Hello,
I performed a luciferase assay to quantify the possible interaction of a Tbox factor on a putative Tbox binding site/enhancer element of the gene to be transcribed (identified using ChiP seq). based on results from ChIP seq and the Tbox phenotype, my PI - I believe - was expecting to see a result. I have never performed ChiP seq myself and am unaware of the possible disagreements between results obtained with this method and what would be seen in vivo.
I am an undergrad writing my report at the moment so any lines of thought are welcomed!
Hi Mohammad, thanks for that.
zebrafish embryos were injected at 1-4 cell stage and needed to be assessed early on in development, so I presume my PI did something similar for ChIP seq.
T-box redundancy/masked results is interesting. Would you mind elaborating as to why I would knock out the gene? I am not sure I have understood correctly. Do you mean that endogenous activation of my reporter gene may be taking place? If that were the case would it not be activating by the same proportion in negative control and injected? (I also think the concentration of Tbox RNA injected was pretty high). would that not also mean that I would expect the firefly luciferase readings (experimental) to be higher than the renilla luciferase (internal), even if firefly readings showed no difference between injected sample and negative control?
Or do you mean that my Tbox RNA may have accumulated mutations? In which case, how does that compare with the positive control, which did show fold activation?
just to clarify - i used the dual luciferase with renilla as an internal control + a positive control and negative water control for each injection.
thanks again, very helpful!
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