Where have we been? Where are we going?

Thesis has been going well! In essence, I’m trying to understand the role of nfe2 and alas2 in the genetic pathway for the blood development of zebrafish.

Nuclear Factor Erythroid, 2 (NFE2) is a transcription factor that is responsible for the regulation of erythropoiesis and oxidative stress in vertebrates. Erythroid-specific -aminolevulinate synthase (alas2) gene initiates heme biosynthesis in verterbrates during erythropoiesis. In zebrafish embryos that had Nfe2 knocked out, there was a downregulation of alas2 expression. The minimal expression of alas2 induced a phenotype of hypochromia. I will attempt a gain of function assay where zebrafish with nfe2 knocked out will be microinjected with capped mRNA coding for alas2. If a normal phenotype is observed in the rescued zebrafish, then this would implicate alas2 as the cause of their hypochromia.

Recently, I’ve been practicing my microinjecting and qPCR technique. I’ve been injecting morpholino (with a florescent tag) into the vegetal pole of the zebrafish embryos which causes them to fluoresce. When the microinjection was done successfully, I observed viable zebrafish embryos that were red (when observed under green fluorescence). I have also been practicing my technique when performing qPCR. The goal was to achieve Ct values that had a standard deviation that was less than 0.5. When performing qPCR, Ct values represent the cycles to threshold. This is the amount of PCR cycles required for a fragment to reach a predetermined fluorescence threshold. On my first attempt, my standard deviation was 1.38. On my second attempt, the standard deviation was 2.95. I was advised to alter the procedure such that I created a master mix containing all the required components without water. This drastically decreased my standard deviation to 0.507!

Moving forward, I will be starting a culture of E.coli that contain a plasmid (via transformation) with the gene for alas2 mRNA. E.coli will be used to further amplify the gene. The bacteria will then by lysed to re-isolate the plasmid. We will then be able to synthesize capped mRNA that will be injected into the zebrafish embryos, so we can observe erythroid development. This project is going to be lots of fun, and I’m glad that I’m able to pull from all of the laboratory skills I’ve learned at Bates to possibly produce data of great importance to the science community at large.