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M.E. Ireland; Gene Silencing of the Epidermal Growth Factor Receptor in Cultured Chicken Lens Annular Pad Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):1889.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To determine if the epidermal growth factor receptor (EGFR) is required to promote the accumulation of differentiated traits in cultured chicken lens annular pad cells. Methods: Silencing RNAs (siRNAs) specific for the chicken EGFR were obtained from a commercial source (Dharmacon) and transfected into first passage chicken lens annular pad cells. Cell morphology was monitored with phase contrast or bright field microscopy. After four days, DNA was isolated to quantify cell numbers. In replicate cultures, immunoprecipitation of the EGFR followed by western blotting and densitometry was performed to examine protein levels. Accumulation of the lens fiber specific beaded filament proteins phakinin and filensin was also examined with western blotting. Results: Non–transfected and mock–transfected cultures each elaborated extensive lentoids, morphologic indicators of in vitro fiber differentiation, within the four–day culture period. In a dose–dependent manner between 25 nM and 200 nM of added siRNAs, transfected cultures failed to produce lentoids but instead elaborated extensive areas of multilayering, a precursor step observed prior to lentoid formation. DNA levels were not significantly altered between the various groups at the termination of the experiments. Densitometric analysis of immunoprecipitates showed an approximate 60% reduction in the protein levels of the EGFR between non– or mock–transfected cultures and 100 nM transfected cultures. No differences were noted in phakinin or filensin levels as judged by densitometry of western blots. Conclusions: EGFR protein levels in cultured chicken lens annular pad cells can be readily reduced using siRNA technology. Subsequent effects on the appearance of differentiated traits indicate that fiber cell morphologic differentiation may be regulated by EGFR expression or activity levels.
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