Xenograft in zebrafish

Materials and Reagents

1. Human Cancer Cells (MDA-MB-435, T47D, and TC-1)
2. CellTracker CM-DiI (Thermo Fisher Scientific, C7000, USA)
3. Agarose Low Melting-10G (Mentos, 9012-36-6, Korea)
4. The Tg(kdrl:EGFP) zebrafish embryos at 2dpf
5. Tricaine (Sigma, A5040, USA)
6. Phosphate-buffered saline (PBS)/ Dulbeccoʼs phosphate-buffered saline (DPBS)
7. Matrigel (Corning, 354234)
8. N-phenylthiourea (PTU, Sigma, P7629, USA)
9. E3 Solution and Methylene blue (Sigma-Aldrich)
10. Agarose gel mold trays

Equipment/Software

1. Microinjection system (World Precision Instruments, PV83 Pneumatic PicoPump, SYS-PV830, FL, USA)
2. Confocal microscope (Carl Zeiss, LSM700, Germany)
3. Zeiss ZEN imaging software (Germany)

Procedures

1. Preparing the animal (1 day)

The cep41 AUG MO-injected or mutant zebrafish embryos were dechorinated and immersed in PTU at 12 hour post fertilization (hpf)

2. Labeling cancer cells (2 day)

1) Prepare cancer cells cultured in two of T175 flasks

2) Wash the cells twice with DPBS 

3) Dilute CellTracker CM-DiI in dimethylsulfoxide (DMSO) to a concentration of 1mg/mL

4) Dilute the CM-DiI solution in DPBS to generate a working solution (ranging from 1~2 µg/mL)

5) Incubate the cells in CM-DiI working solution for 5 min at 37°C and then incubate them at 4°C for 15 min

6) Wash the incubated cells with DPBS and count the cells

   * The number of collected cells: MDA-MB-435 (3X10⁷), T47D (2X10⁷), TC-1 (2.5X10⁷) 

7) Resuspend the cells in 20 μl of Matrigel solution

   * One day before xenograft, the Matrigel was thawed overnight at 4°C 

3. Transplantation of cancer cells (2 day)

1) Anesthetize the 2 dpf cep41 AUG MO-injected or mutant zebrafish larvae using Tricaine

2) Transfer them onto agarose gel mold trays for microinjection 

3) Microinject 10 nl of labeled cancer cells near the perivitelline space of zebrafish larvae

  * The number of injected cells: MDA-MB-435 (1.5X10⁴), T47D (1X10⁴), TC-1 (1.25X10⁴) 

  * The zebrafish larvae injected with 10 nl of matrigel were applied as negativecontrol 

4. Tumor growth assessment (3 day)

1) The cells-grafted zebrafish larvae were grown for 24 h in a 28.5°C incubator 

2) Dissolve 1% low melting agarose in E3 solution

3) Place one drop of agarose solution containing single zebrafish larva on a cover slide glass 

4) Observation and photography using a confocal microscope

5) Analysis of additionally generated vessels using Zeiss ZEN imaging software

Recipes

1. Cell culture medium: Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin
2. E3 solution (60X stock solution) pH 7.2: For 2 liters, dissolve 34.8g NaCl, 1.6g KCL, 5.8g CaCl₂.2H₂O, and 9.78g MgCl₂.6H₂O in distilled water and then autoclave
3. E3 solution (1X working solution): Dilute 16.5 ml of 60X E3 stock solution in 1 L of distilled water and add 100 μl of 1% Methylene blue

How to cite 

Ki SM, Kim JH, Won SY, Oh SJ, Lee IY, Bae YK, Chung KW, Choi BO, Park B, Choi EJ, Lee JE. CEP41-mediated ciliary tubulin glutamylation drives angiogenesis through AURKA-dependent deciliation. EMBO Rep. 2020 Feb 5;21(2):e48290. doi: 10.15252/embr.201948290. Epub 2019 Dec 29. PMID: 31885126; PMCID: PMC7001496.