pAAV-hSOX9-IRES-tdTomato recombinant plasmid constructs adeno-related virus package
- VernacularTitle:pAAV-hSOX9-IRES-tdTomato重组质粒构建腺相关病毒包装
- Author:
Zezheng DIAO
;
Guoqing YAN
;
Zhiwei ZHANG
;
Jing FANG
;
Peng XU
;
Yongming XI
;
Shan REN
;
Yongjun LIU
;
Aihua SUI
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2012;16(20):3759-3762
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND: As the preliminary experiment for gene therapy in intervertebral disc degeneration, this study aims to construct a recombinant plasmid containing fluorescent pAAV-hSOX9-IRES-tdTomato for adeno-associated virus packaging, in a broader attempt to lay the foundation for late experiments in vitro and in vivo.OBJECTIVE: To construct human SOX9 gene overexpressing adeno-associated virus, pAAV-hSOX9-IRES-tdTomato, packaging. METHODS: The plasmid pAAV-IRES-tdTomato and plasmid pUC57-hSOX9 were connected into pAAV-hSOX9-IRES-tdTomato by enzyme digestion method. The adeno-associated virus was packaged with plasmid co-transfections method. The recombinant pAAV-hSOX9-IRES-tdTomato was transfected into 293AAV cell by calcium phosphate transfection. The purification and drop of adeno-associated virus was tested by determination of biological titer. RESULTS AND CONCLUSION: The results of BLAST sequence comparison analysis showed that, pAAV-hSOX9-IRES-tdTomato exactly matched the synthetic gene sequence hSOX9. The titer is 1×107 TU/mL. Human gene SOX9 recombinant adenoviruses, pAAV-hSOX9-IRES-tdTomato, have been constructed successfully.
