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

Laura B. Gottschalk, Briana Vecchio-Pagan, Neeraj Sharma, Sangwoo T. Han, Arianna Franca, Elizabeth S. Wohler, Denise Batista, Loyal Goff, Garry R Cutting

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
JournalJournal of Cystic Fibrosis
DOIs
StateAccepted/In press - Sep 9 2015

Fingerprint

Epithelial Cells
Cell Line
Genetic Recombination
Complementary DNA
RNA Sequence Analysis
Transcriptome
Confocal Microscopy
Western Blotting
Genes

Keywords

  • CFBE
  • CFTR
  • Cystic fibrosis model
  • Ivacaftor
  • RNA sequencing

ASJC Scopus subject areas

  • Pulmonary and Respiratory Medicine
  • Pediatrics, Perinatology, and Child Health

Cite this

Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants. / Gottschalk, Laura B.; Vecchio-Pagan, Briana; Sharma, Neeraj; Han, Sangwoo T.; Franca, Arianna; Wohler, Elizabeth S.; Batista, Denise; Goff, Loyal; Cutting, Garry R.

In: Journal of Cystic Fibrosis, 09.09.2015.

Research output: Contribution to journalArticle

@article{4304524437144c67911f85b33693e09e,
title = "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",
author = "Gottschalk, {Laura B.} and Briana Vecchio-Pagan and Neeraj Sharma and Han, {Sangwoo T.} and Arianna Franca and Wohler, {Elizabeth S.} and Denise Batista and Loyal Goff and Cutting, {Garry R}",
year = "2015",
month = "9",
day = "9",
doi = "10.1016/j.jcf.2015.11.010",
language = "English (US)",
journal = "Journal of Cystic Fibrosis",
issn = "1569-1993",
publisher = "Elsevier",

}

TY - JOUR

T1 - Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants

AU - Gottschalk, Laura B.

AU - Vecchio-Pagan, Briana

AU - Sharma, Neeraj

AU - Han, Sangwoo T.

AU - Franca, Arianna

AU - Wohler, Elizabeth S.

AU - Batista, Denise

AU - Goff, Loyal

AU - Cutting, Garry R

PY - 2015/9/9

Y1 - 2015/9/9

N2 - 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.

AB - 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.

KW - CFBE

KW - CFTR

KW - Cystic fibrosis model

KW - Ivacaftor

KW - RNA sequencing

UR - http://www.scopus.com/inward/record.url?scp=84949661376&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84949661376&partnerID=8YFLogxK

U2 - 10.1016/j.jcf.2015.11.010

DO - 10.1016/j.jcf.2015.11.010

M3 - Article

C2 - 26694805

AN - SCOPUS:84949661376

JO - Journal of Cystic Fibrosis

JF - Journal of Cystic Fibrosis

SN - 1569-1993

ER -