Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector
Autor(a) principal: | |
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Data de Publicação: | 2010 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Arquivos brasileiros de oftalmologia (Online) |
Texto Completo: | http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0004-27492010000500012 |
Resumo: | PURPOSE: 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 5th post-transduction day (14.1%) and tended to stabilize after the 8th 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. |
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Arquivos brasileiros de oftalmologia (Online) |
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Gene transfer to primary corneal epithelial cells with an integrating lentiviral vectorGene therapyCorneal diseasesLentivirus/geneticsTransgenes/geneticsStem cellsEpithelium corneal/physiologyGene expressionPURPOSE: 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 5th post-transduction day (14.1%) and tended to stabilize after the 8th 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.Conselho Brasileiro de Oftalmologia2010-10-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://old.scielo.br/scielo.php?script=sci_arttext&pid=S0004-27492010000500012Arquivos Brasileiros de Oftalmologia v.73 n.5 2010reponame:Arquivos brasileiros de oftalmologia (Online)instname:Conselho Brasileiro de Oftalmologia (CBO)instacron:CBO10.1590/S0004-27492010000500012info:eu-repo/semantics/openAccessOliveira,Lauro Augusto deKim,CharlesSousa,Luciene Barbosa deSchwab,Ivan R.Rosenblatt,Mark I.eng2011-01-04T00:00:00Zoai:scielo:S0004-27492010000500012Revistahttp://aboonline.org.br/https://old.scielo.br/oai/scielo-oai.phpaboonline@cbo.com.br||abo@cbo.com.br1678-29250004-2749opendoar:2011-01-04T00:00Arquivos brasileiros de oftalmologia (Online) - Conselho Brasileiro de Oftalmologia (CBO)false |
dc.title.none.fl_str_mv |
Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector |
title |
Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector |
spellingShingle |
Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector Oliveira,Lauro Augusto de Gene therapy Corneal diseases Lentivirus/genetics Transgenes/genetics Stem cells Epithelium corneal/physiology Gene expression |
title_short |
Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector |
title_full |
Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector |
title_fullStr |
Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector |
title_full_unstemmed |
Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector |
title_sort |
Gene transfer to primary corneal epithelial cells with an integrating lentiviral vector |
author |
Oliveira,Lauro Augusto de |
author_facet |
Oliveira,Lauro Augusto de Kim,Charles Sousa,Luciene Barbosa de Schwab,Ivan R. Rosenblatt,Mark I. |
author_role |
author |
author2 |
Kim,Charles Sousa,Luciene Barbosa de Schwab,Ivan R. Rosenblatt,Mark I. |
author2_role |
author author author author |
dc.contributor.author.fl_str_mv |
Oliveira,Lauro Augusto de Kim,Charles Sousa,Luciene Barbosa de Schwab,Ivan R. Rosenblatt,Mark I. |
dc.subject.por.fl_str_mv |
Gene therapy Corneal diseases Lentivirus/genetics Transgenes/genetics Stem cells Epithelium corneal/physiology Gene expression |
topic |
Gene therapy Corneal diseases Lentivirus/genetics Transgenes/genetics Stem cells Epithelium corneal/physiology Gene expression |
description |
PURPOSE: 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 5th post-transduction day (14.1%) and tended to stabilize after the 8th 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. |
publishDate |
2010 |
dc.date.none.fl_str_mv |
2010-10-01 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0004-27492010000500012 |
url |
http://old.scielo.br/scielo.php?script=sci_arttext&pid=S0004-27492010000500012 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
10.1590/S0004-27492010000500012 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
text/html |
dc.publisher.none.fl_str_mv |
Conselho Brasileiro de Oftalmologia |
publisher.none.fl_str_mv |
Conselho Brasileiro de Oftalmologia |
dc.source.none.fl_str_mv |
Arquivos Brasileiros de Oftalmologia v.73 n.5 2010 reponame:Arquivos brasileiros de oftalmologia (Online) instname:Conselho Brasileiro de Oftalmologia (CBO) instacron:CBO |
instname_str |
Conselho Brasileiro de Oftalmologia (CBO) |
instacron_str |
CBO |
institution |
CBO |
reponame_str |
Arquivos brasileiros de oftalmologia (Online) |
collection |
Arquivos brasileiros de oftalmologia (Online) |
repository.name.fl_str_mv |
Arquivos brasileiros de oftalmologia (Online) - Conselho Brasileiro de Oftalmologia (CBO) |
repository.mail.fl_str_mv |
aboonline@cbo.com.br||abo@cbo.com.br |
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1754209026475819008 |