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BRAIN Publication-derived

Genetic Ablation of AXL in Human Neural Progenitor Cells and Cerebral Organoids for Zika Virus Infection Studies

Source Wells et al., 2016 · Harvard University, Harvard Stem Cell Institute; Novartis Institutes for BioMedical Research · 10.1016/j.stem.2016.11.011

👤 Michael F. Wells, Max R. Salick, Ole Wiskow, Daniel J. Ho, Kathleen A. Worringer, Robert J. Ihry, Sravya Kommineni, Bilada Bilican, Joseph R. Klim, Ellen J. Hill, Liam T. Kane, Chaoyang Ye, Ajamete Kaykas, Kevin Eggan ⏱ 549 days 📋 6 phases 🧫 Human iPSC

Abstract

This protocol describes the generation of AXL-knockout human induced pluripotent stem cells (iPSCs) via doxycycline-inducible CRISPR/Cas9, their differentiation into neural progenitor cells (NPCs) and cerebral organoids, and subsequent infection with Zika virus (ZIKV) to investigate the role of AXL as a viral attachment factor in neural tissue susceptibility to ZIKV-mediated infection and cell death.

Cell source
Human iPSC
Application
Disease modeling

Protocol overview

33 steps across 6 phases

Generation of Doxycycline-Inducible Cas9 iPSCs Days 1–14
  1. 1 Electroporate iCas9 donor plasmid with TALENs into WT-GM08402A-SeV-hiPSC cells
  2. 2 Select G418-resistant clones
  3. 3 Maintain dox-iCas9 iPSCs
AXL Knockout Generation via CRISPR/Cas9 Targeting Days 1–10 (post-doxycycline induction)
  1. 1 Design and synthesize guide RNAs targeting AXL exons
  2. 2 Induce Cas9 expression with doxycycline
  3. 3 Prepare cells for transfection
  4. 4 Transfect crRNAs and tracrRNAs using Lipofectamine RNAimax
  5. 5 Maintain Cas9 induction during editing
  6. 6 Transition to standard maintenance medium
  7. 7 Screen edited cells via T7 Endonuclease I assay
  8. 8 Perform Next-Generation Sequencing (NGS) for indel analysis
  9. 9 Single-cell subcloning of highly-mutated pools
Directed Neuronal Differentiation to Neural Progenitor Cells (Days 1–22) Days 1–22 post-differentiation
  1. 1 Initiate neural induction (Days 1–6)
  2. 2 Continue neural differentiation (Days 7–20)
  3. 3 Transition to NPC expansion medium (Days 21+)
  4. 4 Verify NPC identity and differentiation efficiency
  5. 5 Confirm AXL knockout via Western blotting
  6. 6 Immunofluorescence validation of NPC differentiation and AXL knockout
ZIKV Infection of 2D NPC Cultures (Days 21–45) Days 21–45 of differentiation, infection on Days 21–35 or 36–45
  1. 1 Prepare ZIKV stocks (ZIKV-UG and ZIKV-PR)
  2. 2 Infect NPCs at early stage (Days 21–35)
  3. 3 Assess cell viability at 72 hours post-infection (hpi)
  4. 4 Quantify viral RNA in conditioned media (36 and 72 hpi)
  5. 5 Measure ZIKV infectivity via immunofluorescence (48 hpi)
  6. 6 Assess infection in late-stage radial glia (Days 36–45)
Generation and Infection of Cerebral Organoids (Days 1–30+) Days 1–24 (organoid formation) + Days 24–30 (ZIKV infection)
  1. 1 Generate embryoid bodies (Days 1–3)
  2. 2 Initiate neural induction (Days 4–24)
  3. 3 Visualize organoid structure via light sheet microscopy (Day 24)
  4. 4 Infect organoids with ZIKV-PR (Day 24)
  5. 5 Cryosection infected organoids for immunohistochemistry (Days 24–32)
  6. 6 Quantify viral infection and apoptosis in organoids
  7. 7 Quantify organoid growth inhibition
Supplementary Studies: TYRO3 Expression Analysis and A549 siRNA Knockdown Throughout differentiation (TYRO3) and Days 1–2 (A549 siRNA)
  1. 1 Quantify TYRO3 expression during NPC differentiation
  2. 2 Validate AXL importance in non-neural cells via A549 siRNA knockdown

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Wells et al., 2016. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

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