Hello,
I am stuck in a weird situation. I have performed some microinjections on Zebrafishes. I injected a Bovine gene driven by a Zebrafish promoter. I see some phenotype as well. I would like to experimentally demonstrate that the phenotype is because of over (ectopic) expression of this gene. I thought that the simplest way to do this was to perform a qPCR on uninjected, control injected and Bovine gene injected samples. The issue is that the Bovine gene bears 84% similarity with that of Zebrafish gene. The primers I had designed were for Bovine gene and when I use these for amplification , i see bands even in uninjected samples (cDNA of course). The primer although bovine, is binding and amplifying the fish gene. Does anyone have a better assay or remedy for me?
P.S. Antibodies for this gene are not that specific either and if binding to Bovine protein, they also bind to fish.
Any suggestion would be appreciated.
Thanks a lot,
researcher in distress :( :(
Hi!
I have been in a similar situation because I had an experiment where I had to over express a gene from the same species but I wanted to know how much of the transgene was being over expressed without getting it mixed with the basal expression of the gene. In the end I think the best solution is to design a new primer pair that covers the part of the construct that is not, in your case, the bovine gene ORF but the promoter or the AttB border that was used for the vector construction etc... depends on how your construct looks.
Good luck!
Hello Miguel,
Thanks for your reply. The idea is cool, except that if I do this, the expression would again not be different as the control construct has identical structures, just lacks the CDS of this gene. If I want to see the expression of this gene only, then having primers from within it is the only solution, except that these primers are not so specific, as claimed to be theoretically. I have changed 4 primer pairs so far, and I am running out of regions of dissimilarity between bovine gene and fish gene.
Hi Nivedita
You should go for the Taqman approach instead of SYBR. The probe will give you the necessary specificity to differentiate the endogenous from exogenous gene.
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