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LUNG Publication-derived

Functional Gene Correction for Cystic Fibrosis in Lung Epithelial Cells Generated from Patient iPSCs

Source Firth et al., 2015 · The Salk Institute of Biological Studies · 10.1016/j.celrep.2015.07.062

👤 Amy L. Firth, Tushar Menon, Gregory S. Parker, Susan J. Qualls, Benjamin M. Lewis, Eugene Ke, Carl T. Dargitz, Rebecca Wright, Ajai Khanna, Fred H. Gage, Inder M. Verma ⏱ 16 days 📋 7 phases 🧫 Patient-Derived iPSC (Cystic Fibrosis - DF508 CFTR mutation)

Abstract

This protocol describes the generation of cystic fibrosis (CF) patient-derived induced pluripotent stem cells (iPSCs) carrying a homozygous DF508 CFTR mutation, precise CRISPR-mediated correction of the mutation at the endogenous locus using a footprint-free approach with piggyBac transposase-based selection marker excision, and differentiation to functional airway epithelial cells demonstrating recovered CFTR expression and chloride channel activity.

Cell source
Patient-Derived iPSC (Cystic Fibrosis - DF508 CFTR mutation)
Application
Disease modeling and functional gene correction

Protocol overview

52 steps across 7 phases

iPSC Derivation and Characterization Weeks 1-4
  1. 1 Obtain skin biopsy from CF patient
  2. 2 Isolate and expand fibroblasts
  3. 3 Reprogram fibroblasts to iPSCs using Sendai virus
  4. 4 Characterize and validate iPSCs
  5. 5 Verify DF508 CFTR mutation in CF iPSCs
CRISPR Vector Construction and Validation Weeks 2-4
  1. 1 Construct Cas9 expression plasmid
  2. 2 Construct gRNA cassette plasmid
  3. 3 Construct donor vector with selection cassette
  4. 4 Validate gRNA activity in HEK293T cells
  5. 5 Validate gRNA activity in CF iPSCs
CRISPR-Mediated Gene Targeting and Selection Weeks 5-7
  1. 1 Prepare CF iPSCs for nucleofection
  2. 2 Nucleofect CRISPR components into CF iPSCs
  3. 3 Recovery and expansion post-nucleofection
  4. 4 Passage nucleofected iPSCs to single-cell density
  5. 5 Selection with puromycin
  6. 6 Mechanical isolation of puromycin-resistant clones
  7. 7 Expansion of isolated clones
  8. 8 Genomic DNA isolation for screening
  9. 9 Integration-specific PCR screening
  10. 10 Sequencing confirmation of integration
piggyBac Transposase-Mediated Selection Cassette Excision Weeks 8-9
  1. 1 Prepare integration-positive iPSCs for piggyBac nucleofection
  2. 2 Nucleofect piggyBac transposase variants
  3. 3 Recovery post-nucleofection
  4. 4 Selection against unexcised clones using ganciclovir
  5. 5 Mechanical isolation of ganciclovir-resistant colonies
  6. 6 Expand excision candidate clones
  7. 7 Genomic DNA isolation from excision candidates
  8. 8 PCR and ClaI restriction digest screening
  9. 9 Sequencing confirmation of excision
  10. 10 Analysis of off-target CRISPR effects
  11. 11 Confirmation of no ectopic selection cassette integration
Differentiation to Respiratory Epithelial Cells Weeks 10-14
  1. 1 Prepare transwell insert culture system
  2. 2 Prepare single-cell suspension of iPSCs
  3. 3 Plate iPSCs on transwell inserts
  4. 4 Differentiation to definitive endoderm (DE) - Days 1–5
  5. 5 Verify DE differentiation by immunofluorescence
  6. 6 Differentiation to anterior foregut endoderm (AFE) and lung progenitors - Days 6–28
  7. 7 Establish air-liquid interface (ALI) culture - Day 28 onwards
  8. 8 Verify epithelial maturation by immunofluorescence - Day 42–48
  9. 9 Quantitative PCR analysis of differentiation progression
Functional Analysis of Corrected Lung Epithelial Cells Weeks 14–16
  1. 1 Isolation of EPCAM-positive epithelial cells
  2. 2 Whole-cell patch-clamp electrophysiology setup
  3. 3 Patch-clamp recording baseline assessment
  4. 4 CFTR activation with forskolin, genistein, and IBMX
  5. 5 CFTR inhibition with CFTRinh-172
  6. 6 Data analysis and reporting
Western Blot Analysis of CFTR Protein Expression and Glycosylation Weeks 14–16
  1. 1 Protein isolation from differentiated epithelial cells
  2. 2 SDS-PAGE separation
  3. 3 Protein transfer to nitrocellulose membrane
  4. 4 Blocking and antibody probing
  5. 5 Chemiluminescent detection
  6. 6 Data interpretation for CFTR glycosylation status

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Firth et al., 2015. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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