Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants

Abstract

Background: Analysis of the functional consequences and treatment response of rare CFTR variants is challenging due to the limited availability of primary airways cells.

Methods: A Flp recombination target (FRT) site for stable expression of CFTR was incorporated into an immortalized CF bronchial epithelial cell line (CFBE41o-). CFTR cDNA was integrated into the FRT site. Expression was evaluated by western blotting and confocal microscopy and function measured by short circuit current. RNA sequencing was used to compare the transcriptional profile of the resulting CF8Flp cell line to primary cells and tissues.

Results: Functional CFTR was expressed from integrated cDNA at the FRT site of the CF8Flp cell line at levels comparable to that seen in native airway cells. CF8Flp cells expressing WT-CFTR have a stable transcriptome comparable to that of primary cultured airway epithelial cells, including genes that play key roles in CFTR pathways.

Conclusion: CF8Flp cells provide a viable substitute for primary CF airway cells for the analysis of CFTR variants in a native context.

Keywords: CFBE; CFTR; Cystic fibrosis model; Ivacaftor; RNA sequencing.

Fig. 1. pFRT/lacZeo/ΔAGG is stably integrated onto chromosome 8

(A) Southern blot of hygromycin resistant clone using lacZeo specific probe. Arrow indicates band of genomic integration for clone. (B) Diagram of inverse PCR strategy to determine the genomic location of the integrated pFRT/lacZeo. Gray rectangle represents the pFRT/lacZeo plasmid integrated into the genomic DNA depicted by black lines. (C) Chromatogram of the inverse PCR product Sanger sequenced using the InvR primer. The resulting sequence contained a portion of pFRT/lacZeo along with 535 bp of genomic DNA from chromosome 8. (D) Fluorescence in situ hybridization of CF8Flp cells in metaphase. Chromosome 8 was painted and appears green. A probe specific for pFRT/lacZeo is shown in orange. The inset shows a 3× magnification of the chromosome containing the positive signal for the pFRT/lacZeo probe.

Fig. 2. The FRT site of CF8Flp cells is targetable and expresses functional CFTR

(A) PCR of genomic DNA from CF8Flp cells and CF8Flp cells containing GFP-CFTR to confirm integration. Primers to confirm integration were used for lanes one and two while lanes three through seven used primers spanning the length of CFTR cDNA. (B) FISH of CF8Flp cells containing GFP-CFTR using a probe specific to pFRT/lacZeo (C) Western blot of 40 μg of whole cell lysate probed with antibody raised against GFP. CFBE cells transiently transfected with GFP-CFTR construct served as a positive control. (D) XY image of scanning disc laser confocal microscopy. CFTR is in red, GFP in green, and blue indicates DAPI stained nuclei. (E) XZ images of the cells show apical localization of the CFTR. Antibody specific to ZO-1 (red) hybridizes to the tight junctions of the polarized cells. (F) Representative tracing of electrophysiologic assessment of CF8Flp cells expressing WT-CFTR after sequential addition of 10 μM forskolin and 10 μM CFTR inhibitor 172 (G) Representative tracing from cells expressing G551D-CFTR after sequential addition of 10 μM forskolin, 10 μM Ivacaftor and 10 μM CFTR inhibitor 172 (H) Average change in current after addition of forskolin or Ivacaftor plus forskolin (Mean ± SEM, n = 6 independent tracings).

Fig. 3. The transcriptome of CF8Flp cells is stable between independently derived pools

(A) Diagram of timing for cell plating and harvesting for RNA-seq. Transepithelial electrical resistance (TEER) to monitor tight junction formation was measured every day until a resistance of ≥200 Ω/cm² was reached for three consecutive days, at which point cell lysate was collected and placed at −80 °C. RNA was harvested from all frozen lysates simultaneously. B) Heat map of 13 housekeeping genes based on FPKM levels. Each row represents a gene while each column represents a cellular pool. C) Manhattan plots of CF8Flp parental cells compared to CF8Flp cells expressing either WT, F508del, or G551D CFTR. Red points indicate ≥3 SD from the mean of the statistic. CFTR and MUC4 are labeled for each plot. Generated by CuffDuff software [17].

Fig. 4. The CF8Flp transcriptome resembles that of primary bronchial epithelial cells

(A) Dendrogram derived from Jensen–Shannon distances calculated by using the top 50 genes with shared gene ontologies to CFTR. Data were obtained from the Sequence Read Archive and processed via the same RNA-seq pipeline as CF8Flp samples prior to normalization. (B) CFTR FPKM values from RNA-seq data (mean with 95% CI) (C) Manhattan plot of CF8Flp-WT-CFTR compared to FACS sorted lung epithelial cells. Red points indicate ≥3 SD from the mean of the statistic.