Home › Cell Biology › Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin
Cell Biology JoVE (Open Access) Citable · DOI

Live Imaging of Innate Immune and Preneoplastic Cell Interactions Using an Inducible Gal4/UAS Expression System in Larval Zebrafish Skin

DOI: 10.3791/52107-v
What you'll learn
  • ✓ Perform microinjection of plasmid DNA into zebrafish embryos using Gal4/UAS system
  • ✓ Induce conditional epithelial cell transformation using 4-hydroxytamoxifen treatment
  • ✓ Acquire live-imaging data of immune-preneoplastic cell interactions in larval skin
Protocol

Biopharma Insights Studying the earliest events of preneoplastic cell progression and innate immune cell interaction is pivotal to understand and treat cancer. Here we describe a method to conditionally induce epithelial cell transformations and the subsequent live imaging of innate immune cell interaction with HRASG12V expressing skin cells in zebrafish larvae.

Difficulty
advanced
Total time
~3-5 days (embryo injection to imaging; includes larval development and induction period)
Model organism
Zebrafish larva (Danio rerio)
Biosafety
BSL-1

Steps

1
Microinject plasmid DNA into zebrafish embryos

Prepare and inject Gal4/UAS expression plasmids into early-stage zebrafish embryos to establish transgenic reporter lines capable of conditional transformation.

▶ 01:46
2
Induce skin cell transformation with 4-hydroxytamoxifen

Treat larval zebrafish with 4-OHT to conditionally activate HRASG12V expression in epithelial cells, triggering preneoplastic cell phenotype.

▶ 04:16
3
Mount zebrafish larvae for live imaging

Prepare treated larvae on imaging slides or chambers and position under stereomicroscope for sustained live-cell observation.

▶ 05:34
4
Acquire live-imaging data of cell interactions

Capture time-lapse fluorescence and brightfield images documenting innate immune cell recruitment and behavior around transformed skin cells.

▶ 07:06
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