The overall goal of the following experiment is to screen for melanoma modifiers using a zebrafish autogenous tumor model. This is achieved by creating transgenic animals in which the melanocytes express a gene of interest and the fish are predisposed to melanoma. Next melanoma onset is scored to assess whether the gene of interest alters tumor initiation.
Then tumor invasion and cell specific assays are performed to determine any additional impact of the gene of interest on melanomas and pre-malignant melanocytes. Finally, melanoma cells are transplanted to measure how the gene of interest affects the frequency of melanoma initiating cells in established tumors. The results show how a candidate gene affects various properties of melanocytes and melanomas, including onset invasion and transplant ability.
The main advantage of this technique over existing methods like germline transgenesis for surveying candidate cancer genes is that time, effort, and expense are saved, and throughput is increased by assay. Various aspects of tumorgenesis in chime transgenic animals Demonstrating this procedure will be sherania agar, a graduate student in my laboratory To generate constructs for injecting. Begin by creating gateway middle entry clones by PCR, amplifying the full length open reading frame of genes of interest and recombining into P don R 2 21.
Then use multi-site gateway technology to recombine the melanocyte specific mifa promoter gene of interest and polyadenylation signal into the mini Cooper vector to place genes of interest under the control of the mifa promoter inject 25 picograms of each clone, along with 25 picograms of TOL two transposes mRNA into one cell transgenic triple homozygous zebrafish embryos. Mifa BRAF FP 53. Mutant animals are melanocyte deficient and prone to transformation and melanoma formation.
Along with the gene of interest. The mini Cooper vector contains a rescuing MVA mini gene. So successfully injected animals will develop rescued melanocytes, which express the gene of interest.
Incubate the injected embryos at 28.5 degrees Celsius at 24 hours post fertilization or HPF. Remove any dead embryos at 72 hours post fertilization using a dissecting microscope under incident light against a white background. Select transgenic animals with rescued melanocytes.
When the animals reach four days post fertilization, transfer them to three liter tanks in the nursery of the zebrafish facility. At two months of age, select the animals with at least one area of melanocyte rescue greater than four millimeters squared. Screen the selected animals weekly for the presence of tumors.
Isolate tumor bearing animals for study. Draw melanoma free survival curves with age in weeks on the absa and percent melanoma free survival on the ordinate. Compare animals with melanocytes that express a gene of interest to control animals that express EGFP and melanocytes.
Use a log rank test to determine whether the two curves are statistically different. To perform a tumor invasion assay. Begin by selecting isolated zebra fish that develop tumors dorsally in a region between the posterior boundary of the hind brain and anterior border of the dorsal fin.
Two weeks after melanoma onset, use trica to euthanize the fish after fixing and embedding the fish in paraffin. Make five micrometer transverse sections through the lesions.One. Every 50 micrometers cut through the entire lesion so that the point of deepest invasion can be identified.
Use hematin and eoin to stain the sections. Assess tumor invasion by measuring whether tumor cells remain outside the collagenous stratum compactive invade into the dorsal musculature or invade further into the spinal column. Determine the statistical difference between the two sets of samples to perform immuno staining.
Anesthetize the fish in 0.17 milligrams per milliliter of trica with incident light under a dissecting microscope. Use sharp fine tipped forceps to pluck dorsal scales. Taking care not to damage the melanocyte containing half of the scale.
Place the scales directly from the forceps into one milliliter of fixative and incubate at room temperature while turning for greater than or equal to two hours. Transfer the fish into fish water and monitor the fish to confirm successful recovery to bleach pigmented melanocytes. First wash fixed scales in PBST two times for five minutes at room temperature.
Next, add one milliliter of freshly made bleach solution and cover with parfum or cap locks to prevent gas from bursting tubes open. Continue bleaching until pigment is gone. Then wash in PBST four times per five minutes at room temperature.
Incubate for at least 30 minutes in block solution, then incubate with about 400 microliters of primary antibody and block solution overnight At room temperature the following day, wash the scales three times for 30 minutes in PBS at room temperature, incubate with secondary antibody for two hours to overnight at room temperature. After standing with dpi, apply a drop of vector shield to a glass slide and mount the scales so that the concave side of the scales faces down against the slide. Place a glass cover slip over the samples.
Use clear nail polish to seal the edges and observe the slides under a fluorescent microscope. After a radiating recipient, two to three month old Casper fish with 25 grays of gamma irradiation one day prior to transplantation. Allow the fish to recover in fish water.
Use trica to euthanize a tumor bearing fish, cut off the tumor and put it in a Petri dish with approximately five milliliters of filter sterilized 0.9 XPBS with 5%FBS with a razor dice the tumor while in solution working at room temperature and with a P 1000 pipette iterate to produce single cells. Bring up the volume to 25 milliliters. Filter the solution through a 40 micrometer mesh filter and spin in a tabletop centrifuge at 453 RCF for 10 minutes.
Re suspend the pellet in 0.9%PBS 5%FBS to the desired concentration. Using a hemo cytometer, calculate the exact cell number and dilute the cell suspension so that the final injection volume is approximately five microliter. For example, if 50, 000 cells are to be injected, the cell suspension should be diluted to a final concentration of 10, 000 cells per microliter.
Flick the tube containing the cell suspension every few minutes to prevent clumping of the cells with 100%ethanol and 0.9 XPBS. Wash a 26 s gauge bevel tip 7 0 1 and 10 microliter Hamilton syringe two to three times before loading the syringe with five microliters of the cell suspension. After anesthetizing the irradiated recipient fish in trica, place it on its side on a damp Kim wipe.
Stabilize the fish with one hand and insert the needle with the bevel facing up at a 45 degree angle into the flank of the fish above the peritoneal cavity. About halfway between the posterior boundary of the hind brain and anterior border of the dorsal fin. Gently depress the plunger.
Allow the fish to recover in fresh fish water and observe the fish daily for tumor engraftment. If engraftment has occurred, continued growth and disease development can also be observed. One cell BRAF FP 53 MIFA zebrafish embryos were injected with the mini Cooper vector containing the melanoma oncogene set DB one or EGFP each under the control of the MFA promoter as shown here.
Tumor incidence curves for the adults revealed that the SET DB one significantly accelerated melanoma onset as compared to the EGFP control as seen here, hematin and EOSIN staining shows that melanomas expressing SET DB one were more locally invasive than EGFP control melanomas. This figure shows bleached or unbleached zebrafish dorsal scales that were stained with an antibody recognizing the nuclear localized mifa transcription factor to assess transplant ability of the tumor. Melanoma cells were isolated from a BRAF FP 53 mifa fish injected with mini cup R-E-G-F-P 50, 000 cells were subcutaneously injected into a recipient Casper mutant that had been irradiated the day before with 25 grays as can be seen here by two weeks of age.
Pigmented donor derived cells were easily recognized. So after its development, this technique paved the way for researchers in the field of cancer biology to study the functional consequences of melanoma associated genetic changes by using the zebrafish. After watching this video, you should have a good understanding of how to screen for melanoma modifiers by assay, differences in tumor onset invasion, gene expression and transplant ability using a zebrafish auto tumor model.
