Summary
In vitro suspension cultures of embryonal carcinoma or embryonic stem cells (EC/ES) generate cell aggregates termed as embryoid bodies (EBs). EBs have been analyzed to study the mechanisms of cellular differentiation in vitro. The multipotency of EC/ES cells to differentiate into various cell types as well as the expression of many marker genes provides a valuable in vitro model system to study the mechanisms of cellular differentiation. Here we present a procedure for a mRNA detection of a specific gene using double labeling-mRNA probe and an antibody against cellular marker proteins. This double labeling analysis in combination with a culture of EBs provides a useful approach to analyze several mechanisms of cellular differentiation from multipotent EC/ES cells.
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References
Bradley A, Evans MJ, Kaufman MH, et al. (1984) Formation of germ-line chimeras from embryoderived teratocarcinoma cell lines. Nature 309: 255–256.
Capecchi MR (1989). The new mouse genetics: Altering the genome by gene targeting. Trends Genet 5: 70–76.
Doetschman TC, Eistetter H, Katz M, et al. (1985). The in vitro development of blastocyst-derived embryonic stem cell lines: Formation of visceral yolk sac, blood islands and myocardium. J Embryol Exp Morphol 87: 27–45.
Evans MJ, Kaufman MH (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature 292: 154–156.
Friedrich G, Soriano P (1991). Promoter traps in embryonic stem cells: A genetic screen to identify and mutate developmental genes in mice. Genes & Dev 5: 1513–1523.
Kelley J, Fabisiak JP, Hawes K, et al. (1991). Cytokine signalling in lung: transforming growth factor-beta secretion by lung fibrolasts. Am J Physiol 260: 123–128.
Kessel M, Gruss P (1990). The murine development control genes. Science 249: 374–379.
Kioussi C, Gruss P (1994). Differential induction of Pax genes by NGF and BDNF in cerebellar primary cultures. J Cell Bio 125: 417–425.
Martin GR (1980). Teratocarcinomas and mammalian embryogenesis. Science 209: 768–776.
McBurney MW, Jones-Villeneuve EMW, Edwards M, et al. (1982). Controlled differentiation of teratocarcinoma cells in culture. Nature 299: 165–167.
Mortensen RM, Conner DA, Chao S, et al. (1992). Production of homozygous mutant ES cells with a single targeting construct. Mol Cell Biol 12: 2391–2395.
Muramatsu T (1993). Midkine (MK), the product of a retinoic acid responsive gene, and pleiotrophin constitute a new protein family regulating growth and differentiation. Int J Dev Biol 37: 183–188.
O'Kane CJ, Gehring WJ (1987). Detection in situ of genomic regulatory elements in Drosophila. Proc Natl Acad Sci USA 84: 9123–9127.
Ruther U, Wagner EF, Muller R (1985). Analysis of the differentiation-promoting potential of inducible c-fos genes introduced into embryonal carcinoma cells. EMBO J 4: 1775–1781.
Smith AG, Heath JK, Donaldson DD, et al. (1988). Inhibition of pluripotent embryonic stem cell differentiation by purified polypeptides. Nature 336: 688–690.
Smithies O, Gregg RG, Boggs SS, et al. (1985). Insertion of DNA sequences into the human chromosomal beta-globin locus by homologous recombination. Nature 317: 230–234.
Ware LM, Axelrad AA (1972). Inherited resistance to N- and B-tropic murine leukemia viruses in vitro. Virology 50: 339–348.
Yamada G, Kioussi C, Schubert FR, et al. (1994). Regulated expression of Brachyury (T), Nkx1.1 and Pax genes in embryoid bodies. Biochem Biophys Res Com 199: 552–563.
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Kioussi, C., Yamada, G. Double labeling of mRNA and protein markers in cultured embryoid bodies. Journal of Tissue Culture Methods 16, 11–16 (1994). https://doi.org/10.1007/BF01404831
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DOI: https://doi.org/10.1007/BF01404831