z-TRAP Say Whaaaaat?
My project is super cool, but very high stakes since I am the only person working on making a zebrafish TRAP transgenic to isolate for the specific cell line of red blood cells in the United States.
The TRAP construct I need to develop the transgenic is scarce (some microliters) because it is all that’s left from a deceased scientists’ lab. That being said, I have been very nervous in executing each step to get to a complete z-TRAP transgenic specific to erythrocytes. At this point in time I have successfully completed a bacterial transformation using ampicillin resistant heat competent E. Coli bacteria cells. By injecting my TRAP construct into them and plating them on ampicillin plates, I was able to let the bacteria quickly replicate my plasmid with certainty that the bacteria that survived contained my plasmid. I did this with a transposase construct as well. Next, I was able to use a miniprep plasmid kit to lyse the bacteria and get my TRAP plasmid as well as the transposase plasmid in enough quantity to later microinject into zebrafish embryo.
Why am I doing this? Well, I’m glad you asked. I am interested in a gene called biliverdin reductase b that is known to bind to Gata-1 sites in red blood cells specifically. I have designed primers that (hopefully only amplify a portion of blvrb that has Gata-1 binding sites in it) are specific enough to only bind Gata-1. If I can’t clear this stage then I wouldn’t be able to inject my construct into zebrafish embryo because I wouldn’t be certain if I’m only amplifying biliverdin, thus I won’t be sure that my TRAP construct is in red blood cells when the ribosomal protein is isolated using affinity purification. So far, I have not been successful at getting primers specific enough to only bind to biliveridin, so I have redesigned new ones that should be here by Friday. If I can get my PCR product to have one band at the size I need soon (about 600 base pairs) then I can send it out to a company for sequences to confirm it is indeed biliverdin and I can begin growing my transgenic fish. Otherwise, my thesis will conclude with obtaining the TRAP plasmid. The next person will be who gets to put it into wild type and Nfe2 knockout embryos to see the role of Nfe2 in primitive erythropoiesis for zebrafish embryo that have been exposed to pro-oxidants.
Science is fun, unpredictable, and frustrating, but immensely rewarding nonetheless! Wish me luck!!!