title: Maintenance of pluripotency in human embryonic stem cells stably over-expressing enhanced green fluorescent protein.
abstract: The availability of human embryonic stem (HES) cells with a readily evaluated genetic marker such as green fluorescent protein (GFP) could facilitate a number of experimental opportunities. We constructed a novel plasmid with two elongation factor-1alpha (EF-1alpha) promoters (YPL2) to obtain a vector with mammalian promoters for simultaneous transgene expression in HES cells. An enhanced green fluorescent protein (EGFP) cDNA was inserted under the control of the first EF-1alpha promoter to construct plasmid YPL2-EGFP. The second EF1-alpha promoter was upstream of the neomycin resistance gene. H1 HES cells were transfected with YPL2-EGFP using Fugene 6. Following 100 microg/ml neomycin selection, individual colonies demonstrating stable EGFP expression were observed. After 4 months of passage under neomycin selection, the cells continued to maintain typical HES cell morphology. Undifferentiated cells showed no change in EGFP expression as determined by FACS analysis. Immunostaining demonstrated maintenance of Oct-3/4 expression in undifferentiated H1EGFP cells that was indistinguishable from wild-type HES cells. Addition of 10 ng/ml bone morphogenic protein-4 (BMP-4) to the cells provoked morphological and functional differentiation to trophoblasts, but no loss of EGFP expression. Following injection of EGFP-HES cells into immunodeficient mice, there was robust formation of teratomas that demonstrated a broad range of morphological pluripotency with widespread EGFP expression. EGFP expression was also maintained in differentiating embryoid bodies formed from EGFP-HES cells. This report demonstrates that ES cells carrying EGFP will be useful in diverse areas of embryonic stem cell research.
authors.affiliation: Department of Obstetrics and Gynecology and Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI 53715-1299, USA.
authors.forename: Yi-Ping
authors.initials: YP
authors.lastname: Liu
authors.forename: Oksana V
authors.initials: OV
authors.lastname: Dovzhenko
authors.forename: Mark A
authors.initials: MA
authors.lastname: Garthwaite
authors.forename: Svetlana V
authors.initials: SV
authors.lastname: Dambaeva
authors.forename: Maureen
authors.initials: M
authors.lastname: Durning
authors.forename: Leah M
authors.initials: LM
authors.lastname: Pollastrini
authors.forename: Thaddeus G
authors.initials: TG
authors.lastname: Golos
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D002478:Cells, Cultured; D003094:Collagen / Q000494:pharmacology; D003470:Culture Media / Q000494:pharmacology; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004338:Drug Combinations; D004351:Drug Resistance; D004622:Embryo, Mammalian / Q000166:cytology*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006801:Humans; D007797:Laminin / Q000494:pharmacology; D051379:Mice; D009355:Neomycin / Q000494:pharmacology; D020648:Peptide Elongation Factor 1 / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology*; D011401:Promoter Regions, Genetic; D011509:Proteoglycans / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013234:Stem Cells / Q000166:cytology; D013724:Teratoma / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D019076:Transgenes
chemical_list: D003470:Culture Media; D017931:DNA Primers; D018076:DNA, Complementary; D004338:Drug Combinations; D007797:Laminin; D020648:Peptide Elongation Factor 1; D011509:Proteoglycans; C059485:matrigel; D049452:Green Fluorescent Proteins; D003094:Collagen; D009355:Neomycin