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INTESTINAL Publication-derived

Traditional Serrated Adenoma Colonic Organoids

Full SOP with Academic or a plan

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

Authors: Kenta Kawasaki, Masayuki Fujii, Shinya Sugimoto, Keiko Ishikawa, Mami Matano, Yuki Ohta, Kohta Toshimitsu, Sirirat Takahashi, Naoki Hosoe, Shigeki Sekine, Takanori Kanai, Toshiro Sato Duration: 60 days 9 phases Cell source: 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

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

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Attribution

This SOP was written by Organthis based on the published method in Kawasaki et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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