Construction of human GDF-5 on adenovirus vector and its expression in MSCs.
- Author:
Xiangjun CHENG
1
;
Hao LIN
;
Jianxin XUE
;
Ting ZHANG
Author Information
1. Department of Orthopaedics, West China Hospital of Sichuan University, Chengdu 610041, China.
- Publication Type:Journal Article
- MeSH:
Adenoviridae;
genetics;
metabolism;
Genetic Vectors;
genetics;
Growth Differentiation Factor 5;
biosynthesis;
genetics;
Humans;
Mesenchymal Stromal Cells;
metabolism;
Recombinant Proteins;
biosynthesis;
genetics;
Transfection
- From:
Journal of Biomedical Engineering
2010;27(1):120-125
- CountryChina
- Language:Chinese
-
Abstract:
This experimental study was aimed to construct the recombinant adenovirus vector containing human GDF-5 gene, and to use it for infecting human MSCs and detecting the expression of the gene GDF-5. The core sequence of human GDF-5 was amplified by PCR from pCMV-SPORT6, and then was cloned to pAdtrack-CMV. The linearized shuttle plasmid pAdtrack-CMV-GDF-5 was homogenously recombined with pAdeasy-1 in BJ5183. The potential clone was analyzed by restriction endonuclease digestion. The correct clone was linearized and transfected into QBI-293 cells for packing and amplifying so as to obtain adenovirus pAd-GDF-5 and identify it, while the titer was also determined by TCID50. MSCs were infected by the harvested virus, and the expression of GDF-5 was detected by RT-PCR. The recombinant adenovirus vector containing human GDF-5 gene was constructed successfully, its titer was 1 x 10(9) PFU/ml, and it could infect MSCs efficiently. The human MSCs infected by constructed adenovirus vector could continue expressing GDF-5 in a certain time, and the transgenic MSCs would be much potential on tissue regeneration.
