Construction and identification of bicistronic retrovirus expression vector for Delta1 and EGFP gene coexpression
- VernacularTitle:Delta1-IRES-EGFP双顺反子逆转录病毒表达载体的构建及鉴定
- Author:
Fei HE
;
Yinghui TAN
;
Zhengron YANG
- Publication Type:Journal Article
- Keywords:
Delta;
GFP;
IRES;
retrovirus;
gene transfer;
gene expression
- From:Journal of Third Military Medical University
2002;0(12):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To construct a bicistronic retrovirus expression vector to coexpress Delta1 and EGFP genes. Methods Delta1-IRES-EGFP bicistronic retrovirus vector was constructed by linking full-length Delta1 cDNA with IRES-EGFP retrovirus vector. Transfected with the retrovirus vector by electroporation, the ecotropic- and amphotropic-packaging cell lines GP+E86 and PA317 were selected by addition of puromycin. Positive cell clones were obtained and tested for the efficiency of the retroviral supernatants. Two positive cell clones with relatively highest titers were picked up and used for ping-pong amplification assay to improve the retroviral supernatants titer. At day 2 after transfection, GFP expression in NIH3T3 was detected by FACS, and Delta1 expression was determined by RT-PCR assay. Transfected or wild type NIH3T3 were co-cultured with myoblasts cell line C2C12 to test whether the exogenous Delta1 protein was functional or not. Results The bicistronic retrovirus expression vector coexpressing Delta1 and EGFP genes was successfully constructed and verified by PCR and DNA sequencing. A high-level expression of functional Delta protein was detected in transfected NIH3T3 cells. Conclusion Retrovirus is a safe, highly efficient gene transfer system.
