Abstract
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.
| Original language | English |
|---|---|
| Article number | 31180 |
| Journal | Scientific reports |
| Volume | 6 |
| DOIs | |
| Publication status | Published - Aug 8 2016 |
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All Science Journal Classification (ASJC) codes
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Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing. / Benakanakere, Manjunatha R.; Finoti, Livia S.; Tanaka, Urara; Grant, Gregory R.; Scarel-Caminaga, Raquel M.; Kinane, Denis F.
In: Scientific reports, Vol. 6, 31180, 08.08.2016.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing
AU - Benakanakere, Manjunatha R.
AU - Finoti, Livia S.
AU - Tanaka, Urara
AU - Grant, Gregory R.
AU - Scarel-Caminaga, Raquel M.
AU - Kinane, Denis F.
PY - 2016/8/8
Y1 - 2016/8/8
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84981214270&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84981214270&partnerID=8YFLogxK
U2 - 10.1038/srep31180
DO - 10.1038/srep31180
M3 - Article
C2 - 27499075
AN - SCOPUS:84981214270
VL - 6
JO - Scientific Reports
JF - Scientific Reports
SN - 2045-2322
M1 - 31180
ER -