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Influence of 3' untranslated region of vascular endothelial growth factor-C gene on the expression of luciferase / 解放军医学杂志
Medical Journal of Chinese People's Liberation Army ; (12)2001.
Article in Chinese | WPRIM | ID: wpr-562090
ABSTRACT
Objective To construct a luciferase gene expression vector containing full-length 3' untranslated region(3'UTR)of mouse vascular endothelial growth factor-C(VEGF-C)gene,and to observe the effects of VEGF-C 3'UTR on luciferase gene expression by a double-fluorescence report system.Methods Polymerase chain reaction(PCR)was used to amplify VEGF-C 3'UTR and a 312bp VEGF-C coding region(CR)fragment from full-length VEGF-C cDNA in mouse Lewis lung cancer cells.The luciferase expression vectors containing VEGF-C 3'UTR or VEGF-C CR were constructed by subcloning the PCR products to luciferase reporter vector pGL3-Promoter using gene engineering technology,and then they were transfected to mouse Lewis lung carcinoma cells by LipofectamineTM 2000.The activities and mRNA expression of luciferase were detected by Dual-Luciferase Reporter System and quantitative RT-PCR,respectively.Results Mouse VEGF-C 3'UTR(429bp)and VEGF-C CR(312bp)were successfully amplified by PCR.The VEGF-C 3'UTR and VEGF-C CR fragments were successfully inserted into pTA2 vector,and then subcloned to pGL3-Promoter vector at Xba Ⅰ site by using restriction endonucleases analysis.The DNA sequences and insertion orientation of PCR products were all correct by sequencing analysis.The resulted luciferase expression plasmids were named pGL3-VEGF-C 3'UTR and pGL3-VEGF-C CR,respectively.Dual-Luciferase Reporter System detection and quantitative RT-PCR showed that in Lewis lung carcinoma cells,the activities of luciferase and expression of luciferase mRNA in the pGL3-Promoter group were higher than that in the pGL3-VEGF-C 3'UTR group,and there was no significant difference between pGL3-VEGF-C CR group and pGL3-Promoter group.Conclusion VEGF-C 3'UTR can inhibit luciferase gene expression.

Full text: Available Index: WPRIM (Western Pacific) Language: Chinese Journal: Medical Journal of Chinese People's Liberation Army Year: 2001 Type: Article

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Full text: Available Index: WPRIM (Western Pacific) Language: Chinese Journal: Medical Journal of Chinese People's Liberation Army Year: 2001 Type: Article