Use of the cytomegalovirus promoter for transient and stable transgene expression in mouse embryonic stem cells.
Embryonic stem (ES) cells are pluripotent cells derived from the epiblast of preimplantation embryos. These cells are emerging as a key model system for elucidating mechanisms involved in development and disease as well as having a unique potential as a source of unlimited somatic cells for transplantation therapies. ES cells can be easily manipulated at the DNA level, allowing both transient and stable expression of complementary DNA encoding transgenes of interest. The human cytomegalovirus (CMV) immediate-early enhancer and promoter is commonly used for transient expression of transgenes in ES cells. However, its use in the formation of stable cell lines is less common. We demonstrate an electroporator transformation technique that results in up to 90% transfection efficiency of CMV-encoding vectors in ES cells. Furthermore, we describe the design of vectors and cloning techniques that allow stable expression of transgenes under control of the CMV promoter and a fluorescent microscopy method for detecting protein expression in ES cells in situ.
Barrow
Katie M
KM
Centre for Molecular Medicine, Faculty of Medical and Human Sciences, The University of Manchester, Manchester, UK.
Perez-Campo
Flor M
FM
Ward
Christopher M
CM
D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D002469:Cell Separation; D003587:Cytomegalovirus / Q000235:genetics*; D004622:Embryo, Mammalian / Q000166:cytology*; D005434:Flow Cytometry; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D051379:Mice; D008856:Microscopy, Fluorescence; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D014162:Transfection
C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins