High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors
High-level sustained transgene expression in human embryonic stem cells using lentiviral vectors
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DOI：
10.1634/stemcells.21-1-111
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发表时间：
2003-01-01
期刊：
影响因子：
5.2
通讯作者：
Thomson, JA
中科院分区：
文献类型：
--
作者：
Ma, Y;Ramezani, A;Thomson, JA
在这里，我们描述了持续表达的转基因引入人胚胎干细胞（ES）使用自失活慢病毒载体。在低感染复数下，含有在人延伸因子Ia启动子控制下的绿色荧光蛋白（GFP）转基因的水泡性口炎病毒假型载体以高效率转导人ES细胞。大多数转导的ES细胞，其具有低数目的整合载体，在培养60天后继续表达GFP。将来自人干扰素-P基因的支架附着区（SAR）并入慢病毒载体骨架中增加了GFP表达的平均水平，并且将SAR与来自鸡β-珠蛋白基因座5'端的染色质绝缘子一起纳入降低了GFP表达的可变性。当体外诱导转导的ES细胞分化为CD 34（+）造血前体细胞时，GFP表达保持最小沉默。有效地将活性转基因导入人ES细胞的能力将促进人类系统中早期发育过程的功能获得研究。这些结果也有重要的意义，基因修饰的人胚胎干细胞在移植和组织再生应用的未来可能使用。
Here we describe the sustained expression of transgenes introduced into human embryonic stem (ES) cells using self-inactivating lentiviral vectors. At low multiplicity of infection, vesicular stomatitis virus-pseudotyped vectors containing a green fluorescent protein (GFP) transgene under the control of a human elongation factor la promoter transduced human ES cells at high efficiency. The majority of the transduced ES cells, which harbored low numbers of integrated vectors, continued to express GFP after 60 days of culture. Incorporation of a scaffold attachment region (SAR) from the human interferon-P gene into the lentiviral vector backbone increased the average level of GFP expression, and inclusion of the SAR together with a chromatin insulator from the 5' end of the chicken beta-globin locus reduced the variability in GFP expression. When the transduced ES cells were induced to differentiate into CD34(+) hematopoietic precursors in vitro, GFP expression was maintained with minimal silencing. The ability to efficiently introduce active transgenes into human ES cells will facilitate gain-of-function studies of early developmental processes in the human system. These results also have important implications for the possible future use of gene-modified human ES cells in transplantation and tissue regeneration applications.