Noisa Parinya, Urrutikoetxea-Uriguen Alai, Cui Wei
Institute of Reproductive and Developmental Biology, Imperial College London, London, UK.
Methods Mol Biol. 2011;690:95-106. doi: 10.1007/978-1-60761-962-8_6.
The distinctive properties of human embryonic stem cells (hESCs) enable them to provide unique models to study the network of signaling pathways that regulate organogenesis, generate disease models, produce cells and tissues for therapies, and identify new drugs for treatment. Genetic modification of hESCs is a powerful tool to assist the above studies. Generation of lineage-specific fluorescent protein reporter hESC lines will greatly benefit investigators to monitor specific cell lineages in a live, easy, and timely manner. This technique will facilitate high throughput screening to identify molecules important in regulating specific cell fate commitment. In addition, such reporter cell lines enable researchers to enrich certain cell populations by fluorescent activated cell sorting (FACS) for either downstream biological analysis or in vivo applications. We have shown that hESCs can be stably transfected with a plasmid in which expression of the green fluorescent protein (GFP) is under the control of the Oct-4 promoter using chemical transfection. The expression pattern of transgenic Oct-4-GFP reflects that of endogenous Oct-4.
人类胚胎干细胞（hESCs）的独特特性使其能够提供独特的模型，用于研究调节器官发生的信号通路网络、生成疾病模型、生产用于治疗的细胞和组织以及鉴定新的治疗药物。对hESCs进行基因改造是辅助上述研究的有力工具。生成谱系特异性荧光蛋白报告基因hESC系将极大地有助于研究人员以实时、简便和及时的方式监测特定细胞谱系。这项技术将有助于高通量筛选，以鉴定在调节特定细胞命运决定中起重要作用的分子。此外，这种报告细胞系使研究人员能够通过荧光激活细胞分选（FACS）富集某些细胞群体，用于下游生物学分析或体内应用。我们已经表明，使用化学转染法，hESCs可以被稳定转染一种质粒，其中绿色荧光蛋白（GFP）的表达受Oct-4启动子的控制。转基因Oct-4-GFP的表达模式反映了内源性Oct-4的表达模式。
