[Enhancement of exogenous gene expression by artificial transcription factor in CHO cells].
Using the amino acids 1-147 of the yeast transcriptional activator GAL4 as the DNA-binding domain and four tandem repeats of the 12-aa peptide (DALDDFDLDMLG) of the herpesvirus as the activation domain, an artificial transcription factor, GVP4,was constructed via the linkage of the nuclear localization signal sequence of SV40. And then, GVP4 was cloned into expression vector pcDNA3 . 1/Hygro ( + ) . Various amounts of targeting sites of artificial transcription factor were linked to the upstream of promoter CMV in exogenous gene expression vector pcDNA3.1 ( + ) that separately harbored EGFP cDNA and t-PA cDNA.The CHO cells were then co-transfected with GVP4 expression vector and EGFP or t-PA expression vector. The effect of GVP4 on exogenous gene expression was evaluated by measuring the fluorescence intensity of EGFP in CHO cells and the concentration of t-PA in the supernatant. GVP4 showed positive effect on the enhancement of exogenous gene expression in CHO cells integrated with targeting sites of artificial transcription factor. And, CHO cells integrated with 10 targeting sites of GVP4 was more favorable to foreign gene expression, which resulted in 2-3-fold increase in both EGFP and t-PA expressions. These results indicated that artificial transcription factor is potent in the enhancement of exogenous gene expression in mammalian cells.
Li
Shi-Chong
SC
Institute of Biotechnology , Academy of Military Medical Sciences, Beijing 100071, China.
Ye
Ling-Ling
LL
Yang
Hai
H
Liu
Hong
H
Xu
Jian
J
Wu
Ben-Chuan
BC
Huang
Pei-Tang
PT
Chen
Zhao-Lie
ZL
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection
D014157:Transcription Factors; D049452:Green Fluorescent Proteins; D010959:Tissue Plasminogen Activator