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W. Dailey, K. Drenser, M. Trese; Analysis of Wnt Signal Transduction Pathway Gene Expression in Mouse Embryonic Stem Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4068.
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© ARVO (1962-2015); The Authors (2016-present)
Many pediatric vitreoretinopathies ( Norrie disease, FEVR, Coats disease, ROP, and OPS) have been associated with mutations occurring in genes associated with the Wnt receptor:ß-catenin signal transduction pathway. Our purpose was to better understand the expression of three key genes (NDP, Fzd4 and LRP5) that activate this pathway in Mouse Embryonic Stem Cells (MES).
Initially, MES were grown on feeder MEF cells and supplemented with LIF to prevent differentiation. The hanging drop method was then used to form embroid bodies that were seeded into tissue culture plates and allowed to differentiate. mRNA was purified from the undifferentiated cells and cells that were allowed to differentiate for either four or nine days. RT-PCR was performed using an ABI PRISM 7000 Sequence Detection System and gene specific primers for NDP, Fzd4, LRP5 and GADPH housekeeping gene.
Expression of NDP mRNA was extremely low (<100 copies/µg of total RNA) at all three time points. For both Fzd4 and LRP5, an increase in expression at subsequent time points was seen. LRP5 expression in undifferentiated cells and day 9 cells was (200 and 6000) copies/µg of total RNA, respectively. Fzd4 expression in undifferentiated cells and day 9 cells was (100 and 2000) copies/µg of total RNA, respectively.
Regulation of the Wnt receptor:ß-catenin signal transduction pathway varies with cell differentiation, as demonstrated by changing expression levels of key components of this pathway. This indicates that this pathway plays a dynamic role in retinal development.
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