I have found a mutation that probably decreases the level of transcription of my gene of interest.
I would like to clone a part of the gene into a plasmid that would contain a reporter gene, so that I can have a control gene for my Real-Time PCR expression quantification.
I should note - I DON'T want to fuse the gene with a reporter protein.
Which plasmids can you recommend for me (as most of them are offered by companies I am not familiar with)? The fragment I would like to clone is quite big - 6kb.
Hello,
as I understood, what you would like to do is to clone the promoter region of your gene upstream of a reporter its expression you can mesure. With this same objective I've used the Dual-luciferase reporter assay system, from promega. It is really useful, since you use two different luciferases that you read from the same sample, so it's the best way to normalize the luciferase activity of your promoter gene to the amount of transfected cells. Look at the protocol, and see if it is optimal for your needs, and if you have all the necessary equipment.
Hope it helps.
best regards.
Your question is not completely clear for me. Do you need a promoter-probe vector for mycobacteria?
if yes you can read Microbiology. 1995 Aug;141 ( Pt 8):1785-92.
Activity of mycobacterial promoters during intracellular and extracellular growth.
Dellagostin OA1, Esposito G, Eales LJ, Dale JW, McFadden J. or another paper using GFP as reporter.
No, I don't mean promoter. It's a part of an intron that is essential for mRNA stability. Hence, I would like to coexpress my gene and another gene for control (e.g. GFP) and compare the amount of mutated vs wild-type through Real-Time.
Alternatively I can transfect the cells with a plasmid containing my gene of interest only. However I have tried that in the past and got very inconsistent results. But maybe this is the way to go?
Have you tried to characterize your gene on protein level instead of RT-PCR? Sometimes it is difficult to get rid of all plasmid DNA before the PCR, which might explain your inconsistent results.
Michael,
it is a tedious task to construct a plasmid that would contain the whole cDNA of the gene and insert the mutated intron between the exons. Therefore what I'm describing here is a minigene assay, with no intention for the cloned gene fragment to be translated.
Hey Filip, could you please clarify? Among your skills you have all bacterial molecular biology and now you want to insert an intron. So you are not working on becteria but on eukaryotic cells. Going with that, does your gene have a mouse homologue? If so, I would try the allelic exchange in mice embryonic stem cells and then quantification through RT-PCR comparing to the wild type expression and not some viral promoter driven expression when using a plasmid. If so, then I would write an email to Dr. Robert Lenartowski in UMK Toruń. I had classes with him in that subject, so maybe he could help you with his experience. But of course I am totally unsure if I understood you completly. Your question is very chaotic;)
http://www.lifetechnologies.com/au/en/home/life-science/cloning/topo/long-fragment-cloning.html
cloning 6kb can be pretty rough, good luck on that.
btw if you know where the mutant is, why not just targeting it? Real-time PCR can only amplify ~100 bp amplicons anyway. I dont see the point cloning 6kb as template as a control.
Hi Filip,
if I understood you properly, you want to find a plasmid that contains your GOI and a reporter gene because you want to check RNA transciption of your GOI after cloning an intronic sequence and you want to use the reporter gene as an internal reference. If wrong, please clarify.
I am currently using a plasmid with two promoters, one driving the expression of my GOI and the other driving the expression of EGFP (http://www.addgene.org/23027/). I do not know the size of your GOI, including the mini-intron you want to clone. Depending on its size you could have some problems. My final constructs are about 9 kbp and the ones tested so far are working fine for checking protein levels after transiently transfecting HEK293T.
I have very recently cloned a small intron (about 100 bp) into my GOI for checking also RNA expression of mutants that could activate NMD, but I have not tested if the intron is processed properly yet, so I cannot tell you much about how it is working. Hope to get an answer to this soon.
Another problem you might face is that it could be tricky to remove completely the plasmid on the RNA preparation, so look for a good strategy to differentiate the levels of DNA contaminating your RNA prep.
Hope this helps.
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