Maintenance of pluripotency in human embryonic stem cells stably over-expressing enhanced green fluorescent protein.

摘要: The availability of human embryonic stem (HES) cells with a readily evaluated genetic marker such as green fluorescent protein (GFP) could facilitate number experimental opportunities. We constructed novel plasmid two elongation factor-1alpha (EF-1alpha) promoters (YPL2) to obtain vector mammalian for simultaneous transgene expression in HES cells. An enhanced (EGFP) cDNA was inserted under the control first EF-1alpha promoter construct YPL2-EGFP. second EF1-alpha upstream neomycin resistance gene. H1 were transfected YPL2-EGFP using Fugene 6. Following 100 microg/ml selection, individual colonies demonstrating stable EGFP observed. After 4 months passage continued maintain typical cell morphology. Undifferentiated showed no change determined by FACS analysis. Immunostaining demonstrated maintenance Oct-3/4 undifferentiated H1EGFP that indistinguishable from wild-type Addition 10 ng/ml bone morphogenic protein-4 (BMP-4) provoked morphological and functional differentiation trophoblasts, but loss expression. injection EGFP-HES into immunodeficient mice, there robust formation teratomas broad range pluripotency widespread also maintained differentiating embryoid bodies formed This report demonstrates ES carrying will be useful diverse areas research.