Barriers to productive transfection of trabecular meshwork cells

Emely A. Hoffman, Shannon M. Conley, W. Daniel Stamer, Brian S Mckay

Research output: Contribution to journalArticle

7 Citations (Scopus)

Abstract

Purpose: A critical function of trabecular meshwork cells is to degrade cellular debris, including DNA. We hypothesize that low transfection efficiencies of primary human trabecular meshwork (HTM) cell cultures with plasmid DNA are a function of retained capacity to efficiently degrade exogenous DNA in vitro. Methods: To determine mechanisms responsible for low transfection efficiencies of cultured HTM cells, steps of DNA entry into cytoplasm and nucleus were characterized. Following synchronization with sequential serum starvation and serum reintroduction, the HTM cell cycle was characterized using 5-bromo-2-deoxyuridine incorporation into replicating DNA. HTM cells were transfected during S-phase with plasmid DNA encoding green fluorescence protein (GFP) or plasmid DNA conjugated with Cy3. In some experiments, cells were treated with a DNase I inhibitor, 100 nM aurintricarboxylic acid. Uptake of plasmid DNA was measured by intracellular fluorescence of Cy3 and productive transfection efficiency was measured by intracellular fluorescence of GFP. Results: HTM cells enter S-phase between 18 and 20 h after synchronization. Plasmid DNA reached the cytosolic compartment in 95% of transfected cells, regardless of synchronization. Synchronization dramatically increased productive transtection efficiency of in HTM cells, from 3.0 to 9.0%. DNase I inhibition increased productive transfection efficiency of HTM cells two fold. Conclusions: Cultured HTM cells have a lower transfection efficiency than other primary ocular cell cultures, likely due partially to cytoplasmic digestion of DNA. We suggest that the difficulties in transfecting cultured HTM cells may be related to the filter function of the cells in vivo where the cells must degrade exogenous DNA.

Original languageEnglish (US)
Pages (from-to)869-875
Number of pages7
JournalMolecular Vision
Volume11
StatePublished - Oct 26 2005

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Trabecular Meshwork
Transfection
DNA
Plasmids
Fluorescence
Deoxyribonuclease I
S Phase
Aurintricarboxylic Acid
Primary Cell Culture
Bromodeoxyuridine
Starvation
Serum
Digestion
Cell Cycle
Cytoplasm
Proteins

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Hoffman, E. A., Conley, S. M., Stamer, W. D., & Mckay, B. S. (2005). Barriers to productive transfection of trabecular meshwork cells. Molecular Vision, 11, 869-875.

Barriers to productive transfection of trabecular meshwork cells. / Hoffman, Emely A.; Conley, Shannon M.; Stamer, W. Daniel; Mckay, Brian S.

In: Molecular Vision, Vol. 11, 26.10.2005, p. 869-875.

Research output: Contribution to journalArticle

Hoffman, EA, Conley, SM, Stamer, WD & Mckay, BS 2005, 'Barriers to productive transfection of trabecular meshwork cells', Molecular Vision, vol. 11, pp. 869-875.
Hoffman EA, Conley SM, Stamer WD, Mckay BS. Barriers to productive transfection of trabecular meshwork cells. Molecular Vision. 2005 Oct 26;11:869-875.
Hoffman, Emely A. ; Conley, Shannon M. ; Stamer, W. Daniel ; Mckay, Brian S. / Barriers to productive transfection of trabecular meshwork cells. In: Molecular Vision. 2005 ; Vol. 11. pp. 869-875.
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