Transient high-level expression of B-galactosidase after transfection of fibroplasts from GM1 gangliosidosis patients with plasmid DNA
Autor(a) principal: | |
---|---|
Data de Publicação: | 2008 |
Outros Autores: | , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UFRGS |
Texto Completo: | http://hdl.handle.net/10183/21223 |
Resumo: | GM1 gangliosidosis is an autosomal recessive disorder caused by the deficiency of lysosomal acid hydrolase ß-galactosidase (ß-Gal). It is one of the most frequent lysosomal storage disorders in Brazil, with an estimated frequency of 1:17,000. The enzyme is secreted and can be captured by deficient cells and targeted to the lysosomes. There is no effective treatment for GM1 gangliosidosis. To determine the efficiency of an expression vector for correcting the genetic defect of GM1 gangliosidosis, we tested transfer of the ß-Gal gene (Glb1) to fibroblasts in culture using liposomes. ß-Gal cDNA was cloned into the expression vectors pSCTOP and pREP9. Transfection was performed using 4 μL lipofectamine 2000 and 1.5-2.0 μg DNA. Cells (2 x 105/well) were harvested 24 h, 48 h, and 7 days after transfection. Enzyme specific activity was measured in cell lysate and supernatant by fluorometric assay. Twenty-four hours after transfection, treated cells showed a higher enzyme specific activity (pREP9-ß- Gal: 621.5 ± 323.0, pSCTOP-ß-Gal: 714.5 ± 349.5, pREP9-ß-Gal + pSCTOP-ß-Gal: 1859.0 ± 182.4, and pREP9-ß-Gal + pTRACER: 979.5 ± 254.9 nmol·h-1·mg-1 protein) compared to untreated cells (18.0 ± 3.1 for cell and 32.2 ± 22.2 nmol·h-1·mg-1 protein for supernatant). However, cells maintained in culture for 7 days showed values similar to those of untreated patients. In the present study, we were able to transfect primary patients’ skin fibroblasts in culture using a non-viral vector which overexpresses the ß-Gal gene for 24 h. This is the first attempt to correct fibroblasts from patients with GM1 gangliosidosis by gene therapy using a non-viral vector. |
id |
UFRGS-2_2f2462d8f36899356ca39b6579c35991 |
---|---|
oai_identifier_str |
oai:www.lume.ufrgs.br:10183/21223 |
network_acronym_str |
UFRGS-2 |
network_name_str |
Repositório Institucional da UFRGS |
repository_id_str |
|
spelling |
Balestrin, Raquel CristinaBaldo, GuilhermeVieira, Matheus BarbosaSano, RenataCoelho, Janice CarneiroGiugliani, RobertoMatte, Ursula da Silveira2010-04-24T04:15:50Z20080100-879Xhttp://hdl.handle.net/10183/21223000654639GM1 gangliosidosis is an autosomal recessive disorder caused by the deficiency of lysosomal acid hydrolase ß-galactosidase (ß-Gal). It is one of the most frequent lysosomal storage disorders in Brazil, with an estimated frequency of 1:17,000. The enzyme is secreted and can be captured by deficient cells and targeted to the lysosomes. There is no effective treatment for GM1 gangliosidosis. To determine the efficiency of an expression vector for correcting the genetic defect of GM1 gangliosidosis, we tested transfer of the ß-Gal gene (Glb1) to fibroblasts in culture using liposomes. ß-Gal cDNA was cloned into the expression vectors pSCTOP and pREP9. Transfection was performed using 4 μL lipofectamine 2000 and 1.5-2.0 μg DNA. Cells (2 x 105/well) were harvested 24 h, 48 h, and 7 days after transfection. Enzyme specific activity was measured in cell lysate and supernatant by fluorometric assay. Twenty-four hours after transfection, treated cells showed a higher enzyme specific activity (pREP9-ß- Gal: 621.5 ± 323.0, pSCTOP-ß-Gal: 714.5 ± 349.5, pREP9-ß-Gal + pSCTOP-ß-Gal: 1859.0 ± 182.4, and pREP9-ß-Gal + pTRACER: 979.5 ± 254.9 nmol·h-1·mg-1 protein) compared to untreated cells (18.0 ± 3.1 for cell and 32.2 ± 22.2 nmol·h-1·mg-1 protein for supernatant). However, cells maintained in culture for 7 days showed values similar to those of untreated patients. In the present study, we were able to transfect primary patients’ skin fibroblasts in culture using a non-viral vector which overexpresses the ß-Gal gene for 24 h. This is the first attempt to correct fibroblasts from patients with GM1 gangliosidosis by gene therapy using a non-viral vector.application/pdfengBrazilian Journal of medical and biological research. Vol. 41, n. 4 (2008), p. 283-288Gangliosidose GM1GM1 gangliosidosisß-galactosidase deficiencyGene therapyLysosomal storage disorderLipofectamineTransient high-level expression of B-galactosidase after transfection of fibroplasts from GM1 gangliosidosis patients with plasmid DNAinfo:eu-repo/semantics/articleinfo:eu-repo/semantics/otherinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFRGSinstname:Universidade Federal do Rio Grande do Sul (UFRGS)instacron:UFRGSORIGINAL000654639.pdf000654639.pdfTexto completo (inglês)application/pdf480399http://www.lume.ufrgs.br/bitstream/10183/21223/1/000654639.pdfff109d1690188f5074bdb78ecc4e8c51MD51TEXT000654639.pdf.txt000654639.pdf.txtExtracted Texttext/plain26961http://www.lume.ufrgs.br/bitstream/10183/21223/2/000654639.pdf.txt869c786c576b442533a6c0fdb28fd28bMD52THUMBNAIL000654639.pdf.jpg000654639.pdf.jpgGenerated Thumbnailimage/jpeg2023http://www.lume.ufrgs.br/bitstream/10183/21223/3/000654639.pdf.jpgb3705c5280826efd7286069bc48f89b2MD5310183/212232018-10-08 08:12:52.591oai:www.lume.ufrgs.br:10183/21223Repositório de PublicaçõesPUBhttps://lume.ufrgs.br/oai/requestopendoar:2018-10-08T11:12:52Repositório Institucional da UFRGS - Universidade Federal do Rio Grande do Sul (UFRGS)false |
dc.title.pt_BR.fl_str_mv |
Transient high-level expression of B-galactosidase after transfection of fibroplasts from GM1 gangliosidosis patients with plasmid DNA |
title |
Transient high-level expression of B-galactosidase after transfection of fibroplasts from GM1 gangliosidosis patients with plasmid DNA |
spellingShingle |
Transient high-level expression of B-galactosidase after transfection of fibroplasts from GM1 gangliosidosis patients with plasmid DNA Balestrin, Raquel Cristina Gangliosidose GM1 GM1 gangliosidosis ß-galactosidase deficiency Gene therapy Lysosomal storage disorder Lipofectamine |
title_short |
Transient high-level expression of B-galactosidase after transfection of fibroplasts from GM1 gangliosidosis patients with plasmid DNA |
title_full |
Transient high-level expression of B-galactosidase after transfection of fibroplasts from GM1 gangliosidosis patients with plasmid DNA |
title_fullStr |
Transient high-level expression of B-galactosidase after transfection of fibroplasts from GM1 gangliosidosis patients with plasmid DNA |
title_full_unstemmed |
Transient high-level expression of B-galactosidase after transfection of fibroplasts from GM1 gangliosidosis patients with plasmid DNA |
title_sort |
Transient high-level expression of B-galactosidase after transfection of fibroplasts from GM1 gangliosidosis patients with plasmid DNA |
author |
Balestrin, Raquel Cristina |
author_facet |
Balestrin, Raquel Cristina Baldo, Guilherme Vieira, Matheus Barbosa Sano, Renata Coelho, Janice Carneiro Giugliani, Roberto Matte, Ursula da Silveira |
author_role |
author |
author2 |
Baldo, Guilherme Vieira, Matheus Barbosa Sano, Renata Coelho, Janice Carneiro Giugliani, Roberto Matte, Ursula da Silveira |
author2_role |
author author author author author author |
dc.contributor.author.fl_str_mv |
Balestrin, Raquel Cristina Baldo, Guilherme Vieira, Matheus Barbosa Sano, Renata Coelho, Janice Carneiro Giugliani, Roberto Matte, Ursula da Silveira |
dc.subject.por.fl_str_mv |
Gangliosidose GM1 |
topic |
Gangliosidose GM1 GM1 gangliosidosis ß-galactosidase deficiency Gene therapy Lysosomal storage disorder Lipofectamine |
dc.subject.eng.fl_str_mv |
GM1 gangliosidosis ß-galactosidase deficiency Gene therapy Lysosomal storage disorder Lipofectamine |
description |
GM1 gangliosidosis is an autosomal recessive disorder caused by the deficiency of lysosomal acid hydrolase ß-galactosidase (ß-Gal). It is one of the most frequent lysosomal storage disorders in Brazil, with an estimated frequency of 1:17,000. The enzyme is secreted and can be captured by deficient cells and targeted to the lysosomes. There is no effective treatment for GM1 gangliosidosis. To determine the efficiency of an expression vector for correcting the genetic defect of GM1 gangliosidosis, we tested transfer of the ß-Gal gene (Glb1) to fibroblasts in culture using liposomes. ß-Gal cDNA was cloned into the expression vectors pSCTOP and pREP9. Transfection was performed using 4 μL lipofectamine 2000 and 1.5-2.0 μg DNA. Cells (2 x 105/well) were harvested 24 h, 48 h, and 7 days after transfection. Enzyme specific activity was measured in cell lysate and supernatant by fluorometric assay. Twenty-four hours after transfection, treated cells showed a higher enzyme specific activity (pREP9-ß- Gal: 621.5 ± 323.0, pSCTOP-ß-Gal: 714.5 ± 349.5, pREP9-ß-Gal + pSCTOP-ß-Gal: 1859.0 ± 182.4, and pREP9-ß-Gal + pTRACER: 979.5 ± 254.9 nmol·h-1·mg-1 protein) compared to untreated cells (18.0 ± 3.1 for cell and 32.2 ± 22.2 nmol·h-1·mg-1 protein for supernatant). However, cells maintained in culture for 7 days showed values similar to those of untreated patients. In the present study, we were able to transfect primary patients’ skin fibroblasts in culture using a non-viral vector which overexpresses the ß-Gal gene for 24 h. This is the first attempt to correct fibroblasts from patients with GM1 gangliosidosis by gene therapy using a non-viral vector. |
publishDate |
2008 |
dc.date.issued.fl_str_mv |
2008 |
dc.date.accessioned.fl_str_mv |
2010-04-24T04:15:50Z |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/other |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/10183/21223 |
dc.identifier.issn.pt_BR.fl_str_mv |
0100-879X |
dc.identifier.nrb.pt_BR.fl_str_mv |
000654639 |
identifier_str_mv |
0100-879X 000654639 |
url |
http://hdl.handle.net/10183/21223 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.ispartof.pt_BR.fl_str_mv |
Brazilian Journal of medical and biological research. Vol. 41, n. 4 (2008), p. 283-288 |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.source.none.fl_str_mv |
reponame:Repositório Institucional da UFRGS instname:Universidade Federal do Rio Grande do Sul (UFRGS) instacron:UFRGS |
instname_str |
Universidade Federal do Rio Grande do Sul (UFRGS) |
instacron_str |
UFRGS |
institution |
UFRGS |
reponame_str |
Repositório Institucional da UFRGS |
collection |
Repositório Institucional da UFRGS |
bitstream.url.fl_str_mv |
http://www.lume.ufrgs.br/bitstream/10183/21223/1/000654639.pdf http://www.lume.ufrgs.br/bitstream/10183/21223/2/000654639.pdf.txt http://www.lume.ufrgs.br/bitstream/10183/21223/3/000654639.pdf.jpg |
bitstream.checksum.fl_str_mv |
ff109d1690188f5074bdb78ecc4e8c51 869c786c576b442533a6c0fdb28fd28b b3705c5280826efd7286069bc48f89b2 |
bitstream.checksumAlgorithm.fl_str_mv |
MD5 MD5 MD5 |
repository.name.fl_str_mv |
Repositório Institucional da UFRGS - Universidade Federal do Rio Grande do Sul (UFRGS) |
repository.mail.fl_str_mv |
|
_version_ |
1801224711110131712 |