• Home
  • Search Results
  • CFTR inactivation by lentiviral vector-mediated RNA interference and CRISPR-Cas9 genome editing in human airway epithelial cells.

CFTR inactivation by lentiviral vector-mediated RNA interference and CRISPR-Cas9 genome editing in human airway epithelial cells.

Current gene therapy (2015-08-13)
Jessica Bellec, Marc Bacchetta, Davide Losa, Ignacio Anegon, Marc Chanson, Tuan Huy Nguyen
ABSTRACT

Polarized airway epithelial cell cultures modelling Cystic Fibrosis Transmembrane conductance Regulator (CFTR) defect are crucial for CF and biomedical research. RNA interference has proven its value to generate knockdown models for various pathologies. More recently, genome editing using CRISPR-Cas9 artificial endonuclease was a valuable addition to the toolbox of gene inactivation. Calu-3 cells and primary HAECs were transduced with HIV-1-derived lentiviral vectors (LVV) encoding small hairpin RNA (shRNA) sequence or CRISPR-Cas9 components targeting CFTR alongside GFP. After sorting of GFP-positive cells, CFTR expression was measured by RT-qPCR and Western blot in polarized or differentiated cells. CFTR channel function was assessed in Ussing chambers. Il-8 secretion, proliferation and cell migration were also studied in transduced cells. shRNA interference and CRISPRCas9 strategies efficiently decreased CFTR expression in Calu-3 cells. Strong CFTR knockdown was confirmed at the functional level in CRISPR-Cas9-modified cells. CFTR-specific shRNA sequences did not reduce gene expression in primary HAECs, whereas CRISPR-Cas9-mediated gene modification activity was correlated with a reduction of transepithelial secretion and response to a CFTR inhibitor. CFTR inactivation in the CRISPR-Cas9-modified Calu-3 cells did not affect migration and proliferation but slightly increased basal interleukin-8 secretion. We generated CFTR inactivated cell lines and demonstrated that CRISPR-Cas9 vectorised in a single LVV efficiently promotes CFTR inactivation in primary HAECs. These results provide a new protocol to engineer CF primary epithelia with their isogenic controls and pave the way for manipulation of CFTR expression in these cultures.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium chloride, BioXtra, ≥99.5% (AT)
Sigma-Aldrich
Sodium chloride, for molecular biology, DNase, RNase, and protease, none detected, ≥99% (titration)
Sigma-Aldrich
Potassium phosphate monobasic, powder, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99.0%
Sigma-Aldrich
Sodium chloride, BioReagent, suitable for cell culture, suitable for insect cell culture, suitable for plant cell culture, ≥99%
Sigma-Aldrich
Sodium chloride solution, 0.9% in water, BioXtra, suitable for cell culture
Sigma-Aldrich
Sodium chloride solution, 5 M in H2O, BioReagent, for molecular biology, suitable for cell culture
Sigma-Aldrich
Potassium phosphate monobasic, ReagentPlus®
Sigma-Aldrich
Sodium chloride solution, 5 M
SAFC
Sodium chloride solution, 5 M
Sigma-Aldrich
Potassium phosphate monobasic, for molecular biology, ≥98.0%
Sigma-Aldrich
Sodium chloride, meets analytical specification of Ph. Eur., BP, USP, 99.0-100.5%
Sigma-Aldrich
Sodium chloride, BioUltra, for molecular biology, ≥99.5% (AT)
Sigma-Aldrich
Sodium chloride solution, 0.85%
Sigma-Aldrich
Sodium chloride solution, BioUltra, for molecular biology, ~5 M in H2O
Sigma-Aldrich
Anti-Glyceraldehyde-3-Phosphate Dehydrogenase Antibody, clone 6C5, clone 6C5, Chemicon®, from mouse
Sigma-Aldrich
Sodium chloride, 99.999% trace metals basis
Sigma-Aldrich
Potassium phosphate monobasic, BioUltra, for molecular biology, anhydrous, ≥99.5% (T)
Sigma-Aldrich
Sodium chloride, BioPerformance Certified, ≥99% (titration), suitable for insect cell culture, suitable for plant cell culture
Sigma-Aldrich
Potassium phosphate monobasic, 99.99% trace metals basis
Sigma-Aldrich
Sodium chloride, tablet
Sigma-Aldrich
Sodium chloride, AnhydroBeads, −10 mesh, 99.999% trace metals basis
Sigma-Aldrich
Sodium chloride, random crystals, optical grade, 99.9% trace metals basis
Sigma-Aldrich
Sodium chloride, AnhydroBeads, −10 mesh, 99.99% trace metals basis
Sigma-Aldrich
Sodium chloride-35Cl, 99 atom % 35Cl