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Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Name: Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish
Uploaded: 2026-06-21
Duration: 8 min 1 s
Description: Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

DOI: 10.3791/2790-v

What you'll learn

✓ Perform DNA microinjection of zebrafish embryos to generate transgenic leukemia models
✓ Isolate and purify fluorescently-labeled leukemia cells from larval zebrafish
✓ Execute limiting dilution transplantation assays into adult zebrafish peritoneum
✓ Quantify tumor-propagating cell frequency through statistical analysis of transplant outcomes

Protocol

Limiting dilution cell transplantation assays are used to determine the frequency of tumor-propagating cells. This protocol describes a method for generating syngeneic zebrafish that develop fluorescently-labeled leukemia and details how to isolate and transplant these leukemia cells at limiting dilution into the peritoneal cavity of adult zebrafish.

Difficulty

advanced

Total time

~4-6 weeks (embryo injection to final analysis; transplantation readout ~2-4 weeks)

Model organism

Zebrafish (Danio rerio)

Biosafety

BSL-1

Steps

Microinject DNA into zebrafish embryos

Inject transgenic DNA constructs into one-cell-stage zebrafish embryos to establish syngeneic leukemia models. Monitor survival and integration of injected genetic material.

▶ 01:40

Screen larval zebrafish for primary T-ALL

Examine developing zebrafish larvae for fluorescent leukemia markers and phenotypic signs of T-cell acute lymphoblastic leukemia (T-ALL) to identify disease-positive individuals.

▶ 02:47

Isolate and purify fluorescent leukemia cells

Extract leukemia cells from diseased larvae and perform fluorescence-activated cell sorting (FACS) or density-based purification to obtain homogeneous populations of fluorescently-labeled tumor cells.

▶ 03:25

Transplant leukemia cells at limiting dilution

Inject serially diluted leukemia cell suspensions into the peritoneal cavity of syngeneic adult zebrafish recipients at defined cell doses to establish dose-response transplantation assays.

▶ 04:57

Analyze leukemia-initiating cell frequency

Monitor transplant recipients for leukemia development and use limiting dilution statistical models (e.g., Poisson regression) to calculate the frequency of tumor-propagating cells in the original population.

▶ 05:31

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