Efficient recombinase-mediated cassette exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors.
Efficient recombinase-mediated cassette exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors.
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DOI：
10.1093/nar/gkr409
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发表时间：
2011-09-01
影响因子：
14.9
通讯作者：
Wang S
中科院分区：
文献类型：
--
作者：
Ramachandra CJ;Shahbazi M;Kwang TW;Choudhury Y;Bak XY;Yang J;Wang S
将转基因插入人胚胎干细胞（hESCs）的特定基因组位点对于防止随机整合诱导的插入突变至关重要，并且可能在hESC扩增过程中及其分化后代中实现转基因的持续表达。在这里，我们在hESCs中采用同源重组将异源loxP位点引入AAVS1位点，AAVS1位点具有开放的染色质结构，可以避免转基因沉默现象。然后，我们使用杆状病毒载体进行Cre重组酶介导的盒式交换，将转基因插入修饰的AAVS1位点。在具有loxp对接位点的主hESC系中，靶向效率高达100%。在hESC扩增过程中，插入的转基因基因的表达持续了至少20代，并保留在转基因hESCs衍生的分化细胞中。因此，本研究证明了通过同源重组对AAVS1位点进行基因操作，并在hESCs中利用病毒转导促进重组酶介导的卡式交换的可行性。该方法的发展将是有用的重复基因靶向在hESC基因组的一个确定的位点。
Insertion of a transgene into a defined genomic locus in human embryonic stem cells (hESCs) is crucial in preventing random integration-induced insertional mutagenesis, and can possibly enable persistent transgene expression during hESC expansion and in their differentiated progenies. Here, we employed homologous recombination in hESCs to introduce heterospecific loxP sites into the AAVS1 locus, a site with an open chromatin structure that allows averting transgene silencing phenomena. We then performed Cre recombinase mediated cassette exchange using baculoviral vectors to insert a transgene into the modified AAVS1 locus. Targeting efficiency in the master hESC line with the loxP-docking sites was up to 100%. Expression of the inserted transgene lasted for at least 20 passages during hESC expansion and was retained in differentiated cells derived from the genetically modified hESCs. Thus, this study demonstrates the feasibility of genetic manipulation at the AAVS1 locus with homologous recombination and using viral transduction in hESCs to facilitate recombinase-mediated cassette exchange. The method developed will be useful for repeated gene targeting at a defined locus of the hESC genome.
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影响因子：
3.8
作者：
Ho, YC;Chen, HC;Hu, YC
通讯作者：
Hu, YC
影响因子：
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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
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Jaenisch, Rudolf
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5.2
作者：
Ma, Y;Ramezani, A;Thomson, JA
通讯作者：
Thomson, JA
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作者：
Ogata, T;Kozuka, T;Kanda, T
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Kanda, T
影响因子：
3.4
作者：
HEIMPEL, AM;BUCHANAN, LK
通讯作者：
BUCHANAN, LK