Details of My Project
This summer I am investigating different genes in the GSH pathway in zebrafish. This pathway aids in the detoxification of cells, which my lab wants to study in relation to heavy metal toxins. Zebrafish are used because they are transparent and have a similar genetic code to humans. My area of the project focuses on the genes gclc, gclm, and gstp1/gstp2, which are all involved in the GSH pathway. We make mutated lines of zebrafish without these genes, typically causing a knockout line. Knockout means that the fish no longer has a copy of this gene. We cause the gene to get knocked out because we want to see which gene has the biggest influence on detoxification. Each of these lines are at different steps right now, so my partner and I are going to be running multiple experiments simultaneously.
For the gclm line, we are sending off samples to Next Gen Sequencing (NGS) to see if our F1 generation is heterozygous. Heterozygous means that the fish has one mutated copy of the gene and one normal copy. We want the offspring to be heterozygous because we bred mutated fish with wild type (normal) fish. For our gstp1 line, we are working on creating and testing primers for NGS in preparation for sending samples off for sequencing. Our gstp2 line is currently being bred to an F1 generation, and we will start sampling once we have a good number of those. Overall, my summer is going to involve experimenting with a few different protocols to work towards these goals.
One of the skills I have been practicing is microinjections. Microinjections involve making a solution of the gene of interest, cas9 (which helps insert our gene into the fish's DNA), water, and dye so I can see it under the microscope. Then I use a microscopic needle to inject this mix into embryos minutes after they are fertilized. My lab does this soon after fertilization because we want the gene to infiltrate the germ cells (cells involved in reproduction).
The main analysis I run is High Resolution Melt Analysis (HRMA), which includes melting down isolated DNA with synthetic DNA to see if it melts faster or slower than normal. Melting at a different rate can indicate that the gene has more or less DNA, which tells us that a mutation likely took place. With these tests, we can decide which fish we should sample and send to NGS to confirm our findings.
June Progress
During June, I have been practicing my skills with some control fish and tyrosinase, which shows a loss of pigment in the fish. I completed two clutches before running out of compressed air, which is needed for microinjection. My professor, Dr. Carter, and I were also emailing BioRad, who made our HRMA machine, because it had been acting weird for about a month. We talked through our process and found a few steps that weren't being completed correctly, and once we corrected those we started getting good reads again.
Right now I am working on reading results from the first samples we sent to NGS. I have to send them through a program called Galaxy, which is free to use. Sorting through thousands of reads can take hours, so I am trying to figure out how to condense the data into an easier format. My lab has not done this with NGS data before, so I am making a brand new protocol for our lab that will be used for a while. Thankfully, I had one read that showed a mutation, so I am able to continue with the sequencing.
Plans for July and August
July and August are going to be spent on breeding the four lines of fish and experimenting with our protocols. For the gclm line, the main goals include defining the alleles of our mutated gene, crossing confirmed heterozygotes to create mutants with two copies of the gene (homozygous), and raising the homozygous generation. The gstp2 line’s goals include ordering the NGS primers and testing them, breeding the injected generation to make a heterozygous generation, and using sequencing and HMRA to confirm and identify the alleles. Lastly, we will be resolving issues with the gstp1 primers so that we can continue that line. Overall, I am very excited for the rest of this summer and I can't wait to see what we find!
