Institute of Bioengineering and Nanotechnology and Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore.
Nucleic Acids Res. 2011 Sep 1;39(16):e107. doi: 10.1093/nar/gkr409. Epub 2011 Jun 17.
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.
将转基因插入人胚胎干细胞 (hESC) 中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，并且可能能够在 hESC 扩增及其分化后代中持续表达转基因。在这里，我们在 hESC 中利用同源重组将异源 loxP 位点引入 AAVS1 基因座，该基因座具有开放染色质结构，可避免转基因沉默现象。然后，我们使用杆状病毒载体进行 Cre 重组酶介导的盒交换，将转基因插入修饰后的 AAVS1 基因座。带有 loxP 对接位点的主 hESC 系的靶向效率高达 100%。在 hESC 扩增过程中，插入的转基因表达至少持续 20 代，并在源自基因修饰 hESC 的分化细胞中保留。因此，本研究证明了在 hESC 中利用同源重组和病毒转导在 AAVS1 基因座进行遗传操作的可行性，以促进重组酶介导的盒交换。所开发的方法将有助于在 hESC 基因组的特定基因座上进行重复的基因靶向。
