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Anne Terrell, Stephanie Waters, Christine Dang, David C Beebe, Salil Anil Lachke; Molecular characterization of mouse lens epithelial cell lines and their applicability to study lens RNA granules. Invest. Ophthalmol. Vis. Sci. 2014;55(13):743.
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© ARVO (1962-2015); The Authors (2016-present)
The discovery of RNA granule proteins associated with mammalian cataract has initiated an interest in investigating post-transcriptional control mechanisms in the lens. However, deleting the core components (e.g. Dcp1a, Ddx6, etc.) of RNA granules like Processing Bodies (PBs) and Stress Granules (SGs) is expected to cause early embryonic lethality in mice, and conditional null mutant alleles for these genes are as yet unavailable. Mouse lens epithelial cell lines offer an alternate reagent, but have not been characterized for their suitability in these studies. Therefore, we investigated several mouse lens epithelial cell lines for their expression of lens marker genes as well as their potential to form distinct RNA granules.
Mouse lens epithelial cell lines (LECs) 21EM15, 17EM15 and αTN4 were characterized in this study. Mouse fibroblast cell line NIH3T3 was analyzed as a non-lens derived reference. Comparative gene expression analysis between 21EM15 cell line microarrays and iSyTE expression datasets were used to determine lens-enriched genes that are potentially expressed in these cell lines. Expression of candidate genes was validated using regular and quantitative RT-PCR. For testing their potential to form PBs and SGs, cells were stressed with sodium arsenite and were examined by confocal microscopy.
We find that LECs 21EM15, 17EM15 and αTN4 exhibit significantly higher expression of the following key lens genes when compared to NIH3T3 cells: Pax6, Foxe3, Prox1, Jag1, Aldh1a1 and Trpm3. Additionally, these LECs also expressed 35 other lens marker genes. When tested for RNA granules, all three LECs were found to have significantly higher (1.5 to 6-fold) levels of PBs when compared to NIH3T3 cells in unstressed conditions. In stress conditions, all cell lines exhibited elevated levels of both classes of RNA granules, but the extent of elevation was higher for LECs when compared to the reference cell line.
In sum, our data demonstrate that lens epithelial cell lines 21EM15, 17EM15 and αTN4 retain the expression of several important lens markers genes. Significantly, these LECs exhibit elevated levels of P-bodies and Stress granules when compared to a non-lens cell line in both normal and stress conditions, indicating their suitability for investigating the function of these cytosolic ribonucleoprotein complexes in lens cells.
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