Expression from cell type-specific enhancer-modified retroviral vectors after transduction: influence of marker gene stability
Expression from cell type-specific enhancer-modified retroviral vectors after transduction: influence of marker gene stability
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DOI：
10.1016/s0378-1119(01)00857-5
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发表时间：
2002-01-23
期刊：
影响因子：
3.5
通讯作者：
Amadori, A
中科院分区：
文献类型：
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作者：
Indraccolo, S;Roni, V;Amadori, A
增强型绿色荧光蛋白 (EGFP) 越来越多地用作病毒载体中的报告基因，用于多种应用。为了建立一个研究顺式作用细胞调节序列活性的系统，A,e删除了携带EGFP的逆转录病毒载体中的病毒增强子，并用细胞类型特异性元件取代它。在本研究中，我们使用该系统来证明人 CD2 淋巴样特异性和 Tie2 内皮细胞类型特异性增强剂在细胞系和逆转录病毒载体转导的原代细胞中的活性。此外，我们将使用 EGFP 作为报告基因获得的结果与用 d2EGFP 替换 EGFP 获得的结果进行比较，d2EGFP 是 EGFP 的不稳定变体，其特征是与 EGFR 相比半衰期短得多，并且细胞中的积累减少。产生的携带d2EGFP的载体的滴度与携带EGFP的cot应答载体产生的滴度没有不同。此外，用 EGFP 或 d2EGFP 携带载体转导靶细胞后，可以很容易地检测到莫洛尼鼠白血病病毒增强子的活性。然而，相对较弱的 CD2 和 Tie2 增强子的活性只能使用 EGFP 作为报告基因来检测。这些发现表明，增强子替换是解决细胞功能的可行且有前景的方法。 携带 EGFP 基因的逆转录病毒载体中的类型特异性调控元件。 (C) 2002 Elsevier Science B.V. 保留所有权利。
The enhanced green fluorescent protein (EGFP) is increasingly used as a reporter gene in viral vectors for a number of applications. To establish a system to study the activity of cis-acting cellular regulatory sequences,,A,,e deleted the viral enhancer in EGFP-carrying retroviral vectors and replaced it with cell type-specific elements. In this study, we use this system to demonstrate the activity of the human CD2 lymphoid-specific and the Tie2 endothelial cell type-specific enhancers in cell lines and in primary cells transduced by retroviral vectors. Furthermore, we compare findings obtained with EGFP as the reporter gene to those obtained replacing EGFP with d2EGFP, an unstable variant of EGFP characterized by a much shorter half-life compared to EGFR and by reduced accumulation in the cells. d2EGFP-carrying vectors were generated at titers which Acre not different front those generated by the cot-responding vectors carrying EGFP. Moreover, the activity of a Moloney murine leukemia virus enhancer could be readily detected following transduction of target cells with either EGFP- or d2EGFP-carrying vectors, However, the activity of the relatively weak CD2 and Tie2 enhancers was exclusively detected using EGFP as the reporter gene.These findings indicate that enhancer replacement is a feasible and promising approach to address the function of cell type-specific regulatory elements in retroviral vectors carrying the EGFP gene. (C) 2002 Elsevier Science B.V. All rights reserved.