Spatiotemporal control of porcine p65RHD expression by advanced Tet-On system in PIEC cells helps regulate NF kappa B activity

文献类型： 外文期刊

第一作者： Ruan, Jinxue

作者： Ruan, Jinxue;Ouyang, Hongsheng;Ruan, Jinxue;Liu, Nan;Yang, Shulin;Li, Kui;Liu, Nan

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关键词： Porcine p65RHD;PIEC;Tet-On advanced system;Spatiotemporal expression;NFкB activity

期刊名称：MOLECULAR BIOLOGY REPORTS （ 影响因子：2.316； 五年影响因子：2.357 ）

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收录情况： SCI

摘要： (NFB)-B-kappa transcription activation leads to malfunction of endothelial cells, which is the main reason for pig xenograft rejection. Overexpression of a dominant negative mutant of porcine NF kappa B p65 (pp65RHD) could inhibit (NFDB)-B-0 activation in endothelial cells. This study presents an advanced tetracycline-regulated system for pp65RHD spatiotemporal expression in porcine iliac endothelial cell line. In this system, an endothelial specific promoter ICAM-2 is used to improve pTet-On and internal ribosome entry site as well as enhanced green fluorescent protein (EGFP) elements are used to facilitate the result observation in pTRE-Tight. Through transfection and drug selection, we obtained 7 single cell clones containing the advanced Tet-On system, in which pp65RHD expression is under tight regulated by doxycycline and can be visualized easily through EGFP. The distribution of induced pp65RHD was verified by immunocytochemical assays test. Then, (NFB)-B-kappa activity was tested. Luciferase reporter assays showed that (NFB)-B-kappa activity in two clones was influenced by the Dox-induced pp65RHD expression, but other clones weren't influenced. Therefore, we picked up 2 cell clones from the uninfluenced clones for further investigation by immunocytochemical assays and RT-PCR detection. The final results supported the overexpression of pp65RHD in one clone could successfully inhibit (NFDB)-B-0 activity. The success of pp65RHD spatiotemporal expression system is helpful to regulate (NFB)-B-kappa activity and conquer cell-mediated immunity and could be used for preparation of transgenic pig, contributing to xenotransplantation.

分类号： Q7

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