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INTESTINAL Publication-derived

Loss of Syntaxin 3 Causes Variant Microvillus Inclusion Disease: Patient-Derived Organoid and Caco-2 Cell Culture Protocol

Source Wigerninck et al., 2014 · University Medical Center Utrecht, Department of Pediatric Gastroenterology, Wilhelmina Children's Hospital · 10.1053/j.gastro.2014.04.002

👤 Caroline L. Wigerninck, Andreas R. Janecke, Kerstin Schneeberger, Georg F. Vogel, Johanna C. Escher, Thomas Müller, Sabine Middendorp ⏱ 230 days 📋 9 phases 🧫 Patient-Derived iPSC (Microvillus Inclusion Disease)

Abstract

This protocol describes the establishment and culture of human intestinal organoids derived from patient duodenal biopsy tissue, as well as lentiviral-mediated expression of wild-type and truncated syntaxin 3 (STX3) in Caco-2 intestinal epithelial cells. The methods enable recapitulation of variant microvillus inclusion disease (MVID) phenotypes in vitro to validate loss-of-function mechanisms of STX3 mutations.

Cell source
Patient-Derived iPSC (Microvillus Inclusion Disease)
Application
Disease modeling and cellular mechanism investigation

Protocol overview

48 steps across 9 phases

Patient Material and Genetic Characterization Day 1-14
  1. 1 Obtain Duodenal Biopsy Specimens
  2. 2 Perform Whole-Exome Sequencing on Patient 1
  3. 3 Perform Whole-Exome Sequencing on Patient 2
  4. 4 Comparative Variant Analysis
  5. 5 Confirm STX3 Mutations by Sanger Sequencing
  6. 6 Screen Additional MYO5B-Negative Samples
Organoid Culture Establishment Day 15-70
  1. 7 Isolate Crypts from Duodenal Biopsy Specimens
  2. 8 Culture Organoids in Expansion Medium (Weeks 1-5 to 10)
  3. 9 Induce Differentiation
Plasmid Construction and Lentiviral Preparation Day 71-100
  1. 10 Amplify STX3 cDNA
  2. 11 Ligate STX3 into pENTR4-mCitrine Vector
  3. 12 Clone Fusion Constructs into Lentiviral Expression Vector
  4. 13 Transfect HEK293LTV Cells for Lentiviral Production
  5. 14 Harvest Lentiviral Supernatant
Caco-2 Cell Culture and Lentiviral Transduction Day 101-120
  1. 15 Culture Caco-2 Cells Under Standard Conditions
  2. 16 Infect Caco-2 Cells with Lentiviral Particles
  3. 17 Select Transduced Caco-2 Cells
  4. 18 Culture Transduced Cells on Transwell Filters
Histological Analysis and Sample Preparation Day 121-140
  1. 19 Fix Biopsy Tissue for Histology
  2. 20 Prepare Paraffin Sections for Light Microscopy
  3. 21 Prepare Semithin Resin Sections for Toluidine Blue Staining
  4. 22 Prepare Tissue for Transmission Electron Microscopy
  5. 23 Prepare Organoids for Histological Analysis
Immunofluorescence and Immunohistochemistry Day 141-165
  1. 24 Prepare Duodenal Biopsy Sections for Immunofluorescence
  2. 25 Block Nonspecific Binding in Biopsy Sections
  3. 26 Incubate Biopsy Sections with Primary Antibodies
  4. 27 Apply Secondary Antibodies and Phalloidin in Biopsy Sections
  5. 28 Prepare Caco-2 Cells on Filters for Immunofluorescence
  6. 29 Incubate Caco-2 Cells with Primary Antibodies
  7. 30 Apply Secondary Antibodies and Phalloidin to Caco-2 Cells
  8. 31 Nuclear Counterstaining and Mounting
  9. 32 Image Sections using Epifluorescence Microscopy
  10. 33 Image Organoids using Confocal Microscopy
  11. 34 Image Caco-2 Cells using Confocal Microscopy
Electron Microscopy Analysis Day 166-190
  1. 35 Prepare Biopsy Tissue for Transmission Electron Microscopy
  2. 36 Prepare Organoids for Transmission Electron Microscopy
  3. 37 Prepare Caco-2 Cells for Transmission Electron Microscopy
  4. 38 Perform Scanning Electron Microscopy of Caco-2 Cells
Protein Analysis and Quantification Day 191-210
  1. 39 Lyse Caco-2 Cells for Western Blotting
  2. 40 Perform Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
  3. 41 Incubate Membranes with Primary Antibodies
  4. 42 Apply Secondary Antibodies and Develop Blots
  5. 43 Lyse Organoids for Western Blotting
  6. 44 Detect Organoid Proteins using Odyssey System
Quantitative Morphometry and Statistical Analysis Day 211-230
  1. 45 Quantify Microvillus Inclusions and Basolateral Microvilli
  2. 46 Count Ring- and Dot-like Actin and CD26/DPPIV Structures
  3. 47 Use Transmission Electron Microscopy to Distinguish Microvillus Inclusions from Basolateral Microvilli
  4. 48 Perform Statistical Analysis

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Wigerninck et al., 2014. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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