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Z. Yin, Y. Tong, H. Zhu, M.A. Watsky; ClC–3 Knockdown and Overexpression in a Human Corneal Keratocyte Cell Line . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2981.
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To determine the effects of ClC–3 knockdown and overexpression on cell morphology and growth in a human corneal keratocyte cell line.
We used a hairpin siRNA (shRNA) expression system based on a mouse mU6 promoter to knock down ClC–3 in our human keratocyte cell line. The ClC–3 gene sequence was scanned with Genscript siRNA Target Finder software to select 5 candidate shRNAs. Designed shRNA sequences were synthesized as two complementary DNA oligonucleotides, annealed from 91oC to 22oC and ligated to the mU6 vector. To test the efficiency of the candidate shRNA constructs, they were introduced into an eGFP reporter system, which fused the CLC–3 gene cDNA with eGFP DNA into the pcDNA3 vector to construct a mammalian cell eGFP expression plasmid. Cells expressing ClC–3 were thus fluorescent. To screen the efficiency of candidate shRNA sequences, the shRNA vector and the reporter eGFP vector were co–transfected into HK293 cells, using cells expressing the eGFP reporter vector alone as controls. Using this system, HK293 cells with the lowest eGFP expression were those with the highest degree of ClC–3 knockdown. The most efficient shRNA was then introduced into keratocytes by electroporation. ClC–3 overexpression was achieved by electroporating full–length hClC3 within a pcDNA3.1 vector, into keratocytes.
shRNA was successful at knocking down ClC–3. Cells expressing shRNA ClC–3 shRNA had reduced survival as compared to control shRNA expressing cells, as did cells overexpressing ClC–3. Compared to controls, shRNA transfected cell morphology was differed significantly, with shRNA cells being rounder with fewer cell extensions. ClC–3 overexpressing cells were more spindle–shaped than controls.
ClC–3 knockdown and overexpression affect keratocyte survival and morphology.
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