Nicholas Cory R, Gaur Meenakshi, Wang Shaohui, Pera Renee A Reijo, Leavitt Andrew D
Human Embryonic Stem Cell Center, Department of Obstetrics, Gynecology and Reproductive Sciences, San Francisco, CA 94143-0556, USA.
Stem Cells Dev. 2007 Feb;16(1):109-17. doi: 10.1089/scd.2006.0059.
Genetic modification of human embryonic stem (hES) cells is essential for studies of gene function and differentiation. The expression of transgenes may direct tissue-specific differentiation and aid in the identification of various differentiated cell types. Stable genomic integration of transgenes is optimal because hES cell differentiation can span several days to weeks and include numerous cell divisions, and establishing homogeneous modified cell lines will facilitate research studies. Herein we provide a method for producing and expanding hES cell lines from single cells that have been isolated by fluorescence-activated cell sorting (FACS) following genetic modification by lentivirus vectors. Using this method, we have established enhanced green fluorescent protein (eGFP)-expressing hES cell lines that are pluripotent, contain a diploid chromosomal content, and stably express eGFP following more than 2 months of routine culture and in vivo differentiation.
人类胚胎干细胞（hES）的基因改造对于基因功能和分化研究至关重要。转基因的表达可指导组织特异性分化，并有助于鉴定各种分化细胞类型。转基因的稳定基因组整合是最佳选择，因为hES细胞分化可能持续数天至数周，包括多次细胞分裂，建立同质的修饰细胞系将有助于研究。在此，我们提供了一种从经慢病毒载体基因改造后通过荧光激活细胞分选（FACS）分离的单细胞中产生和扩增hES细胞系的方法。使用该方法，我们建立了表达增强型绿色荧光蛋白（eGFP）的hES细胞系，这些细胞系具有多能性，含有二倍体染色体含量，并且在常规培养和体内分化超过2个月后仍稳定表达eGFP。
