Construction and identification of eukaryotic co-expression vector carrying Myod1 and Myog
10.3969/j.issn.2095-4344.2013.37.017
- VernacularTitle:Myod1和Myog真核共表达载体构建及鉴定
- Author:
Hongfei GAO
;
Bingsheng LIANG
;
Weibing SHUANG
- Publication Type:Journal Article
- From:
Chinese Journal of Tissue Engineering Research
2013;(37):6645-6651
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUND:Now it has cooperation and facilitative effete between myogenic regulatory factors through a long time study. So, gene therapy of double genes of Myod1 and Myog can obtain better effect, and can provide a new way for preventing denervated skeletal muscle atrophy.
OBJECTIVE:To construct eukaryotic co-expression vector carrying Myod1 and Myog genes.
METHODS:Ful-length Myod1 gene and Myog gene cDNA were amplified by reverse transcription PCR, and then inserted into pVAX1 vector after digested to establish the recombined Myod1 and Myog eukaryotic co-expression vector pVAX1-Myod1-IRES2-Myog-IRES2-EGFP, and then identified with gene sequencing. The in vitro cultured 3T3 cel s were transfected with pVAX1-Myod1-IRES2-Myog-IRES2-EGFP, and the expressions of Myod1 and Myog genes in the 3T3 cel s were detected with western blot assay in order to identify whether the 3T3 cel s could express the target protein correctly.
RESULTS AND CONCLUSION:The sequencing results showed that the sequence length and base sequence of Myod1 and Myog cDNA in eukaryotic co-expression vector pVAX1-Myod1-IRES2-Myog-IRES2-EGFP were identical with the reported sequences. Myod1 and Myog protein band expressions were detected in 3T3 cel s by western blot after transient transfection. The pVAX1-Myod1-IRES2-Myog-IRES2-EGFP, a eukaryotic co-expression vector of Myod1 gene and Myog gene is successful y constructed.
