Home › Biochemistry › Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5
Biochemistry JoVE (Open Access) Citable · DOI

Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5

DOI: 10.3791/53594-v
What you'll learn
  • ✓ Transfect Drosophila S2 cells with humanized kinesin-5 constructs
  • ✓ Apply RNA interference to achieve target gene knockdown
  • ✓ Perform live cell imaging and error correction assays
  • ✓ Evaluate kinesin-5 inhibitor sensitivity in engineered cell lines
Protocol

This protocol describes how to generate a Drosophila S2 cell line that is sensitive to small molecule inhibitors of kinesin-5. The use of these cells in a cell-based error correction assay is also outlined.

Difficulty
advanced
Total time
~3–5 days (transfection, selection, and assay execution)
Model organism
Drosophila melanogaster S2 cells
Biosafety
BSL-1

Steps

1
Transfect S2 cells with kinesin-5 constructs

Introduce humanized kinesin-5 expression plasmids into Drosophila S2 cells using standard transfection methods. Allow cells to recover and select stable transfectants.

▶ 01:01
2
Perform RNA interference knockdown

Apply RNAi targeting endogenous Drosophila kinesin-5 to reduce background expression and increase sensitivity to pharmacological inhibitors in the engineered cell line.

▶ 03:29
3
Conduct live cell imaging experiments

Acquire time-lapse microscopy data of transfected and RNAi-treated S2 cells to visualize kinesin-5 localization and mitotic dynamics.

▶ 04:32
4
Execute error correction assay

Treat humanized S2 cells with kinesin-5 inhibitors and quantify mitotic errors (lagging chromosomes, aneuploidy) as readout of on-target pharmacological effect.

▶ 06:03
5
Analyze inhibitor response and data

Evaluate dose-dependent kinesin-5 inhibition phenotypes in the engineered cell line and compare to wild-type S2 cells to validate humanized model sensitivity.

▶ 08:59
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