Transfection and promoter analysis in embryonic stem cells.
Embryonic stem (ES) cells are useful for the molecular analysis of developmental pathways when combined with efficient genetic manipulation and in vitro differentiation procedures. To facilitate the genetic modification of ES cells, we describe how to analyze the transcriptional activities of different promoter systems. The DNA segments from a number of promoters are subcloned into the multiple cloning sites of the green fluorescent protein (GFP) reporter plasmid. The resulting recombinant plasmids are used to transfect ES cells by lipofection. Using luciferase-normalized GFP assays, we observed differing promoter activities in transient transfections of ES cells. The judicious selection of the optimum promoter system for specific genetic modification of ES cells will ensure the successful generation of transgenic ES cell lines.
Chung
Sangmi
S
Molecular Neurobiology Laboratory, McLean Hospital, Harvard Medical School, Belmont, MA, USA.
Kim
Kwang-Soo
KS
D000818:Animals; D004622:Embryo, Mammalian / Q000166:cytology*; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics; D008156:Luciferases; D051379:Mice; D039904:Pluripotent Stem Cells / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics; D014162:Transfection
D005456:Fluorescent Dyes; D011994:Recombinant Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D008156:Luciferases