Engineering of Human Pluripotent Stem Cells by AAV-mediated Gene Targeting
Engineering of Human Pluripotent Stem Cells by AAV-mediated Gene Targeting
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DOI：
10.1038/mt.2010.55
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发表时间：
2010-06-01
影响因子：
12.4
通讯作者：
Russell, David W.
中科院分区：
文献类型：
--
作者：
Khan, Iram F.;Hirata, Roli K.;Russell, David W.
人类多能干细胞的精确遗传操作将需要实现其科学和治疗潜力。在这里，我们表明，腺相关病毒（AAV）基因靶向载体可用于遗传工程的人胚胎干细胞（ESC）和诱导多能干细胞（iPSC）。不同类型的序列特异性变化，包括突变的产生和校正，被引入人HPRT 1和HMGA 1基因（HPRT 1突变是Lesch-Nyhan综合征的原因）。基因靶向在ESC和iPSC中都以高频率发生，在一些实验中超过1%的所有集落形成单位（CFU）经历靶向。AAV载体也可用于靶向人成纤维细胞中的基因，所述基因随后用于衍生iPSC。准确和有效的靶向发生最小或没有细胞毒性，并且产生的大多数基因靶向干细胞是整倍体和多能性的。
Precise genetic manipulation of human pluripotent stem cells will be required to realize their scientific and therapeutic potential. Here, we show that adeno-associated virus (AAV) gene targeting vectors can be used to genetically engineer human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Different types of sequence-specific changes, including the creation and correction of mutations, were introduced into the human HPRT1 and HMGA1 genes (HPRT1 mutations being responsible for Lesch-Nyhan syndrome). Gene targeting occurred at high frequencies in both ESCs and iPSCs, with over 1% of all colony-forming units (CFUs) undergoing targeting in some experiments. AAV vectors could also be used to target genes in human fibroblasts that were subsequently used to derive iPSCs. Accurate and efficient targeting took place with minimal or no cytotoxicity, and most of the gene-targeted stem cells produced were euploid and pluripotent.