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INTESTINAL Publication-derived

Chromosome Engineering of Human Colon-Derived Organoids to Develop a Model of Traditional Serrated Adenoma

Source Kawasaki et al., 2020 · Keio University School of Medicine · 10.1053/j.gastro.2019.10.009

👤 Kenta Kawasaki, Masayuki Fujii, Shinya Sugimoto, Keiko Ishikawa, Mami Matano, Yuki Ohta, Kohta Toshimitsu, Sirirat Takahashi, Naoki Hosoe, Shigeki Sekine, Takanori Kanai, Toshiro Sato ⏱ 60 days 📋 9 phases 🧫 Human normal colon-derived organoid cells

Abstract

This protocol details the chromosome engineering of human colonic organoids using CRISPR-Cas9 to introduce R-spondin gene fusions, BRAF V600E mutations, and GREM1 overexpression, enabling the generation of human organoid-derived xenografts that phenocopy Traditional Serrated Adenoma histopathology. The engineered organoids can be orthotopically transplanted into NOG mice to form tumors with TSA-characteristic features.

Cell source
Human normal colon-derived organoid cells
Application
Disease modeling - Traditional Serrated Adenoma (TSA) genetic reconstruction

Protocol overview

51 steps across 9 phases

Organoid Culture and Maintenance Ongoing
  1. 1 Prepare basal culture medium
  2. 2 Supplement basal medium with growth factors and niche factors
  3. 3 Embed organoids in Matrigel
  4. 4 Perform niche-based selection (optional)
  5. 5 Culture organoids in refined IGF-1/FGF-2 medium (alternative condition)
CRISPR-Cas9 Plasmid Vector Construction 1–7
  1. 1 Design and synthesize sgRNA sequences
  2. 2 Clone sgRNA sequences into pX330 plasmid
  3. 3 Construct BRAF V600E donor vector
  4. 4 Construct GREM1 overexpression vector
CRISPR-Cas9–Mediated Gene Engineering 1–21
  1. 1 Prepare organoids for electroporation
  2. 2 Co-deliver CRISPR-Cas9 plasmids and donor/overexpression vectors by electroporation
  3. 3 Post-electroporation recovery at reduced temperature
  4. 4 Puromycin selection
  5. 5 Apply niche-based selection
  6. 6 Single organoid cloning
  7. 7 Genomic DNA extraction and genotyping
  8. 8 Knockin of LGR5 reporter (for LGR5-ablation experiments)
  9. 9 Cre-mediated excision of selection cassette
GREM1 Overexpression Engineering 1–21
  1. 1 Co-electroporate GREM1 overexpression PiggyBAC vector
  2. 2 Post-electroporation culture at 30°C
  3. 3 Puromycin selection
  4. 4 Noggin-withdrawal-based functional selection
  5. 5 Single organoid cloning and genomic validation
Orthotopic Xenotransplantation and Tumor Monitoring 1–60
  1. 1 Mouse preparation and anesthesia
  2. 2 Removal of luminal contents
  3. 3 Epithelial denudation with hot EDTA
  4. 4 Epithelial abrasion
  5. 5 Organoid transplantation
  6. 6 Post-transplantation retention
  7. 7 Endoscopic monitoring and tumor size assessment
  8. 8 Fluorescent endoscopic examination (optional)
  9. 9 Tissue collection at endpoint
LGR5 Ablation Experiment (In Vivo) 28–42
  1. 1 Tumor establishment and randomization
  2. 2 Dimerizer or vehicle administration
  3. 3 EdU labeling of proliferating cells
  4. 4 Tissue collection and fixation
Real-Time PCR and Gene Expression Analysis 1–3
  1. 1 RNA extraction
  2. 2 Reverse transcription
  3. 3 Real-time PCR setup
  4. 4 Calculate relative gene expression
Microarray Analysis 1–7
  1. 1 High-quality RNA extraction
  2. 2 Microarray hybridization
  3. 3 Data normalization and analysis
Immunohistochemistry and In Situ Hybridization 1–7
  1. 1 Tissue fixation and processing
  2. 2 Hematoxylin and eosin (H&E) staining
  3. 3 Immunohistochemistry with primary antibodies
  4. 4 In situ hybridization (ISH) with RNAscope
  5. 5 Nuclear counterstaining
  6. 6 EdU detection
  7. 7 Image acquisition
  8. 8 Image analysis and quantification

Full SOP

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Attribution

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

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