Department of Genetics, Stanford University, Stanford, CA 94305, USA, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA 94305, USA, Howard Hughes Medical Institute and Department of Biology, Stanford University, Stanford, CA 94305, USA, Parkinson's Institute and Clinical Center, Sunnyvale, CA 94085, USA and Stanford Transgenic Research Facility, Stanford University, Stanford, CA 94305, USA.
Nucleic Acids Res. 2014 Mar;42(5):e34. doi: 10.1093/nar/gkt1290. Epub 2013 Dec 4.
To reveal the full potential of human pluripotent stem cells, new methods for rapid, site-specific genomic engineering are needed. Here, we describe a system for precise genetic modification of human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We identified a novel human locus, H11, located in a safe, intergenic, transcriptionally active region of chromosome 22, as the recipient site, to provide robust, ubiquitous expression of inserted genes. Recipient cell lines were established by site-specific placement of a 'landing pad' cassette carrying attP sites for phiC31 and Bxb1 integrases at the H11 locus by spontaneous or TALEN-assisted homologous recombination. Dual integrase cassette exchange (DICE) mediated by phiC31 and Bxb1 integrases was used to insert genes of interest flanked by phiC31 and Bxb1 attB sites at the H11 locus, replacing the landing pad. This system provided complete control over content, direction and copy number of inserted genes, with a specificity of 100%. A series of genes, including mCherry and various combinations of the neural transcription factors LMX1a, FOXA2 and OTX2, were inserted in recipient cell lines derived from H9 ESC, as well as iPSC lines derived from a Parkinson's disease patient and a normal sibling control. The DICE system offers rapid, efficient and precise gene insertion in ESC and iPSC and is particularly well suited for repeated modifications of the same locus.
为了充分发挥人类多能干细胞的潜力，需要开发新的方法来实现快速、定点的基因组工程。在这里，我们描述了一种用于精确遗传修饰人类胚胎干细胞（ESCs）和诱导多能干细胞（iPSCs）的系统。我们鉴定了一个位于 22 号染色体上安全、基因间、转录活跃区域的新的人类基因座 H11，作为受体位点，以提供插入基因的强大、普遍表达。通过自发或 TALEN 辅助同源重组，在 H11 基因座上定点放置携带 phiC31 和 Bxb1 整合酶 attP 位点的“着陆垫”盒，从而建立受体细胞系。通过 phiC31 和 Bxb1 整合酶介导的双整合酶盒交换（DICE），将侧翼带有 phiC31 和 Bxb1 attB 位点的目的基因插入 H11 基因座，取代着陆垫。该系统提供了对插入基因的内容、方向和拷贝数的完全控制，特异性为 100%。一系列基因，包括 mCherry 和神经转录因子 LMX1a、FOXA2 和 OTX2 的各种组合，被插入来源于 H9 ESC 的受体细胞系，以及来源于帕金森病患者和正常同胞对照的 iPSC 系。DICE 系统为 ESC 和 iPSC 中的快速、高效和精确基因插入提供了一种方法，特别适合于同一基因座的重复修饰。
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