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INTESTINAL Publication-derived

iPSC-Derived Intestinal Organoids from Cystic Fibrosis Patients: TALEN-Mediated Repair of p.F508del CFTR Mutation and Functional Validation

Source Fleischer et al., 2020 · Consejo Superior de Investigaciones Científicas (CSIC/IMEDEA) · 10.1016/j.omtm.2020.04.005

👤 Aarne Fleischer, Sara Vallejo-Díez, José María Martín-Fernández, Almudena Sánchez-Gilabert, Mónica Castresana, Angel del Pozo, Amaia Esquisabel, Silvia Ávila, José Luis Castrillo, Eusebio Gaínza, José Luis Pedraz, Miguel Viñas, Daniel Bachiller ⏱ 95 days 📋 16 phases 🧫 Patient-Derived iPSC (Cystic Fibrosis p.F508del)

Abstract

This protocol describes the seamless TALEN-mediated homologous recombination correction of the p.F508del CFTR mutation in patient-derived cystic fibrosis iPSCs, followed by directed differentiation into intestinal organoids and functional assessment of restored CFTR activity via forskolin-induced swelling assays.

Cell source
Patient-Derived iPSC (Cystic Fibrosis p.F508del)
Application
Disease modeling and functional validation of gene-editing repair in CF organoids

Protocol overview

50 steps across 16 phases

Generation and Validation of CFTR-Specific TALENs Pre-experiment
  1. 1 Design and synthesis of TALEN expression vectors
  2. 2 Test TALEN cleavage activity in K562 and HeLa cells
Design and Preparation of Targeting Vector Pre-experiment
  1. 1 Design targeting vector with homology arms and selection cassette
  2. 2 Verify targeting vector design by in silico analysis
  3. 3 Assemble final targeting vector pMC3.1
iPSC Culture and Preparation for Gene Editing Days 1–3
  1. 1 Culture CF iPSCs (IMEDEAi001-F line) on feeder-free plates
  2. 2 Prepare iPSCs for nucleofection
TALEN-Mediated Homologous Recombination and Clone Selection Days 3–21
  1. 1 Nucleofect CF-iPSCs with TALEN and donor vector
  2. 2 Apply puromycin selection
  3. 3 Manually pick puromycin-resistant clones
  4. 4 Expand clones and prepare genomic DNA
PCR Screening of Recombinant Clones Days 19–24
  1. 1 First-round PCR screening: 5′ and 3′ recombination sites
  2. 2 Confirm 5′ integration by BglII restriction digestion
  3. 3 Screen for random integration events
piggyBac-Mediated Cassette Excision Days 24–42
  1. 1 Nucleofect positive clones with piggyBac transposase
  2. 2 Apply FIAU selection for transposase-expressing clones
  3. 3 Manually pick FIAU-resistant clones
  4. 4 Screen for cassette excision by allele-specific PCR
Sequence Verification and Clone Selection Days 42–50
  1. 1 Amplify complete CFTR target region for sequencing
  2. 2 Purify and clone PCR amplicons
  3. 3 Sequence CFTR alleles
Genomic Characterization of Corrected iPSCs Days 50–70
  1. 1 Perform G-banding karyotyping
  2. 2 Perform array comparative genomic hybridization (aCGH)
  3. 3 Predict and analyze off-target TALEN sites
  4. 4 Validate predicted off-target sites by Surveyor assay
Characterization of iPSC Stemness and Pluripotency Days 70–95
  1. 1 Immunofluorescence staining for stemness markers
  2. 2 Generate and characterize embryoid bodies (EBs)
  3. 3 Immunofluorescence staining for germ layer differentiation
Directed Differentiation toward Definitive Endoderm (DE) Days 0–3
  1. 1 Plate iPSCs and apply DMSO pretreatment
  2. 2 Initiate definitive endoderm differentiation (Day 1–2)
  3. 3 Verify DE formation by immunofluorescence (Day 3)
Directed Differentiation toward Hindgut Endoderm (HGE) Days 3–8
  1. 1 Initiate hindgut specification with WNT3A agonist (Day 3–8)
  2. 2 Verify hindgut specification by immunofluorescence (Day 8)
Maturation to Intestinal Organoids (HIOs) Days 8–35
  1. 1 Embed HGE spheroids in Matrigel and culture with growth factors (Day 8–35)
  2. 2 Verify intestinal organoid identity by immunofluorescence (Day 35)
Characterization of CFTR Expression Days 35–42
  1. 1 Prepare organoid lysates for Western blot
  2. 2 SDS-PAGE and Western blotting for CFTR
  3. 3 Include appropriate controls
Immunofluorescence Localization of CFTR Days 42–49
  1. 1 Fix organoids and prepare sections
  2. 2 Immunofluorescence for CFTR and apical/basolateral markers
  3. 3 Expected immunofluorescence results
Functional Assessment: Forskolin-Induced Swelling (FIS) Assay Days 49–56
  1. 1 Prepare organoids for FIS assay
  2. 2 Pre-treat organoids with CFTR inhibitor and corrector (16 hours before assay)
  3. 3 Label organoids with calcein green (30 minutes before imaging)
  4. 4 Initiate FIS assay with CFTR activation
  5. 5 Acquire time-lapse fluorescence images
  6. 6 Quantify organoid swelling
  7. 7 Test CFTR dependency with inhibitor washout
Pharmacological Modulation Testing Days 49–56
  1. 1 Test response of mutant organoids to VX-770/VX-809 combination
  2. 2 Test response of corrected organoids to VX-770/VX-809 combination

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Fleischer et al., 2020. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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