Catch Me In The Lab

My Research Project

This summer I am testing the metabolism and toxicity of methylmercury in zebrafish with a mutation in the target gclm gene. Methylmercury (MeHg) is a common environmental contaminant in Wisconsin; the impact of this contaminant on prenatal development is not well characterized. My research involves investigating gene-environment interactions of methylmercury metabolism in the context of embryonic development. This project aims to validate and produce a mutant zebrafish line with homozygous loss-of-function mutations in the glcm gene, genes the are involved in oxidative stress metabolism and clearance of methylmercury from the body. My summer research will create the mutant line using CRISPR reagents. I will use this mutant line to document the effects of gclm knockout on early development, and I will examine the response to methylmercury exposure in comparison to wild type zebrafish using microscopy experiments. My hypotheses are that zebrafish lacking gclm will (1) have changes in early development and (2) be more sensitive to MeHg toxicity.

---

Summer Goals

• My summer goals include successfully completing a CRISPR experiment individually

• Identifying and confirming a mutation using high resolution melt analysis (HRMA) and genotype sequencing

• Creating a mutation in the target gclm gene

---

Current Goals

• Having a proof-of-concept for CRISPR reagents and HRMA for creating DNA mutations in zebrafish

• Independence in techniques for microinjection, HRMA, and microscopy

• Learn fin clipping

---

Challenges I've Faced

The past week I have been practicing microinjections with phenol red to prepare myself to inject zebrafish embryos with CRISPR reagents. However, when trying to inject, the needle would not go through the chorion. I later learned the injection mold I was using to hold the embryos in place was the wrong one. After I made a new injection mold the needle was able to pierce the chorion easier and the process was more efficient.

Another challenge I have faced is not getting embryos to use for microinjections or DNA isolation experiments. I believe this is due to the tank cleaning, reorganizing the fish, and lack of breeding for the weeks following the end of the semester. Cleaning the fish tanks and reorganizing the fish is a huge stressor for the fish because they have to be netted and moved into new tanks. The lack of breeding after the end of the semester can get the fish out of the breeding cycle. The fish then need a period of adjustment when breeding to be able to produce embryos.

Although I cannot control this, there are ways to help the fish produce more embryos. I can ease the fish back into the breeding cycle and I can set up multiple breeding tanks to maximize embryo output.