Purpose: To transfect human lens epithelial cells (HLE B-3) with a cDNA construct encoding human aA-crystallin and isolate cells expressing aAcrystallin. Methods: An 828 bp fragment containing the coding sequence of the human aA-crystallin cDNA was obtained from Dr. Mark Petrash. This fragment was ligated into the pCI-neo' mammalian expression vector, which contains a CMV-immediate-early promoter/enhancer and a neomycin-resistant cassette, leading to pci-neoctA. This construct was transfected into HLE B-3 cells using standard calcium phosphate transfection protocols. Geneticin-resistant colonies were isolated and expanded into mass cultures. Expression of aA-crystallin in the transfectants was examined by western blot analysis. Results: Cells transfected with pCI-neoctA and cultured in selective medium maintained a normal morphology. Before transfection, cells had no detectable expression of a-crystallin. Western blot analysis indicated that transfected cells expressed a -20 kDa protein that co-migrated with native human a-crystallin. Conclusions: The use of stably transfected HLE B-3 cells expressing aA-crystallin should provide a model system to probe the chaperone-like effect of aA-crystallin under physiological conditions of stress.
|Investigative Ophthalmology and Visual Science
|Published - Dec 1 1997