新基因工具可用于分析人类胚胎干细胞分化过程
来自明尼苏达大学干细胞研究所的科学家最近描述了如何将一种基因工具应用于分析人类胚胎干细胞的分化过程。研究结果发表在11月份的《实验生物学和医学》（Experimental Biology and Medicine）上。
科学家对于人类胚胎干细胞（hESC）如何实现自我更新并不清楚。为了全面了解这些细胞的自我更新能力、多能性和调节过程，研究小组需要得到经过充分的基因改造的细胞，并分析对这些基因表达进行增强或是减弱之后的结果。
由明尼苏达大学的Meri Firpo博士领导的研究小组和洛杉矶儿童医院的基因治疗学家以及密歇根大学的发展生物学家进行了合作。科学家们利用基因抑制技术（knockdown）来降低oct4基因的表达，这是一种对老鼠和人类中胚胎干细胞进行自我更新非常关键的基因。通过观察经过基因抑制和其它基因手段处理后的老鼠细胞，科学家发现降低人类胚胎干细胞中oct4的含量可以诱导分化的发生。
科学家接着使用质粒载体来增加hESC中的oct4含量。它同样也会造成预期中的细胞分化，并且分化的模式和基因抑制中观察到的类似。这出乎科学家的预料，因为当在老鼠的胚胎干细胞中提高oct4的表达时，其分化成的细胞类型和oct4表达被抑制时并不一样。（ 教育部科技发展中心）
原文链接：http://www.physorg.com/news112439146.html
原始出处:
Experimental Biology and Medicine 232:1368-1380 (2007)
doi: 10.3181/0703-RM-63
Manipulation of OCT4 Levels in Human Embryonic Stem Cells Results in Induction of Differential Cell Types
Ryan T. Rodriguez*,1, J. Matthew Velkey
, Carolyn Lutzko
, Rina Seerke*, Donald B. Kohn, K. Sue O’Shea and Meri T. Firpo*,3,2
* Department of Obstetrics, Gynecology, and Reproductive Sciences, University of California San Francisco, San Francisco, California 94143; Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109; and Division of Research Immunology/BMT Children’s Hospital Los Angeles, Department of Pediatrics, University of Southern California Keck School of Medicine, Los Angeles, California 90027
To whom requests for reprints should be addressed at 3 2001 6th Street SE, Minneapolis, MN 55455. E-mail: firpo001@umn.edu
To fully understand self-renewal and pluripotency and their regulation in human embryonic stem cells (hESCs), it is necessary to generate genetically modified cells and analyze the consequences of elevated and reduced expression of genes. Genes expressed in hESCs using plasmid vectors, however, are subject to silencing. Moreover, hESCs have a low plating efficiency when dissociated to single cells, making creation of subcloned lines inefficient. In addition to overexpression experiments, it is important to perform loss-of-function studies, which can be achieved rapidly using RNA interference (RNAi). We report stable long-term expression of enhanced green fluorescent protein (eGFP) in hESCs using a lentiviral vector, and establishment of an eGFP-expressing subline (RG6) using manual dissection. To demonstrate the efficacy of RNAi in hESCs, an RNAi expression vector was used to achieve reduced expression of eGFP in hESCs. To evaluate the role of OCT4 in the regulation of hESC self-renewal and differentiation, a vector expressing a hairpin RNA targeting endogenous expression of OCT4 was constructed. In a novel experiment in hESCs, the OCT4 cDNA sequence was cloned into an expression vector to allow for the transient upregulation of OCT4 in hESCs. The ability to manipulate levels of OCT4 above and below enodogenous levels allows the determination of OCT4 function in hESCs. Specifically, reduced expression of OCT4 in hESCs promoted upregulation of markers indicative of mesoderm and endoderm differentiation, and elevated levels of OCT4 in hESCs promoted upregulation of markers indicative of endoderm derivatives. Thus, both upregulation and downregulation of Oct4 in hESCs results in differentiation, but with patterns distinct from parallel experiments in mice.
Key Words: stem cell • self-renewal • differentiation • knockdown • overexpression
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