Gene targeting in human pluripotent stem cells with adeno-associated virus vectors
Gene targeting in human pluripotent stem cells with adeno-associated virus vectors
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DOI：
10.1016/j.bbrc.2009.08.075
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发表时间：
2009-10-30
影响因子：
3.1
通讯作者：
Mitani, Kohnosuke
中科院分区：
文献类型：
--
作者：
Mitsui, Kaoru;Suzuki, Keiichiro;Mitani, Kohnosuke
人类多能干细胞，如胚胎干细胞(HESCs)和诱导多能干细胞(HiPSCs)，具有分化为多种细胞类型的能力，将成为再生医学的潜在细胞材料来源。为了充分利用hESCs或hiPSCs进行基础和临床研究，遗传学。阳离子，特别是通过同源重组(HR)的基因打靶将是一项重要的技术。本文报道了用腺相关病毒(AAV)载体在人多能干细胞中成功地打靶次黄嘌呤磷酸核糖转移酶1(HPRT1)和NANOG基因座。在HPRT1基因座，多达1%的稳定转化子通过HR与AAV-HPRT1靶向载体打靶，而不会失去多能性。另一方面，使用为启动子-陷阱策略设计的AAV-NANOG靶向载体，20-87%的稳定转化子被靶向。在对实验操作表现出特别高脆弱性的KhES-3细胞系中，仅使用AAV载体而不是电穿孔可以成功地进行基因打靶。除了hESC，在HiPSC系中也以相似的频率实现了基因打靶。这些数据表明，AAV载体可能因此成为引入遗传Modi的有用工具。HESCs和HiPSCs中的阳离子。(C)2009 Elsevier Inc.保留所有权利。
Human pluripotent stem cells, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the ability to differentiate into various cell types, and will become a potential source of cellular materials for regenerative medicine. To make full use of hESCs or hiPSCs for both basic and clinical research, genetic modi. cation, especially gene targeting via homologous recombination (HR), would be an essential technique. This report describes the successful gene targeting of the hypoxanthine phosphoribosyl transferase 1 (HPRT1) and the NANOG loci in human pluripotent stem cells with adeno-associated virus (AAV) vectors. At the HPRT1 locus, up to 1% of stable transformants were targeted via HR with an AAV-HPRT1 targeting vector, without loss of pluripotency. On the other hand, 20-87% of stable transformants were targeted using an AAV-NANOG-targeting vector designed for the promoter-trap strategy. In the KhES-3 cell line, which shows particularly high fragility to experimental manipulation, gene targeting was successful only by using an AAV vector but not by electroporation. In addition to hESC, gene targeting was achieved in hiPSC lines at similar frequencies. These data indicate that AAV vectors may therefore be a useful tool to introduce genetic modi. cations in hESCs and hiPSCs. (C) 2009 Elsevier Inc. All rights reserved.