Working in the lab!
We are four weeks into the semester now and last week was my first lab week. I was very excited and nervous to start but so far everything has been going smoothly.
Apart from Nabil, Sophie and Nick who are investigating the effect of Nfe-2 gene on zebra fish across development, I am working on a totally separate project. My thesis focuses on the regulating role of Aryl Hydrocarbon Receptor (Ahr) on the Nuclear factor erythroid 2-related factor (Nrf) family in zebrafish embryos using Chromatin Immunoprecipitation (ChIP). Both Ahr and Nrf are families of transcription factors that are involved in the oxidative stress response. In an unpublished study by Dr. Williams, knockdowns of Ahr1b and Ahr2 have altered expression levels of all six genes in the Nrf family. Additionally, Ahr often binds to a conserved sequence upstream of the transcription start site to activate gene expression. This well-known conserved binding pattern is referred to as the xenobiotic response element (XRE); they are found on the promoter sequences of all the genes in the Nrf family. From these preliminary understandings of the crosstalk between Nrf and Ahr, we hypothesize that Ahr activates the transcription of 6 genes in the Nrf family by binding to their XRE sequence.
To test our hypothesis, we employ a very novel technique called Chromatin Immunoprecipitation (ChIP). First, we crosslink DNA and protein together with formaldehyde. Next, we shear DNA to many small strands, and use the antibody for Ahr1b to precipitate Ahr1b from the solution. Next, we harvest the precipitated product, and de-crosslink Ahr1b and any DNA sequences that it binds to. This product is then subjected to Polymerase Chain Reaction (PCR) amplification using the primers for the 6 Nrf promoters. If there is any binding between Ah1b and any of the Nrf gene promoters, we should be able to obtain DNA from the PCR reactions. Alumnus James Meyo’14 has worked on refining the first part of this research for his thesis and my job is to pick up from what he has left and finish it.
After studying James’ thesis and reading other related articles for two weeks, I started the wet lab for my thesis. The first challenge I encountered was fish husbandry. My procedure called for fresh 24 hours post fertilization embryos and the first thing I had to do was breeding the fish. I have worked with zebrafish embryos before for my Molecular Biology lab but they were prepared by AI Beth Whalon and Professor Larissa Williams so I really did not understand the effort behind fish breeding. On my first attempt, I got no embryos. I even mistakenly returned a female fish to the male tank and left the water current on too strongly. As the result, some fish escaped the tank and went down the drain and Professor Williams had to resolve the consequences for me. I was a bit dismayed, so I treated myself to a cup of smoothie. But I quickly recovered and moved on to my second attempt for breeding, which ended up with no embryos as well. On my third try, after spending sometimes getting to know the fish and gaining trust from them, I finally got embryos!!! Third time the charm!!!
I am on the process of crosslinking protein and DNA and getting ready for ChIP. More challenges are ahead of me, but I am ready for it!!!! Thesis is always challenging but at the same time rewarding!!!