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Hongyu Ying, Ye Qiu, Xiang Shen, Rajalekshmy Shyam, Beatrice Y.J.T. Yue; Establishment of Inducible Wild Type And Mutant Myocilin-GFP-Expressing RGC5 Cell Lines. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4597.
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© ARVO (1962-2015); The Authors (2016-present)
To establish tetracycline regulated (Tet-on) inducible RGC5 cell lines for stable expression of wild type and mutant (P370L and Q368X) myocilin-GFP fusion proteins following doxycycline (Dox) induction.
A plasmid vector pTRE-MYOC-EGFP-INS-rtTA-IRES-hyg-pcDNA3.1z which contains both tetracycline regulatory and responsive components based on the Clontech's Tet-on advance system was constructed. Two other plasmid vectors containing glaucoma related P370L and Q368X mutations were similarly prepared. RGC5 cells were transfected with these constructs and selected in hygromycin (100 µg/ml)-containing medium for about 2 weeks until colonies grew out. The cells were then trypsinized and induced with 1 µg/ml of Dox for 24 h. GFP positive cells sorted using DakoCytomation MoFlo into 96 well plates (1 cell/well) were incubated with maintenance medium (containing 50 µg/ml of hygromycin but without Dox) for another 2 weeks. Cells were screened for high, moderate, or low myocilin-GFP expression after induction by fluorescence microscopy. The level of induced wild type and mutant myocilin-GFP proteins was assessed by Western blotting. The high, moderate, and low expressers were allowed to multiply and were banked in liquid nitrogen.
After several rounds of selection, clones that displayed strong, moderate or faint wild type, P370L, or Q368X myocilin-GFP expression upon Dox induction were obtained. The levels of wild type and mutant myocilin-GFP in the clones were confirmed by Western blotting. Microscopic examination of the inducible, stable cell lines indicated that the wild type myocilin-GFP had a more spread out, cytoplasmic distribution pattern similar to that of the endogenous myocilin. Cytoplasmic aggregates were seen in P370L- and Q368X-GFP-expressing cells.
Tet-on inducible, stable RGC5 cell lines were established. These cell lines, expressing wild type and mutant myocilin-GFP proteins upon Dox induction, are valuable in facilitating studies such as cellular processing of myocilins.
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