农业生物技术学报2012，Vol.20Issue(8)：936-941,6.DOI:10.3969/j.issn.1674-7968.2012.08.013
四环素诱导型单载体的构建、体外表达与鉴定
Construction and Identification of EGFP Eukaryotic Expression Vetor Regulated by Tetracyline
摘要
Abstract
Currently used pTet-on-Advanced regulatory system contains two plasmids, and when it is applied to transgenic animals, two types of genetically modified animals are required to be established and hybridized. This results are in high cost and low efficiency. To address this question, this study aims to construct and functionally characterize tetracycline-inducible single plasmid system. The rtTA and EGFP genes were amplified by PCR from plasmid pTet-on-Advanced and pEGFP-N1, and then sub-cloned into the pTRE-Tight plasmid. The pTRE-Tight-rtTA -EGFP was transfected to fetal porcine fibroblast using liposome transfection. The expression of EGFP under different concentration of doxycyline (Dox)(0.0001,0.001, 0.01, 0.1 and 1 g/L) were assayed by fluorescence inverted microscope, as the results displayed at 48 h after transfection, there were no expressed green fluorescent in the cells before Dox added, and there were still no expressed green fluorescent in the cells under the concentration of Dox were 0.0001, 0.001, 0.01 and 0.1 g/L until the concentration reached 1 g/L. This phenomenon revealed that the regulation effect of Dox was stimulated with the concentration of 1 g/L. The results showed that the recombinant vector pTRE-Tight-rtTA-EGFP which induced with Tet-on was successfully constructed, and its expression could be induced in fetal porcine fibroblast by Dox. This study provides a novel tool for quantitative transgene expression, and also produces important experimental data to prepare transgenic animals under controlled expression.关键词
pTet-on-Advanced/诱导表达载体/真核表达/猪成纤维细胞Key words
pTet-on-Advanced/ Inducible expression vector/ Eukaryotic expression/ Fetal porcine fibroblast引用本文复制引用
乔宪凤,马卓,毕延震,华再东,华文君,肖红卫,周荆荣,郑新民..四环素诱导型单载体的构建、体外表达与鉴定[J].农业生物技术学报,2012,20(8):936-941,6.基金项目
国家转基因生物新品种培育重大专项(No.2011ZX08006-002 （）
No.2011ZX08011-030B （）
No.2011ZX08006-003 （）
No.2011ZX08010-3)和湖北省重点实验室(No.2012ZD120) （No.2012ZD120）
