1. Hypothesis Statements
We are testing the efficiency of an in vivo tool for screening the effects of anti-cancer drugs on zebrafish embryos and comparing the results and their cytotoxicity with MTT assay of the MCF7 breast cancer cell line. The results will indicate if the drugs have similar effects on both breast cancer cells and zebrafish embryos. It is given that embryogenesis and carcinogenesis have similar molecular mechanisms, therefore it is expected that these cell types act somewhat similar. Their developmental process is expected to support the same results as the MTT assay results for the cancer cells.
2. Methods and Materials
A. Zebrafish strains:
1) Acquire AB and Tg (fli1:GFP) zebrafish from the Zebrafish International Resource Center (Eugene, Oregon, USA).
2) …show more content…
3) Let the embryos grow for 24 hours postfertilization
4) Add the compounds to the embryos and let it sit for 24 hours.
5) Observe the embryos under the Microscope (Olympus SZX9 Zoom Ztereo, Japan)
B. Compounds
Five hundred and two compounds were assessed in the primary screen.
1) Dissolved the compounds in DMSO
2) Diluted the solution at a final concentration of 50 μM with Hanks for zebrafish
3) Dilute respectively for cell culture with DMEM
C. Real-time RT-PCR
1) Treat the embryos with compounds for 3 h
2) Extract the RNA
3) Isolate the total RNA from ten treated embryos using an RNeasy Mini kit (QIAgen)
4) Then treat with DNase,
5) Using Moloney murine leukemia virus (MMLV) Reverse Transcriptase (Promega) reverse-transcribe 2 μg of RNA.
6) Quantify the mRNA level using an Eppendorf Real-Time Cycler plus β-actin as a reference.
The primer sequences were as follows: p53: 50-ATGTGGTGCCTGCCTCAGA-30 (sense)
50-CTTCGTCCTTCACCATCAGCTT-30 (antisense) β -actin:
50-TGACAACGGCTCCGGTATG-30 (sense)
50-TTCTGTCCCATGCCAACCAT-30 (antisense)
We are testing the efficiency of an in vivo tool for screening the effects of anti-cancer drugs on zebrafish embryos and comparing the results and their cytotoxicity with MTT assay of the MCF7 breast cancer cell line. The results will indicate if the drugs have similar effects on both breast cancer cells and zebrafish embryos. It is given that embryogenesis and carcinogenesis have similar molecular mechanisms, therefore it is expected that these cell types act somewhat similar. Their developmental process is expected to support the same results as the MTT assay results for the cancer cells.
2. Methods and Materials
A. Zebrafish strains:
1) Acquire AB and Tg (fli1:GFP) zebrafish from the Zebrafish International Resource Center (Eugene, Oregon, USA).
2) …show more content…
3) Let the embryos grow for 24 hours postfertilization
4) Add the compounds to the embryos and let it sit for 24 hours.
5) Observe the embryos under the Microscope (Olympus SZX9 Zoom Ztereo, Japan)
B. Compounds
Five hundred and two compounds were assessed in the primary screen.
1) Dissolved the compounds in DMSO
2) Diluted the solution at a final concentration of 50 μM with Hanks for zebrafish
3) Dilute respectively for cell culture with DMEM
C. Real-time RT-PCR
1) Treat the embryos with compounds for 3 h
2) Extract the RNA
3) Isolate the total RNA from ten treated embryos using an RNeasy Mini kit (QIAgen)
4) Then treat with DNase,
5) Using Moloney murine leukemia virus (MMLV) Reverse Transcriptase (Promega) reverse-transcribe 2 μg of RNA.
6) Quantify the mRNA level using an Eppendorf Real-Time Cycler plus β-actin as a reference.
The primer sequences were as follows: p53: 50-ATGTGGTGCCTGCCTCAGA-30 (sense)
50-CTTCGTCCTTCACCATCAGCTT-30 (antisense) β -actin:
50-TGACAACGGCTCCGGTATG-30 (sense)
50-TTCTGTCCCATGCCAACCAT-30 (antisense)