CRISPR-Cas9-mediated gene knockout in primary human airway epithelial cells reveals a proinflammatory role for MUC18.
Gene Ther
; 22(10): 822-9, 2015 Oct.
Article
en En
| MEDLINE
| ID: mdl-26043872
ABSTRACT
Targeted knockout of genes in primary human cells using CRISPR-Cas9-mediated genome-editing represents a powerful approach to study gene function and to discern molecular mechanisms underlying complex human diseases. We used lentiviral delivery of CRISPR-Cas9 machinery and conditional reprogramming culture methods to knockout the MUC18 gene in human primary nasal airway epithelial cells (AECs). Massively parallel sequencing technology was used to confirm that the genome of essentially all cells in the edited AEC populations contained coding region insertions and deletions (indels). Correspondingly, we found mRNA expression of MUC18 was greatly reduced and protein expression was absent. Characterization of MUC18 knockout cell populations stimulated with TLR2, 3 and 4 agonists revealed that IL-8 (a proinflammatory chemokine) responses of AECs were greatly reduced in the absence of functional MUC18 protein. Our results show the feasibility of CRISPR-Cas9-mediated gene knockouts in AEC culture (both submerged and polarized), and suggest a proinflammatory role for MUC18 in airway epithelial response to bacterial and viral stimuli.
Texto completo:
1
Base de datos:
MEDLINE
Asunto principal:
Lentivirus
/
Mucosa Respiratoria
/
Vectores Genéticos
Límite:
Humans
Idioma:
En
Revista:
Gene Ther
Asunto de la revista:
GENETICA MEDICA
/
TERAPEUTICA
Año:
2015
Tipo del documento:
Article
País de afiliación:
Estados Unidos