May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
p21WAF1/cip1 siRNA-Induced Proliferative Activity of Rat Corneal Endothelial Cells
Author Affiliations & Notes
  • D. L. Harris
    Ophthalmology, Schepens Eye Res Inst/Harvard Med Sch, Boston, Massachusetts
  • N. C. Joyce
    Ophthalmology, Schepens Eye Res Inst/Harvard Med Sch, Boston, Massachusetts
  • Footnotes
    Commercial Relationships  D.L. Harris, None; N.C. Joyce, None.
  • Footnotes
    Support  NIH Grant EY12700 (NCJ)
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 3938. doi:https://doi.org/
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    • Get Citation

      D. L. Harris, N. C. Joyce; p21WAF1/cip1 siRNA-Induced Proliferative Activity of Rat Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3938. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : To determine whether siRNA down-regulation of p21WAF1/cip1 will overcome G1-phase arrest and promote cell cycle progression in rat corneal endothelial cells (RCEC).

Methods: : Confluent cultures of RCEC were trypsinzed and electroporated using a Bio-rad Gene Pulser system. Proper conditions were determined for voltage (150-450V) and pulse length (100-400µs) by viewing Cy3-labeled non-silencing siRNA electroporation efficiency with a fluorescent microscope. Optimal number of pulses (1-3) was determined using G3PDH siRNA and 3 different p21WAF1/cip1 siRNAs (Ambion, Austin, TX). Controls included cells not electroporated or electroporated with non-silencing siRNA. To determine the most efficient downregulation of the corresponding protein, cells were electroporated, seeded in dishes, maintained for 72 hrs in 10% FBS, followed by western blot analysis. To determine the effect of p21WAF1/cip1 siRNA treatment on proliferation, cells were electroporated and then incubated for 24, 72, or 120 hours in 10% FBS. Cell numbers were determined at each time-point using WST-8-based spectrophotometric analysis.

Results: : The following conditions were determined to be optimal for siRNA electroporation in RCEC: voltage of 300V, pulse length of 400 µs, and 2 or 3 pulses. Two of the three p21WAF1/cip1 siRNAs significantly reduced p21WAF1/cip1 protein expression. Treatment of RCEC with one of the p21WAF1/cip1 siRNAs resulted in significantly higher cell numbers by 120 hours compared with controls electroporated with non-silencing siRNA.

Keywords: cornea: endothelium 
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