title: Barriers to productive transfection of trabecular meshwork cells.
abstract: OBJECTIVE
  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 transfection efficiency 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.
authors.affiliation: Department of Ophthalmology and Vision Science, The University of Arizona, Tucson, AZ 85711, USA.
authors.forename: Emely A
authors.initials: EA
authors.lastname: Hoffman
authors.forename: Shannon M
authors.initials: SM
authors.lastname: Conley
authors.forename: W Daniel
authors.initials: WD
authors.lastname: Stamer
authors.forename: Brian S
authors.initials: BS
authors.lastname: McKay
mesh_terms: D000328:Adult; D000368:Aged; D001312:Aurintricarboxylic Acid / Q000494:pharmacology; D001973:Bromodeoxyuridine / Q000378:metabolism; D002232:Carbocyanines / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology*; D002455:Cell Division; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D004261:DNA Replication; D003850:Deoxyribonuclease I / Q000037:antagonists & inhibitors / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007223:Infant; D010857:Pigment Epithelium of Eye / Q000187:drug effects / Q000201:enzymology; D010957:Plasmids / Q000235:genetics*; D014129:Trabecular Meshwork / Q000166:cytology* / Q000378:metabolism; D014162:Transfection
chemical_list: D002232:Carbocyanines; C089187:cyanine dye 3; D049452:Green Fluorescent Proteins; D001312:Aurintricarboxylic Acid; D004247:DNA; D003850:Deoxyribonuclease I; D001973:Bromodeoxyuridine