title: Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector.
abstract: OBJECTIVE
  To evaluate the transfer of heterologous genes carrying a Green Fluorescent Protein (GFP) reporter cassette to primary corneal epithelial cells ex vivo.
  
  
  METHODS
  Freshly enucleated rabbit corneoscleral tissue was used to obtain corneal epithelial cell suspension via enzymatic digestion. Cells were plated at a density of 5×10³ cells/cm² and allowed to grow for 5 days (to 70-80% confluency) prior to transduction. Gene transfer was monitored using fluorescence microscopy and fluorescence activated cell sorter (FACS). We evaluated the transduction efficiency (TE) over time and the dose-response effect of different lentiviral particles. One set of cells were dual sorted by fluorescence activated cell sorter for green fluorescent protein expression as well as Hoechst dye exclusion to evaluate the transduction of potentially corneal epithelial stem cells (side-population phenotypic cells).
  
  
  RESULTS
  Green fluorescent protein expressing lentiviral vectors were able to effectively transduce rabbit primary epithelial cells cultured ex vivo. Live cell imaging post-transduction demonstrated GFP-positive cells with normal epithelial cell morphology and growth. The transduction efficiency over time was higher at the 5(th) post-transduction day (14.1%) and tended to stabilize after the 8(th) day. The number of transduced cells was dose-dependent, and at the highest lentivirus concentrations approached 7%. When double sorted by fluorescence activated cell sorter to isolate both green fluorescent protein positive and side population cells, transduced side population cells were identified.
  
  
  CONCLUSIONS
  Lentiviral vectors can effectively transfer heterologous genes to primary corneal epithelial cells expanded ex vivo. Genes were stably expressed over time, transferred in a dose-dependence fashion, and could be transferred to mature corneal cells as well as presumable putative stem cells.
authors.affiliation: Department of Ophthalmology, Universidade Federal de São Paulo, SP, Brazil. laopadilha@hotmail.com
authors.forename: Lauro Augusto de
authors.initials: LA
authors.lastname: Oliveira
authors.forename: Charles
authors.initials: C
authors.lastname: Kim
authors.forename: Luciene Barbosa de
authors.initials: LB
authors.lastname: Sousa
authors.forename: Ivan R
authors.initials: IR
authors.lastname: Schwab
authors.forename: Mark I
authors.initials: MI
authors.lastname: Rosenblatt
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D019573:Epithelium, Corneal / Q000166:cytology / Q000378:metabolism*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000008:administration & dosage*; D049452:Green Fluorescent Proteins / Q000008:administration & dosage / Q000235:genetics*; D016086:Lentivirus / Q000235:genetics*; D023421:Models, Animal; D011817:Rabbits; D014161:Transduction, Genetic / Q000379:methods*
chemical_list: D049452:Green Fluorescent Proteins