Investigating the Vasculature of Zebrafish: Orange Observers
On the first day, we were introduced to the program and safety requirements for the rest of the week. After the introductions, we were taught of the two forms of science communication we will produce throughout the week, one being this blog, and the other being a PowerPoint presentation that will be presented on the last day. We were also presented to the science inquiries and projects we were dealing with, which include mounting, drug testing and using CRISPR in zebrafish. We were given a program introduction where Maria and Andrea, collaborators from the Peter MacCallum Centre, explained the vasculature and lymphatic system of the zebrafish, as well as describing how stopping the blood supply can contribute to the prevention of tumours growing. Later on, we were able to experiment and play around with the microscopes, observing the zebrafish under the different fluorescent lights.
The second day started with a presentation on CRISPR, which is a system of mutating a specific section of DNA to disable or weaken the gene targeted. We then used PCR to determine whether the CRISPR had made an impact on the targeted gene. The first step of PCR involved adding a master mix which contained many of the components necessary for the PCR to work properly. Primers were then added which finds the specific section of the DNA that should have been mutated and guides the Taq DNA Polymerase to duplicate it. The mutated fish’s DNA is also added which is what is being duplicated. This takes place in a thermocycler which cycled through three different temperatures, 95°C, 68°C, and 73°C which served to allow the primer to bind, an enzyme then uses the primer to copy the rest of the gene, after that the new DNA detaches from the original strand. These three temperatures were done 34 times, each time the number of DNA strands went up exponentially. These were then separated by gel electrophoresis which used charges to order the DNA by size. We compared the results with a typical zebrafish to see if the gene had been manipulated or not. We did see a slight difference in the size of the DNA caused by CRISPR which the researchers can then use to look at the results of CRISPR on the formation of blood vessels in the zebrafish.
The third day started with a look at how drug design can impact the functions of proteins. We looked at how amino acids fold to form proteins using diagrams, and a computer program that gave us an in-depth look at the structure of proteins, starting with the primary structure which is the sequence of amino acids that are in polypeptide chain. Then we moved on to their secondary structures and the different shapes that the chains can fold into, those being alpha helix, beta pleated sheets and random coils. We then looked at the tertiary structure where the protein is folded into its 3-dimensional precise protein structure. Finally, we looked at how some proteins can be formed out of multiple polypeptide chains, meaning two proteins that are in the tertiary structure come together to form the quaternary structure. We used this information to look at the Hex-B protein which was the protein that was being targeted in Andrea’s experiment. After we finished looking at the structure of proteins, we then moved on to focus on the vasculature of the 48-hour old zebrafish that had been given different drugs that affected their vascular systems. To this we started by adding 2 drops of tricaine to the water the fish were in so that the fish would be still while we looked at them under a microscope. Then a few fish were transferred into individual petri-dishes (these dishes had been labelled with the drug). The fish were then set with agarose gel to further ensure that they didn’t move while being looked at under the microscope. Once the set up was finished two members of the group at a time would observe each sample of fish, focusing on a different drug.
The fourth day Maria and Andrea came back to demonstrate how to process our data that we had collected over the previous days. Those of us who had laptops spent their time manipulating the images of the zebrafish at 48 hours old. While the other observed the differences between the control (DMSO) and the two different drug injected fish, AV951 and SL327. Once we had analysed the results we moved onto learning about the animal ethics that are taken into account before an experiment can be conducted. We then did a little activity where we took up a roll in the community to look at different perspectives on ethics.
RESULTS FROM DAY 4:
We observed that the drug SL327 fish had little to no vasculature development throughout the trunk compared to the control (DMSO) but this could also just be because of the lack of pigment that was originally there. Then when we observed the second fish AV951 we can see a clear pathway where the blood vessels should have developed but haven't. This can mean that the drug used has slowed down the development of the vasculature or they are just yet to grow. Some improvements that we would make in further testing would be to remove the original pigment so we can see the vasculature system much more clearly so we will get better images that can be easily analysed.
Kelly: This whole week has been an incredibly unique and valuable learning experience which I would remember for years. Throughout this week, the GTAC SIRE program has been a great opportunity to be able to use different equipment and technology to produce and collect results that I wouldn’t have the opportunity to use otherwise. Overall, this program was an amazing experience where I was able to meet a variety of different people to collaborate on two experimental research projects.
Amra: This experience at GTAC has been great as I was able to meet new people and work with new equipment. I learnt how animals can be used for research and treatment development safely and this opportunity at GTAC introduced me to new views that can definitely be used in the future. The staff and collaborators were passionate and made this experience informative and enjoyable.
Laviyan: This was an interesting and intriguing week, as we got to participate in lots of fun and very educational activities, and experiments, and also got to meet lots of nice people along the way. I love how the workers and staff at GTAC are very kind and generous and made us feel more comfortable with the program. GTAC has organised this program to be one people can walk away from and remember what a great time they had :).
Tess: The GTAC Sire program has been an incredible experience that I was able to participate in over the week. It has given me an opportunity to find out how researchers work and conduct their experiments. I found it very helpful and being able to work with mentors who had hands-on experience in cancer research and biology fields. Although it was tiring, it was overall an amazing experience.
Patrick: GTAC has been a wonderful and unique program for me. It’s allowed me to really see into the inner workings of the science researcher field, and all the interesting results that are gathered. It’s also a great opportunity to meet new people who share a passion for science and to emerge yourself in the experiments and science.
Millie: The GTAC work experience program has by an amazing opportunity for me to work with experienced scientists and mentors. While getting to use high tech equipment that I wouldn’t have gotten to use if I wasn’t in this program. It was really interesting to see how a real lab operates and how Maria and Andrea went about their experiments.