April 2009
Volume 50, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2009
p21WAF1/cip1 siRNA Electroporation of Primary Human Corneal Endothelial Cells
Author Affiliations & Notes
  • D. L. Harris
    Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts
  • N. C. Joyce
    Schepens Eye Research Institute and Department of Ophthalmology, Harvard Medical School, Boston, Massachusetts
  • Footnotes
    Commercial Relationships  D.L. Harris, None; N.C. Joyce, None.
  • Footnotes
    Support  R01 EY12700 (NCJ)
Investigative Ophthalmology & Visual Science April 2009, Vol.50, 1805. doi:
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    • Get Citation

      D. L. Harris, N. C. Joyce; p21WAF1/cip1 siRNA Electroporation of Primary Human Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1805.

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

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Abstract

Purpose: : To test p21WAF1/cip1 siRNA electroporation in primary human corneal endothelial cells (HCEC) from young (< 30yo) and older donors (>50yo).

Methods: : Confluent cultures of HCEC were trypsinzed and electroporated using a Nucleofector II Device from Amaxa, Inc. (Gaithersburg, MD). Optimal conditions were determined using the kit for primary mammalian endothelial cells by electroporating with a pmax GFP plasmid or a Cy3-labeled non-silencing siRNA at 0.5, 1, 2, and 3 ug and viewing efficiency with a fluorescence microscope. Further optimization included testing of 3 different p21WAF1/cip1 siRNAs (Ambion, Austin, TX). Controls included cells not electroporated or electroporated with non-silencing siRNA. To determine the most efficient down-regulation of the corresponding protein, cells were electroporated, seeded in dishes, maintained for 72 hrs in 8% FBS, followed by western blot analysis with p21WAF1/cip1, p16INK4a and beta-actin antibodies.

Results: : The program T-023 using 1 ug of siRNA was the most efficient for siRNA electroporation in HCEC. All three p21WAF1/cip1 siRNAs significantly reduced p21WAF1/cip1 protein expression using beta-actin for normalization. This treatment did not affect expression of p16INK4a. p21WAF1/cip1 protein levels were significantly reduced in HCEC from older donors compared with young donors.

Keywords: cornea: endothelium 
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