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BRAIN Publication-derived

Brain Models for Disease Modelling with Gene Editing

Full SOP with Academic or a plan

Source Blair et al. · University of Michigan, Ann Arbor, MI, USA · 10.1038/s41591-018-0139-y

Authors: Blair JD, Hockemeyer D, Bhatt SM, Bhatt AH Duration: 150 days 10 phases Cell source: Patient-Derived iPSC (TSC2+/-) / CRISPR TSC1/TSC2 Knockout

Abstract

Blair et al. modeled Tuberous Sclerosis Complex using cerebral organoids from TSC2-heterozygous patient iPSCs and CRISPR-engineered biallelic knockouts, recapitulating cortical tuber-like lesions with mTOR hyperactivation and balloon-like cells. Rapamycin treatment rescued the phenotype, validating the model for mTOR-targeted therapy screening.

Cell source
Patient-Derived iPSC (TSC2+/-) / CRISPR TSC1/TSC2 Knockout
Application
Tuberous Sclerosis Modeling

Protocol overview

36 steps across 10 phases

Phase 1 · hESC culture and maintenance Ongoing
  1. a Maintain hESCs on MEF feeder layer
  2. b Passage hESCs every 7 days
Phase 2 · CRISPR/Cas9 genome editing for TSC1 and TSC2 Days 1-30
  1. a Prepare CRISPR/Cas9 constructs
  2. b Select GFP-positive cells by FACS
  3. c Clone and genotype single colonies
  4. d Generate conditional TSC2 allele (for second-hit modeling)
  5. e Remove puromycin cassette via Flp recombinase
  6. f Generate tdTomato Cre-reporter allele
Phase 3 · Two-dimensional neural induction and NPC expansion Days 1-35
  1. a Initiate neural induction
  2. b Transition induction media composition
  3. c Dissociate and expand NPCs
Phase 4 · Neuronal differentiation from NPCs (2D) Days 1-100
  1. a Initiate neuronal differentiation at passage 8
  2. b Plate neurons for analysis
  3. c Mature neurons until harvest
Phase 5 · Three-dimensional cortical spheroid differentiation Days 0-150
  1. a Generate cortical spheroids from hESCs
  2. b Days 1-5: Initial spheroid formation
  3. c Days 6-25: Neural induction phase
  4. d Days 25-43: Neurogenic phase with neurotrophic factors
  5. e Days 43-150: Maturation phase
Phase 6 · Lentiviral infection for Cre-mediated second-hit modeling Days 12-110 (spheroid time)
  1. a Add Cre-GFP or GFP lentivirus at day 12
  2. b Visualize Cre expression via tdTomato reporter
Phase 7 · Rapamycin treatment for mTOR inhibition studies Indicated timepoints during spheroid development
  1. a Prepare rapamycin stock
  2. b Add rapamycin to spheroids at final concentration
  3. c Test treatment windows
Phase 8 · Immunohistochemistry and analysis of cortical spheroids At harvest timepoints
  1. a Fix and cryosection spheroids
  2. b Immunostain sections
  3. c Image and quantify cell phenotypes
Phase 9 · Western blotting for protein analysis At harvest timepoints
  1. a Harvest cells/spheroids
  2. b Quantify total protein
  3. c Transfer and block membranes
  4. d Incubate with primary antibodies
  5. e Incubate with secondary antibodies and detect
  6. f Strip and re-blot
Phase 10 · Quantitative RT-PCR for gene expression At harvest timepoints
  1. a Extract RNA from spheroids
  2. b Reverse transcription
  3. c Perform real-time qPCR

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Attribution

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

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