AAV-mediated gene targeting methods for human cells.
AAV-mediated gene targeting methods for human cells.
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DOI：
10.1038/nprot.2011.301
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发表时间：
2011-04
期刊：
影响因子：
14.8
通讯作者：
中科院分区：
文献类型：
--
作者：
腺相关病毒 (AAV) 载体的基因靶向已在多种人类细胞类型中得到证实，每个感染细胞的靶向频率范围为 10−5 至 10−2。这些靶向频率比传统转染或电穿孔方法获得的频率高 1-4 个对数。可以将多种不同类型的突变高保真度且无遗传毒性地引入染色体位点。在这里，我们提供了使用 AAV 载体在人类细胞中进行基因靶向的详细方案。我们描述了载体设计、储备制备和滴定的方法。为人类多能干细胞、间充质干细胞、成纤维细胞和转化细胞系提供了优化的转导方案，以及通过 Southern blot 鉴定目标克隆的方法。该方案（从载体设计到单轮靶向和筛选）可在约 10 周内完成；随后的每一轮靶向和筛选都需要额外 7 周的时间。
Gene targeting with adeno-associated virus (AAV) vectors has been demonstrated in multiple human cell types, with targeting frequencies ranging from 10−5 to 10−2 per infected cell. these targeting frequencies are 1–4 logs higher than those obtained by conventional transfection or electroporation approaches. a wide variety of different types of mutations can be introduced into chromosomal loci with high fidelity and without genotoxicity. Here we provide a detailed protocol for gene targeting in human cells with AAV vectors. We describe methods for vector design, stock preparation and titration. optimized transduction protocols are provided for human pluripotent stem cells, mesenchymal stem cells, fibroblasts and transformed cell lines, as well as a method for identifying targeted clones by southern blots. this protocol (from vector design through a single round of targeting and screening) can be completed in ~10 weeks; each subsequent round of targeting and screening should take an additional 7 weeks.
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影响因子：
46.9
作者：
Hockemeyer, Dirk;Soldner, Frank;Beard, Caroline;Gao, Qing;Mitalipova, Maisam;DeKelver, Russell C.;Katibah, George E.;Amora, Ranier;Boydston, Elizabeth A.;Zeitler, Bryan;Meng, Xiangdong;Miller, Jeffrey C.;Zhang, Lei;Rebar, Edward J.;Gregory, Philip D.;Urnov, Fyodor D.;Jaenisch, Rudolf
通讯作者：
Jaenisch, Rudolf
影响因子：
11.2
作者：
Cunningham, Steven C.;Gallmeier, Eike;Kern, Scott E.
通讯作者：
Kern, Scott E.
影响因子：
5.4
作者：
Inoue, N;Hirata, RK;Russell, DW
通讯作者：
Russell, DW
影响因子：
56.9
作者：
Chamberlain, JR;Schwarze, U;Russell, DW
通讯作者：
Russell, DW
影响因子：
5.3
作者：
DUBRIDGE, RB;TANG, P;CALOS, MP
通讯作者：
CALOS, MP