Efficient gene transfer into mouse embryonic stem cells with adenovirus vectors
Efficient gene transfer into mouse embryonic stem cells with adenovirus vectors
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DOI：
10.1016/j.ymthe.2005.04.015
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发表时间：
2005-09-01
影响因子：
12.4
通讯作者：
Mizuguchi, H
中科院分区：
文献类型：
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作者：
Kawabata, K;Sakurai, F;Mizuguchi, H
高效、瞬时的基因转移至胚胎干（ES）细胞有望用于发育生物学的基础研究和再生医学的应用。在这里，我们报告了一种腺病毒（Ad）载体的开发，该载体可在小鼠 ES（mES）细胞中有效表达外源基因。我们制备了四个表达 LacZ 的 Ad 载体，每个载体包含以下之一：劳斯肉瘤病毒 (RSV)、巨细胞病毒 (CMV)、β-肌动蛋白启动子/CMV 增强子 (CA) 或 EF-1 α 启动子。虽然 RSV 和 CMV 启动子在 mES 细胞中失活，但 CA 和 EF-1 α 启动子在超过 90% 的 mES 细胞中强烈驱动 LacZ 表达。发现 EF-1 α 启动子比 CA 启动子效率稍高。 mES细胞被发现表达Ad初级受体、柯萨奇病毒和腺病毒受体，这表明虽然Ad载体可以将外源基因引入mES细胞，但合适的启动子的选择对于基因的有效表达至关重要。纤维旋钮上含有 RGD 或多聚赖氨酸肽的纤维突变 Ad 载体不仅在 mES 细胞中而且在饲养细胞中介导有效的 LacZ 表达。通过含有EF-1α启动子的常规Ad载体外源表达Oct-3/4或STAT3显性失活突变体（STAT3F）可促进mES细胞分化为三个胚层细胞，并且通过Nanog的共表达来挽救STAT3F介导的分化。这些结果表明Ad载体可用于使用ES细胞的基础研究，并且它们对于基于ES细胞的基因修饰再生医学的治疗应用可能具有很大的用途。
Efficient and transient gene transfer into embryonic stem (ES) cells is expected to be of use for basic studies in developmental biology and for applications in regenerative medicine. Here, we report the development of an adenovirus (Ad) vector that efficiently expresses foreign genes in mouse ES (mES) cells. We prepared four LacZ-expressing Ad vectors, each of which contained one of the following: Rous sarcoma virus (RSV), cytomegalovirus (CMV), beta-actin promoter/CMV enhancer (CA), or EF-1 alpha promoter. While the RSV and CMV promoters were inactive in mES cells, the CA and EF-1 alpha promoters strongly drove LacZ expression in more than 90% of the mES cells. The EF-1 alpha promoter was found to be slightly more efficient than the CA promoter. mES cells were found to express the Ad primary receptor, coxsackievirus and adenovirus receptor, suggesting that while Ad vectors could introduce the exogenous gene into mES cells, the choice of a suitable promoter was critical for efficient gene expression. Fiber-mutant Ad vectors containing RGD or polylysine peptide on the fiber knob mediated efficient LacZ expression, not only in mES cells, but also in feeder cells. Exogenous expression of Oct-3/4 or the dominant-negative mutant of STAT3 (STAT3F) by conventional Ad vectors containing the EF-1 alpha, promoter promoted the differentiation of mES cells into the cells of three germ layers, and STAT3F-mediated differentiation was rescued by the coexpression of Nanog. These results suggest that Ad vectors can be used for basic research using ES cells and that they may be of great utility for therapeutic applications in gene-modified regenerative medicine based on ES cells.