HHS Public Access Author manuscript Author Manuscript
J Cyst Fibros. Author manuscript; available in PMC 2016 May 25. Published in final edited form as: J Cyst Fibros. 2016 May ; 15(3): 285–294. doi:10.1016/j.jcf.2015.11.010.
Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants Laura B. Gottschalka, Briana Vecchio-Pagana, Neeraj Sharmaa, Sangwoo T. Hana, Arianna Francaa, Elizabeth S. Wohlerb, Denise A.S. Batistab, Loyal A. Goffa,c, and Garry R. Cuttinga,d,* a
Author Manuscript
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States
b
Cytogenetics Laboratory, Kennedy Krieger Institute, Baltimore, MD, United States
c
Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, United States d
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, United States
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.
Author Manuscript
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 WTCFTR 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.
Author Manuscript
Keywords CFBE; CFTR; Cystic fibrosis model; Ivacaftor; RNA sequencing
*
Corresponding author at: Johns Hopkins Medical Institutions, 733 North Broadway, MRB 559, Baltimore, MD 21205, United States. Tel.: +1 410 614 0211; fax: +1 410 614 0213. ; Email: [email protected] (G.R. Cutting). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jcf.2015.11.010.
Gottschalk et al.
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Author Manuscript
1. Introduction Over 2000 different variants have been identified in CFTR [1]. A large majority of these variants are considered rare (~1850) and have yet to be evaluated for their effect on CFTR. An appropriate in vitro model is needed to study these rare variants. Primary cells and tissues provide the most relevant context to determine the consequences of diseaseassociated variants upon epithelial ion transport since mutant CFTR is expressed at endogenous levels in a native context [2]. Both primary airway epithelium and intestinal epithelium [3] have been used for functional studies of mutant CFTR. However, for most CFTR variants, primary tissues are not available due to limited access to the small number of patients carrying these variants.
Author Manuscript
In lieu of primary tissues, cell culture based systems can serve as reasonable proxies for primary cells. Fischer rat thyroid cells have been used extensively to evaluate mutant CFTR function and response to small molecule therapy [4–7]. However, the rat thyroid cells are not of human origin, so interactions with orthologous proteins such as chaperones, kinases, and ion channels may differ from what occurs in human airway epithelial cells. In addition, it has been shown that folding of CFTR is dependent on the cell type in which it is expressed [8]. Therefore, an epithelial cell line of human origin should more closely model the processing and function of CFTR in vivo.
Author Manuscript
CFBE41o− (CFBE) is an immortalized cell line created from the bronchial epithelium of a CF patient homozygous for F508del [9]. CFBE cells have been used to study CFTR function and response to small molecules due to their clinical relevance to CF and their ability to polarize and form tight junctions [10–12]. CFBE cell lines have been transduced to stably express CFTR but this process generates lines with variable numbers of integrated sequences expressing exogenous CFTR at high levels [13,14]. We report the creation of a CF8Flp, a CFBE cell line that contains a single recombination target site for the stable integration and expression of a single cDNA, mini-gene, or complete gene. RNA sequencing was performed on the CF8Flp cells and revealed both the transcriptional background and CFTR expression level to be comparable to native bronchial epithelial cells. Thus, the introduction of a single coding sequence into the CF8Flp line allows for regulated expression of CFTR mutants in a cellular context that approximates native airway cells.1
2. Methodology 2.1. Cell culture
Author Manuscript
Cells were grown in MEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Corning, Corning, NY, USA) and 1% penicillin/streptomycin (Quality Biological, Gaithersburg, MD, USA) in a humidified incubator at 37° in the presence of 5% CO2 on fibronectin/collagen coated plastic ware. For details, see Supplemental materials and methods.
1Flp recombination target (FRT), fragments per kilobase of exon per million fragments mapped (FPKM).
J Cyst Fibros. Author manuscript; available in PMC 2016 May 25.
Gottschalk et al.
Page 3
2.2. Transfection and selection of resistant clones
Author Manuscript
Parental cells were transfected with pFRT/lacZeo plasmid (Thermo Fisher) using Lipofectamine 2000 (Thermo Fisher). Two days after transfection, media was changed to contain 100 μg/mL Zeocin. Individual clones or pools of clones were isolated ~2 weeks post transfection. For details on selection of hygromycin resistant cells, see Supplemental materials and methods. 2.3. Southern blot 10 μg of genomic DNA were blotted and probed with P32 labeled PCR products of approximately 500 bp in length specific to the lacZeo region of pFRT/lacZeo plasmid. For details, see Supplemental materials and methods. 2.4. Inverse PCR
Author Manuscript
Genomic DNA was digested overnight, electrophoresed, and fragments ranging in size from ~3 kb–6 kb were gel extracted. Varying low amounts of DNA, ranging from 10 ng–200 ng, were ligated overnight at 4° using a rapid ligation kit (Roche). Ligated DNA served as a template for PCR using primers Span1R and Span4F. For details, see Supplemental materials and methods. 2.5. FISH
Author Manuscript
Probes were labeled using the Nick Translation Kit (Abbot Molecular/Vysis, Des Plaines, IL, USA). Cells were fixed, washed, and dried on slides. 10 μL of chromosome 8 painting probe (Cytocell, Oxford Gene Technology, Tarrytown, NY, USA) were mixed with 1–2 μL of labeled probe and hybridized for 2 min at 72 °C in LSI/WCP hybridization buffer (Abbott Molecular/Vysis) followed by overnight at 37 °C. The following day, slides were washed and mounted with 10 μL of DAPI with antifade (Cytocell). For details, see Supplemental materials and methods. 2.6. Western blot Whole cell lysates were blotted using mouse monoclonal antibody “596” (UNC antibody distribution program sponsored by Cystic Fibrosis Foundation Therapeutics) to detect CFTR, anti-GFP antibody (#A11122, Thermo Fisher) to detect GFP, and anti-GAPDH antibody (#G9545, Sigma) to detect GAPDH for the loading control. Membrane was incubated with HRP-conjugated secondary antibodies for 1 h at room temp followed by washing with PBST. Membranes were developed using ECL Prime (GE Healthcare, Pittsburgh, PA, USA). For details, see Supplemental materials and methods.
Author Manuscript
2.7. Laser scanning confocal microscopy Fluorescence was imaged using a Zeiss LSM510 confocal microscope at the Johns Hopkins Microscope Facility. For details, see Supplemental materials and methods. 2.8. Short circuit current Short circuit current (Isc) measurements were taken in an Easy-Mount chamber system (Physiologic Instruments, San Diego, CA). Isc was measured with a VCCMC6 multichannel J Cyst Fibros. Author manuscript; available in PMC 2016 May 25.
Gottschalk et al.
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Author Manuscript
voltage–current clamp amplifier (Physiologic Instruments) in the voltage-clamp mode. A Cl− gradient was used to increase measured response. After stabilization, forskolin (10 μM) was added to the basolateral chamber followed by Ivacaftor (10 μM) and CFTR Inhibitor 172 (10 μM) to the apical chamber. For details, see Supplemental materials and methods. 2.9. RNA sequencing
Author Manuscript
See Fig. 3A for plating strategy. RNA-seq libraries were constructed using the TruSEQ RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) and pair-end sequenced using Illumina HiSeq 2000/2500 (Johns Hopkins Medical Institutions Deep Sequencing and Microarray Core Facility). Raw reads were mapped to the reference genome (hg19) using the Bowtie2 algorithm [15] and Tophat2 (v2.0.13) [16] from the Tuxedo software suite. CuffQuant and Cuffdiff (Cufflinks v2.2.1) [17] were then used to determine relative abundance of gene transcripts and differential expression values among samples. For details, see Supplemental materials and methods. RNA-seq analysis example commands and arguments can be found in the Supplemental worksheet.
3. Results 3.1. Integration of a single Flp-In target site into chromosome 8 of CFBE cells
Author Manuscript Author Manuscript
CFBE41o− (CFBE) is an immortalized cell line created from the bronchial epithelium of a CF patient homozygous for F508del that does not express CFTR (Supplemental Fig. 1) [10]. To allow for the targeted integration of heterologous sequences, we elected to incorporate the Flp recombination target (FRT) site into the genomic DNA of CFBE cells using the pFRT/lacZeo plasmid (Thermo Fisher Scientific). To disrupt episomal replication within the Adenovirus 12/SV40 immortalized CFBE cells, the SV40 promoter of the pFRT/lacZeo plasmid was mutated (Supplemental Fig. 2). Following selection for Zeocin resistant cells, Southern blot analysis of genomic DNA revealed one clone with a novel DNA fragment of approximately 4 kb hybridizing to a probe for the lacZ–Zeocin fusion gene (Fig. 1A; highlighted by the arrow). To determine the genomic location of the pFRT/lacZeo plasmid integration, an inverse PCR was performed (Fig. 1B). A DNA fragment of approximately 700 bp, consistent with the size difference between the novel DNA fragment seen on the Southern blot (estimated ~4 kb) and the distance between InvR and InvF (~3.3 kb) was amplified from circularized fragments (Supplemental Fig. 3A). Sequencing of the inverse PCR product revealed 37 bp corresponding to the pFRT/lacZeo plasmid immediately adjacent to 535 bp that mapped to a region on the long arm of chromosome 8 (Ch8:128,569,810) (Fig. 1C). The location of the integration was verified using additional primers designed to encompass the plasmid/genomic DNA junction (Supplemental Fig. 3B/C). The CFBE ‘ΔAGG clone’ with the FRT insertion on chromosome 8 was re-named CF8Flp. Sequencing of the integration region revealed that 4556 bp of the original 8401 bp of pFRT/ lacZeo plasmid DNA was integrated and included the FRT site, modified SV40 promoter, and a full length lacZeo gene for selection. Fourteen bp of the genomic DNA were absent due to the insertion of the plasmid between positions 128,569,810 (hg19) and 128,569,825 (data not shown). Inspection of the site occupied by the Flp-In plasmid in the UCSC genome
J Cyst Fibros. Author manuscript; available in PMC 2016 May 25.
Gottschalk et al.
Page 5
Author Manuscript
browser revealed the nearest annotated gene, CASC8, is located 114 kb upstream. The region has features of open chromatin as it is enriched for the H3K27Ac histone mark, is sensitive to DNAse digestion, and is
J Cyst Fibros. Author manuscript; available in PMC 2016 May 25. Published in final edited form as: J Cyst Fibros. 2016 May ; 15(3): 285–294. doi:10.1016/j.jcf.2015.11.010.
Creation and characterization of an airway epithelial cell line for stable expression of CFTR variants Laura B. Gottschalka, Briana Vecchio-Pagana, Neeraj Sharmaa, Sangwoo T. Hana, Arianna Francaa, Elizabeth S. Wohlerb, Denise A.S. Batistab, Loyal A. Goffa,c, and Garry R. Cuttinga,d,* a
Author Manuscript
McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States
b
Cytogenetics Laboratory, Kennedy Krieger Institute, Baltimore, MD, United States
c
Solomon H. Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, United States d
Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, United States
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.
Author Manuscript
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 WTCFTR 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.
Author Manuscript
Keywords CFBE; CFTR; Cystic fibrosis model; Ivacaftor; RNA sequencing
*
Corresponding author at: Johns Hopkins Medical Institutions, 733 North Broadway, MRB 559, Baltimore, MD 21205, United States. Tel.: +1 410 614 0211; fax: +1 410 614 0213. ; Email: [email protected] (G.R. Cutting). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jcf.2015.11.010.
Gottschalk et al.
Page 2
Author Manuscript
1. Introduction Over 2000 different variants have been identified in CFTR [1]. A large majority of these variants are considered rare (~1850) and have yet to be evaluated for their effect on CFTR. An appropriate in vitro model is needed to study these rare variants. Primary cells and tissues provide the most relevant context to determine the consequences of diseaseassociated variants upon epithelial ion transport since mutant CFTR is expressed at endogenous levels in a native context [2]. Both primary airway epithelium and intestinal epithelium [3] have been used for functional studies of mutant CFTR. However, for most CFTR variants, primary tissues are not available due to limited access to the small number of patients carrying these variants.
Author Manuscript
In lieu of primary tissues, cell culture based systems can serve as reasonable proxies for primary cells. Fischer rat thyroid cells have been used extensively to evaluate mutant CFTR function and response to small molecule therapy [4–7]. However, the rat thyroid cells are not of human origin, so interactions with orthologous proteins such as chaperones, kinases, and ion channels may differ from what occurs in human airway epithelial cells. In addition, it has been shown that folding of CFTR is dependent on the cell type in which it is expressed [8]. Therefore, an epithelial cell line of human origin should more closely model the processing and function of CFTR in vivo.
Author Manuscript
CFBE41o− (CFBE) is an immortalized cell line created from the bronchial epithelium of a CF patient homozygous for F508del [9]. CFBE cells have been used to study CFTR function and response to small molecules due to their clinical relevance to CF and their ability to polarize and form tight junctions [10–12]. CFBE cell lines have been transduced to stably express CFTR but this process generates lines with variable numbers of integrated sequences expressing exogenous CFTR at high levels [13,14]. We report the creation of a CF8Flp, a CFBE cell line that contains a single recombination target site for the stable integration and expression of a single cDNA, mini-gene, or complete gene. RNA sequencing was performed on the CF8Flp cells and revealed both the transcriptional background and CFTR expression level to be comparable to native bronchial epithelial cells. Thus, the introduction of a single coding sequence into the CF8Flp line allows for regulated expression of CFTR mutants in a cellular context that approximates native airway cells.1
2. Methodology 2.1. Cell culture
Author Manuscript
Cells were grown in MEM (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Corning, Corning, NY, USA) and 1% penicillin/streptomycin (Quality Biological, Gaithersburg, MD, USA) in a humidified incubator at 37° in the presence of 5% CO2 on fibronectin/collagen coated plastic ware. For details, see Supplemental materials and methods.
1Flp recombination target (FRT), fragments per kilobase of exon per million fragments mapped (FPKM).
J Cyst Fibros. Author manuscript; available in PMC 2016 May 25.
Gottschalk et al.
Page 3
2.2. Transfection and selection of resistant clones
Author Manuscript
Parental cells were transfected with pFRT/lacZeo plasmid (Thermo Fisher) using Lipofectamine 2000 (Thermo Fisher). Two days after transfection, media was changed to contain 100 μg/mL Zeocin. Individual clones or pools of clones were isolated ~2 weeks post transfection. For details on selection of hygromycin resistant cells, see Supplemental materials and methods. 2.3. Southern blot 10 μg of genomic DNA were blotted and probed with P32 labeled PCR products of approximately 500 bp in length specific to the lacZeo region of pFRT/lacZeo plasmid. For details, see Supplemental materials and methods. 2.4. Inverse PCR
Author Manuscript
Genomic DNA was digested overnight, electrophoresed, and fragments ranging in size from ~3 kb–6 kb were gel extracted. Varying low amounts of DNA, ranging from 10 ng–200 ng, were ligated overnight at 4° using a rapid ligation kit (Roche). Ligated DNA served as a template for PCR using primers Span1R and Span4F. For details, see Supplemental materials and methods. 2.5. FISH
Author Manuscript
Probes were labeled using the Nick Translation Kit (Abbot Molecular/Vysis, Des Plaines, IL, USA). Cells were fixed, washed, and dried on slides. 10 μL of chromosome 8 painting probe (Cytocell, Oxford Gene Technology, Tarrytown, NY, USA) were mixed with 1–2 μL of labeled probe and hybridized for 2 min at 72 °C in LSI/WCP hybridization buffer (Abbott Molecular/Vysis) followed by overnight at 37 °C. The following day, slides were washed and mounted with 10 μL of DAPI with antifade (Cytocell). For details, see Supplemental materials and methods. 2.6. Western blot Whole cell lysates were blotted using mouse monoclonal antibody “596” (UNC antibody distribution program sponsored by Cystic Fibrosis Foundation Therapeutics) to detect CFTR, anti-GFP antibody (#A11122, Thermo Fisher) to detect GFP, and anti-GAPDH antibody (#G9545, Sigma) to detect GAPDH for the loading control. Membrane was incubated with HRP-conjugated secondary antibodies for 1 h at room temp followed by washing with PBST. Membranes were developed using ECL Prime (GE Healthcare, Pittsburgh, PA, USA). For details, see Supplemental materials and methods.
Author Manuscript
2.7. Laser scanning confocal microscopy Fluorescence was imaged using a Zeiss LSM510 confocal microscope at the Johns Hopkins Microscope Facility. For details, see Supplemental materials and methods. 2.8. Short circuit current Short circuit current (Isc) measurements were taken in an Easy-Mount chamber system (Physiologic Instruments, San Diego, CA). Isc was measured with a VCCMC6 multichannel J Cyst Fibros. Author manuscript; available in PMC 2016 May 25.
Gottschalk et al.
Page 4
Author Manuscript
voltage–current clamp amplifier (Physiologic Instruments) in the voltage-clamp mode. A Cl− gradient was used to increase measured response. After stabilization, forskolin (10 μM) was added to the basolateral chamber followed by Ivacaftor (10 μM) and CFTR Inhibitor 172 (10 μM) to the apical chamber. For details, see Supplemental materials and methods. 2.9. RNA sequencing
Author Manuscript
See Fig. 3A for plating strategy. RNA-seq libraries were constructed using the TruSEQ RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) and pair-end sequenced using Illumina HiSeq 2000/2500 (Johns Hopkins Medical Institutions Deep Sequencing and Microarray Core Facility). Raw reads were mapped to the reference genome (hg19) using the Bowtie2 algorithm [15] and Tophat2 (v2.0.13) [16] from the Tuxedo software suite. CuffQuant and Cuffdiff (Cufflinks v2.2.1) [17] were then used to determine relative abundance of gene transcripts and differential expression values among samples. For details, see Supplemental materials and methods. RNA-seq analysis example commands and arguments can be found in the Supplemental worksheet.
3. Results 3.1. Integration of a single Flp-In target site into chromosome 8 of CFBE cells
Author Manuscript Author Manuscript
CFBE41o− (CFBE) is an immortalized cell line created from the bronchial epithelium of a CF patient homozygous for F508del that does not express CFTR (Supplemental Fig. 1) [10]. To allow for the targeted integration of heterologous sequences, we elected to incorporate the Flp recombination target (FRT) site into the genomic DNA of CFBE cells using the pFRT/lacZeo plasmid (Thermo Fisher Scientific). To disrupt episomal replication within the Adenovirus 12/SV40 immortalized CFBE cells, the SV40 promoter of the pFRT/lacZeo plasmid was mutated (Supplemental Fig. 2). Following selection for Zeocin resistant cells, Southern blot analysis of genomic DNA revealed one clone with a novel DNA fragment of approximately 4 kb hybridizing to a probe for the lacZ–Zeocin fusion gene (Fig. 1A; highlighted by the arrow). To determine the genomic location of the pFRT/lacZeo plasmid integration, an inverse PCR was performed (Fig. 1B). A DNA fragment of approximately 700 bp, consistent with the size difference between the novel DNA fragment seen on the Southern blot (estimated ~4 kb) and the distance between InvR and InvF (~3.3 kb) was amplified from circularized fragments (Supplemental Fig. 3A). Sequencing of the inverse PCR product revealed 37 bp corresponding to the pFRT/lacZeo plasmid immediately adjacent to 535 bp that mapped to a region on the long arm of chromosome 8 (Ch8:128,569,810) (Fig. 1C). The location of the integration was verified using additional primers designed to encompass the plasmid/genomic DNA junction (Supplemental Fig. 3B/C). The CFBE ‘ΔAGG clone’ with the FRT insertion on chromosome 8 was re-named CF8Flp. Sequencing of the integration region revealed that 4556 bp of the original 8401 bp of pFRT/ lacZeo plasmid DNA was integrated and included the FRT site, modified SV40 promoter, and a full length lacZeo gene for selection. Fourteen bp of the genomic DNA were absent due to the insertion of the plasmid between positions 128,569,810 (hg19) and 128,569,825 (data not shown). Inspection of the site occupied by the Flp-In plasmid in the UCSC genome
J Cyst Fibros. Author manuscript; available in PMC 2016 May 25.
Gottschalk et al.
Page 5
Author Manuscript
browser revealed the nearest annotated gene, CASC8, is located 114 kb upstream. The region has features of open chromatin as it is enriched for the H3K27Ac histone mark, is sensitive to DNAse digestion, and is
