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Invest Ophthalmol Vis Sci 2004;45: E-Abstract 4432.
© 2004 ARVO


4432—B893

Cell Cycle Synchronization and Transfection Efficiency of Human Trabecular Meshwork Cells

E.A. Hoffman1,A, A. Poncius1,A, B.S. McKay1,B and W.D. Stamer1,C

A Ophthalmology, B Ophthalmology and Cell Biology, C Ophthalmology and Pharmacology, 1 University of Arizona, Tucson, AZ

Commercial Relationships: E.A. Hoffman, None; A. Poncius, None; B.S. McKay, None; W.D. Stamer, None.

Grant Identification: Support: EY12797, Research to Prevent Blindness

Abstract

Purpose: The human trabecular meshwork (HTM) is a likely site of pathology in glaucoma. Studying the role of specific proteins in HTM physiology has been difficult because of inadequate means to transiently express such proteins in HTM cells. The purpose of the current study was to test the hypothesis that cell cycle synchronization increases transfection efficiency of HTM cells.

Methods: After 24 hours of serum starvation, fetal bovine serum (10%) was reintroduced to synchronize cell cycle of HTM cultures. Cells were labeled with bromodeoxyuridine (BrdU) and 4,6–diamidino–2–phenylindole (DAPI) to determine the proportion of cells in S and M phases at specified times after synchronization. Cell cultures were incubated with 10mM BrdU and evaluated from 14 to 28 hours. For transfection experiments, cells were synchronized and transfected with either Cy3 conjugated IgG or Cy3 labeled cDNAs encoding green fluorescent protein (GFP) using Lipofectamine reagent. Forty–eight hours after transfection, cells were fixed, stained with DAPI, and visualized using ultraviolet and fluorescence microscopy.

Results: HTM cell cycle analysis showed that >95% of cells accumulated BrdU, an indicator of S phase during the 18–20 hour incubation interval. The highest percentage of mitotic figures appeared after 24–26 hours. Based on these results, HTM cells were transfected 18 hours after serum reintroduction. Both Cy3 IgG and Cy3/GFP DNA were taken up by >95% of cells. While efficiency of transfection increased about three–fold in synchronized cells compared to controls (n=3), it was still low (~3%).

Conclusions: The large discrepancy between the percentage of cells that take up DNA and express the encoded protein suggests that TM cells efficiently degrade exogenous DNA in the cytosol before it enters the nucleus.

Keywords: gene transfer/gene therapy • trabecular meshwork • microscopy: light/fluorescence/immunohistochemistry

 © 2004, The Association for Research in Vision and Ophthalmology, Inc., all rights reserved. For permission to reproduce any part of this abstract, contact the ARVO Office at arvo{at}arvo.org.





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