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Sci Rep. 2016 Aug 8;6:31180. doi: 10.1038/srep31180.

Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing.

Author information

1
Department of Periodontics, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.
2
Department of Oral Diagnosis and Surgery, School of Dentistry at Araraquara, UNESP- Univ Estadual Paulista, SP, Brazil.
3
Department of Genetics, Perelman School of Medicine, University of Pennsylvania, PA, USA.
4
Department of Morphology, School of Dentistry at Araraquara, UNESP- Univ Estadual Paulista, SP, Brazil.
5
Department of Pathology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.

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.

PMID:
27499075
PMCID:
PMC4976357
DOI:
10.1038/srep31180
[Indexed for MEDLINE]
Free PMC Article

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