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BRAIN Publication-derived

Zika Virus Infection-Induced Microcephaly Brain Organoids

Full SOP with Academic or a plan

Source Liu et al., 2019 · Brain and Spinal Cord Innovative Research Center, Tongji Hospital, Tongji University School of Medicine, Shanghai · 10.1038/s41418-019-0324-7

Authors: Ling Liu, Zhenyu Chen, Xin Zhang, Shun Li, Yi Hui, Hexi Feng, Yanhua Du, Guohua Jin, Xiaohui Zhou, Xiaoqing Zhang Duration: 45 days 12 phases Cell source: Human ESC (H9)

Abstract

This protocol generates forebrain dorsal (FD), forebrain ventral (FV), and hindbrain & spinal cord (H&S) neural progenitor cell (NPC) organoids from human embryonic stem cells (hESCs) for studying Zika virus (ZIKV) infection-induced microcephaly. The protocol establishes regional brain organoids, infects them with ZIKV strain SZ01, and assesses viral pathogenesis through analysis of cell growth arrest, apoptosis, and IFN-independent antiviral gene activation.

Cell source
Human ESC (H9)
Application
Disease modeling

Protocol overview

43 steps across 12 phases

Phase 1 · hESC maintenance and preparation Passages 25-45 (ongoing)
  1. a Culture human ESCs on MEF feeders
Phase 2 · Generation of p65 or IRF3 knockout hESC lines (optional for KO studies) Days 0-17 post-electroporation
  1. a Pre-electroporation treatment with Rho kinase inhibitor
  2. b Prepare single-cell suspension
  3. c Electroporate with CRISPR/Cas9 components
  4. d Puromycin selection
  5. e Clone selection and genotyping
Phase 3 · Neural differentiation to regional NPCs Days 0-23
  1. a EB formation
  2. b Neural induction
  3. c Plating on laminin-coated surface
  4. d Regional patterning (FD identity - no morphogen)
  5. e Regional patterning (FV identity - Shh treatment)
  6. f Regional patterning (H&S identity - RA treatment)
  7. g Organoid maturation
Phase 4 · ZIKV infection of regional organoids Days 17-23
  1. a Virus preparation and titration
  2. b Infection of organoids
  3. c Mock infection control
Phase 5 · Analysis of viral infection and organoid growth Days 17-23 post-infection
  1. a Light microscopy imaging
  2. b Immunostaining for viral envelope protein
  3. c BrdU incorporation assay
  4. d Immunostaining for proliferation markers (Ki67, pH3)
Phase 6 · Assessment of apoptosis and cell death Days 17-23 post-infection
  1. a Annexin V-FITC/PI flow cytometry
  2. b Immunostaining for cleaved caspase-3
Phase 7 · Quantitative RT-PCR analysis of viral RNA and gene expression Days 17-23 post-infection
  1. a RNA extraction
  2. b cDNA synthesis
  3. c Quantitative RT-PCR
Phase 8 · Western blot analysis Days 17-23 post-infection
  1. a Protein extraction
  2. b SDS-PAGE and transfer
  3. c Detection and quantification
Phase 9 · IFNβ treatment studies Days 17-23
  1. a IFNβ or IFNγ treatment
  2. b Assess growth and ISG expression
Phase 10 · Analysis of p65 and IRF3 knockout organoids Days 17-23 post-infection
  1. a Differentiate p65 or IRF3 KO hESCs to regional organoids
  2. b ZIKV infection of KO and WT organoids
  3. c Assess ISG expression in KO organoids
  4. d Measure viral load in KO organoids
  5. e Assess protection from growth arrest in KO organoids
  6. f Assess apoptosis in KO organoids
Phase 11 · TLR3 pathway investigation Optional; days 17-23 post-infection
  1. a Assess basal TLR3 expression
  2. b TLR3 agonist treatment
  3. c Assess ZIKV pathology with TLR3 inhibition
Phase 12 · Transcriptome profiling (RNA-seq) Days 17-23 post-infection; analysis later
  1. a Sample collection and RNA extraction
  2. b RNA-seq library preparation
  3. c Sequencing
  4. d Bioinformatics analysis

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Attribution

This SOP was written by Organthis based on the published method in Liu et al., 2019. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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