapy for reversion of the intervertebral disc degeneration.


METHODS
A newly developed recombinant adenoviral vector construction system was used in the study. The cDNA of hTGFbeta1 was first subcloned into a shuttle plasmid pShuttle-CMV. The resultant plasmid was linearized by digesting with restriction endonuclease PmeI, and subsequently transformed into E.coli. BJ5183 cells with an adenoviral backbone plasmid pAdEasy-1. Recombinants were selected by kanamycin resistance and confirmed by restriction endonuclease analysis. Finally, the recombinant plasmid linearized by PmeI was transfected into 293 cells. Recombinant adenoviruses were generated within 2 weeks.


RESULTS
The recombinant adenoviral plasmids were cut by BamHI and PacI respectively, and the diagnostic fragments appeared in 0.8% agarose electrophoresis. The infected 293 cells showed evident cytopathic effect (CPE). The productions of PCR confirmed the presence of recombinant adenovirus. The expression of hTGFbeta1 was verified by immunohistochemical staining.


CONCLUSIONS
The successful generation of the adenoviral vector Ad-CMV-hTGFbeta1 and the confirmation of the interest gene expression make it possible for the experimental study of the reversion of the intervertebral disc degeneration by gene therapy.
Tan
Jiangwei
J
Institute of Traumatology and Orthopedics of Shandong Province, Affiliated Hospi tal of Qingdao Medical College, Qingdao University, Qingdao 266003, China. gkyj@qdumh.qd.sd.cn
Hu
Yougu
Y
Zheng
Hongjun
H
Li
Shuzhong
S
D000256:Adenoviridae / Q000235:genetics*; D001483:Base Sequence; D002478:Cells, Cultured; D003587:Cytomegalovirus / Q000235:genetics*; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D007403:Intervertebral Disc / Q000166:cytology* / Q000473:pathology*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D012680:Sensitivity and Specificity; D016212:Transforming Growth Factor beta / Q000235:genetics*; D053773:Transforming Growth Factor beta1
C508960:TGFB1 protein, human; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1