Green Geneticists (2022)

Part 1

DNA Extraction

DNA was extracted from the larvae using an extraction buffer. The extraction buffer is used to break down cell membrane and nucleus membrane to give access to the DNA within the nucleus of the larvae’s cells. It contains the enzymes and detergent-like chemicals to dissolve and break down these membranes. Heat aids the enzymes in this task.

In our DNA extraction we transferred the zebrafish into tubes containing the extraction buffer. We then used the vortex and centrifuge to mix these together. We put these samples in the thermocycler to heat them. From this we got our isolated DNA samples which we then used in the PCR.

 

PCR (Polymerase Chain Reaction)

A PCR (polymerase chain reaction) is a technique of copying a gene of interest. In order for a PCR to work, DNA, primers, free nucleotides and DNA polymerase must be present. Primers find and bind to the sections of complementary DNA, free nucleotides make up the copies, DNA polymerase is the enzyme that copies the DNA. There are three steps in the PCR cycle that are repeated.

1. Denaturation, at 95°C, separates the double helix into single strands.
2. Annealing, at 56°C, primer binds to the sequence.
3. Extension, at 72°C, copying starts, polymerase’s optimal temperature.

We used our isolated DNA, from the previous DNA extraction and pipetted it into a new tube, containing primers and the Master Mix. The Master Mix consisted of free nucleotides and taq DNA polymerase. With all of these in the tube, we were able to successfully copy the gene after putting the samples in the PCR machine.

 

Gel electrophoresis

Gel electrophoresis is used to separate DNA fragments based on size or length, we used e-gel to do this. The isolated DNA goes into the wells and gets separated by the electricity that gets passed throughout the gel. The negative charge of the DNA is attracted to the positive electrode and travels through the gel. The shorter fragments travel further through the gel due to the lower number of base pairs and vice versa.

The expected results from this gel electrophoresis were one line from the wild types, and two from the CRISPR-affected zebrafish. However, the primers used in our experiment appeared unable to amplify some of our DNA samples. This resulted in an unclear outcome. Though, other groups were able to achieve the expected results and were the basis for the analysis of our experiment.

 

Results

The aim of this experiment was to observe the effect of a deactivation of the HexB gene, which creates the protein responsible for adding sugars to other proteins. We removed the activity of this gene using CRISPR and observed the following results. Andrea, a working scientist at Peter MacCallum Cancer Centre, was able to analyse these CRISPR-affected fish and was even able to find a visible phenotype. The absence of the HexB gene slowed or potentially stopped the development of vasculature in these zebrafish.

 

Part 2

Fluorescence microscopy of the drug-treated fish

Our research model was a zebrafish which we looked at how proteins act, in order to understand how cancer and tumours work in relation to the vasculature of organisms. We captured images of young zebrafish that have been treated with different drugs (AV951 and SL327) as well as a control specimen (DMSO) with the EVOS microscope to determine if, and how, the drug treatments affect the development of the vascular system in the fish.

 

Results

Using the software ImageJ we analysed the vasculature of the zebrafish +treated with drugs and compared it to the control fish. We could assume that the vasculature of the DMSO sample (control) is more developed than the AV951 and SL327 treated samples as shown by the increased pigmentation in the images. The SL327 and AV951 image appears more hollow than the other two in the red and green fluorescence.

Kayla:

I thoroughly enjoyed my experience at GTAC. I’m glad to have received the opportunities that I have during my time here. Working with the scientists from Peter MacCallum Cancer Centre was very fun and working with the equipment in the lab was cool too. The Green Geneticists were an interesting and creative group of people to work with.

Alexis:

Thanks to this GTAC program, I was able to learn new things about the science I'm fascinated about and to experience what it’s like to be in a lab as a scientist. This experience was really fun and I would do this again at some point in time.

Georges:

During my time at GTAC, I’ve got to experience a working lab, looking at brand new experiments that have never been done before, meeting real cancer researchers at Peter MacCallum as well as a lot of other interesting opportunities. All in all it was a fun experience, I got to use really high-tech equipment, such an EVOS M5000(fluorescence microscope), mechanical pipettes, a PCR machine, and a centrifuge. We were divided into groups and I was put in the fantastic Green Geneticists, we were an untypical group, but hey we took some banger photos.

Vanessa:

Overall, I really adored my experience at GTAC, the hands-on participation in the lab was very valuable and knowing that my team and I’s research could potentially be used in cancer treatment research is out of this world! The Green Geneticists are delightful people I’ve become close to this past week and I'm grateful for the friendships this opportunity has opened for me.

Jacob:

I’ve had a wonderful experience here at GTAC, I am thankful for Peter McCallum for helping us with our research. I am very grateful for working with such bright and brilliant people.

Cody:

I am thankful to GTAC for giving me this chance to experience what it is like to work as a scientist. This gave me the chance to have a deeper insight of the work done in labs.