Hohenstein Kristi A, Pyle April D, Chern Jing Yi, Lock Leslie F, Donovan Peter J
Department of Biological Chemistry, Sue and Bill Gross Stem Cell Research Program, Center for Molecular and Mitochondrial Medicine and Genetics, University of California Irvine, Irvine, California 92697, USA.
Stem Cells. 2008 Jun;26(6):1436-43. doi: 10.1634/stemcells.2007-0857. Epub 2008 Mar 6.
High-efficiency genetic modification of human embryonic stem (hES) cells would enable manipulation of gene activity, routine gene targeting, and development of new human disease models and treatments. Chemical transfection, nucleofection, and electroporation of hES cells result in low transfection efficiencies. Viral transduction is efficient but has significant drawbacks. Here we describe techniques to transiently and stably express transgenes in hES cells with high efficiency using a widely available vector system. The technique combines nucleofection of single hES cells with improved methods to select hES cells at clonal density. As validation, we reduced Oct4 and Nanog expression using siRNAs and shRNA vectors in hES cells. Furthermore, we derived many hES cell clones with either stably reduced alkaline phosphatase activity or stably overexpressed green fluorescent protein. These clones retained stem cell characteristics (normal karyotype, stem cell marker expression, self-renewal, and pluripotency). These studies will accelerate efforts to interrogate gene function and define the parameters that control growth and differentiation of hES cells. Disclosure of potential conflicts of interest is found at the end of this article.
对人类胚胎干细胞（hES细胞）进行高效基因改造，将有助于调控基因活性、实现常规基因靶向，以及开发新的人类疾病模型和治疗方法。对hES细胞进行化学转染、核转染和电穿孔，转染效率都很低。病毒转导虽有效，但存在重大缺陷。在此，我们描述了利用一种广泛可用的载体系统，在hES细胞中高效瞬时和稳定表达转基因的技术。该技术将单个hES细胞的核转染与以克隆密度选择hES细胞的改进方法相结合。作为验证，我们在hES细胞中使用小干扰RNA（siRNAs）和短发夹RNA（shRNA）载体降低了Oct4和Nanog的表达。此外，我们获得了许多hES细胞克隆，它们要么稳定降低了碱性磷酸酶活性，要么稳定过表达绿色荧光蛋白。这些克隆保留了干细胞特征（正常核型、干细胞标志物表达、自我更新和多能性）。这些研究将加速探究基因功能以及确定控制hES细胞生长和分化参数的工作。潜在利益冲突披露见本文末尾。
