Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing
Autor(a) principal: | |
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Data de Publicação: | 2016 |
Outros Autores: | , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.1038/srep31180 http://hdl.handle.net/11449/173330 |
Resumo: | Interleukin-8 (IL-8) gene polymorphisms have been considered as susceptibility factors in periodontal disease. However, the functional roles of IL-8 gene haplotypes have not been investigated. Here, we demonstrate for the first time the use of the CRISPR/Cas9 system to engineer the IL-8 gene, and tested the functionality of different haplotypes. Two sgRNAs vectors targeting the IL-8 gene and the naked homologous repair DNA carrying different haplotypes were used to successfully generate HEK293T cells carrying the AT genotype at the first SNP-rs4073 (alias-251), TT genotype at the second SNP-rs2227307 (alias +396), TC or CC genotypes at the third SNP-rs2227306 (alias +781) at the IL-8 locus. When stimulated with Poly I:C, ATC/TTC haplotype, cells significantly up-regulated the IL-8 at both transcriptional and translational levels. To test whether ATC/TTC haplotype is functional, we used a trans-well assay to measure the transmigration of primary neutrophils incubated with supernatants from the Poly I:C stimulation experiment. ATC/TTC haplotype cells significantly increased transmigration of neutrophils confirming the functional role for this IL-8 haplotype. Taken together, our data provides evidence that carriage of the ATC/TTC haplotype in itself may increase the influx of neutrophils in inflammatory lesions and influence disease susceptibility. |
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Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editingInterleukin-8 (IL-8) gene polymorphisms have been considered as susceptibility factors in periodontal disease. However, the functional roles of IL-8 gene haplotypes have not been investigated. Here, we demonstrate for the first time the use of the CRISPR/Cas9 system to engineer the IL-8 gene, and tested the functionality of different haplotypes. Two sgRNAs vectors targeting the IL-8 gene and the naked homologous repair DNA carrying different haplotypes were used to successfully generate HEK293T cells carrying the AT genotype at the first SNP-rs4073 (alias-251), TT genotype at the second SNP-rs2227307 (alias +396), TC or CC genotypes at the third SNP-rs2227306 (alias +781) at the IL-8 locus. When stimulated with Poly I:C, ATC/TTC haplotype, cells significantly up-regulated the IL-8 at both transcriptional and translational levels. To test whether ATC/TTC haplotype is functional, we used a trans-well assay to measure the transmigration of primary neutrophils incubated with supernatants from the Poly I:C stimulation experiment. ATC/TTC haplotype cells significantly increased transmigration of neutrophils confirming the functional role for this IL-8 haplotype. Taken together, our data provides evidence that carriage of the ATC/TTC haplotype in itself may increase the influx of neutrophils in inflammatory lesions and influence disease susceptibility.National Institute of Dental and Craniofacial ResearchDepartment of Periodontics School of Dental Medicine University of PennsylvaniaDepartment of Oral Diagnosis and Surgery School of Dentistry at Araraquara UNESP-Univ EstadualDepartment of Genetics Perelman School of Medicine University of PennsylvaniaDepartment of Morphology School of Dentistry at Araraquara UNESP-Univ EstadualDepartment of Pathology School of Dental Medicine University of PennsylvaniaDepartment of Oral Diagnosis and Surgery School of Dentistry at Araraquara UNESP-Univ EstadualDepartment of Morphology School of Dentistry at Araraquara UNESP-Univ EstadualUniversity of PennsylvaniaUniversidade Estadual Paulista (Unesp)Benakanakere, Manjunatha R.Finoti, Livia S. [UNESP]Tanaka, UraraGrant, Gregory R.Scarel-Caminaga, Raquel M. [UNESP]Kinane, Denis F.2018-12-11T17:04:42Z2018-12-11T17:04:42Z2016-08-08info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://dx.doi.org/10.1038/srep31180Scientific Reports, v. 6.2045-2322http://hdl.handle.net/11449/17333010.1038/srep311802-s2.0-849812142702-s2.0-84981214270.pdfScopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengScientific Reports1,533info:eu-repo/semantics/openAccess2023-10-03T06:06:15Zoai:repositorio.unesp.br:11449/173330Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462023-10-03T06:06:15Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing |
title |
Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing |
spellingShingle |
Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing Benakanakere, Manjunatha R. |
title_short |
Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing |
title_full |
Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing |
title_fullStr |
Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing |
title_full_unstemmed |
Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing |
title_sort |
Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing |
author |
Benakanakere, Manjunatha R. |
author_facet |
Benakanakere, Manjunatha R. Finoti, Livia S. [UNESP] Tanaka, Urara Grant, Gregory R. Scarel-Caminaga, Raquel M. [UNESP] Kinane, Denis F. |
author_role |
author |
author2 |
Finoti, Livia S. [UNESP] Tanaka, Urara Grant, Gregory R. Scarel-Caminaga, Raquel M. [UNESP] Kinane, Denis F. |
author2_role |
author author author author author |
dc.contributor.none.fl_str_mv |
University of Pennsylvania Universidade Estadual Paulista (Unesp) |
dc.contributor.author.fl_str_mv |
Benakanakere, Manjunatha R. Finoti, Livia S. [UNESP] Tanaka, Urara Grant, Gregory R. Scarel-Caminaga, Raquel M. [UNESP] Kinane, Denis F. |
description |
Interleukin-8 (IL-8) gene polymorphisms have been considered as susceptibility factors in periodontal disease. However, the functional roles of IL-8 gene haplotypes have not been investigated. Here, we demonstrate for the first time the use of the CRISPR/Cas9 system to engineer the IL-8 gene, and tested the functionality of different haplotypes. Two sgRNAs vectors targeting the IL-8 gene and the naked homologous repair DNA carrying different haplotypes were used to successfully generate HEK293T cells carrying the AT genotype at the first SNP-rs4073 (alias-251), TT genotype at the second SNP-rs2227307 (alias +396), TC or CC genotypes at the third SNP-rs2227306 (alias +781) at the IL-8 locus. When stimulated with Poly I:C, ATC/TTC haplotype, cells significantly up-regulated the IL-8 at both transcriptional and translational levels. To test whether ATC/TTC haplotype is functional, we used a trans-well assay to measure the transmigration of primary neutrophils incubated with supernatants from the Poly I:C stimulation experiment. ATC/TTC haplotype cells significantly increased transmigration of neutrophils confirming the functional role for this IL-8 haplotype. Taken together, our data provides evidence that carriage of the ATC/TTC haplotype in itself may increase the influx of neutrophils in inflammatory lesions and influence disease susceptibility. |
publishDate |
2016 |
dc.date.none.fl_str_mv |
2016-08-08 2018-12-11T17:04:42Z 2018-12-11T17:04:42Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.1038/srep31180 Scientific Reports, v. 6. 2045-2322 http://hdl.handle.net/11449/173330 10.1038/srep31180 2-s2.0-84981214270 2-s2.0-84981214270.pdf |
url |
http://dx.doi.org/10.1038/srep31180 http://hdl.handle.net/11449/173330 |
identifier_str_mv |
Scientific Reports, v. 6. 2045-2322 10.1038/srep31180 2-s2.0-84981214270 2-s2.0-84981214270.pdf |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Scientific Reports 1,533 |
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 |
Scopus reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
|
_version_ |
1803649304616763392 |