Me and two of my colleagues from different labs at my institute used three different constructs targeting 3 different genes. All genes seem to get deleted fairly well but unfortunately no one of us got the gene trap LacZ staining to work (except for in bacteria, which got a nice solid blue color), trying different protocols and varying incubation/fixation approaches . We compared the LacZ sequence to the published original sequence and it seems that there are a couple of base pairs missing of the gene. The only answer I got from KOMP was a bunch of protocols which I had already tried. Anyone with the same issue or a possible solution to it? Or maybe an idea of how to check for LacZ by RT-PCR?
I appreciate any input! :)
Sonja, we are experiencing the same thing with newly generated mice from a KOMP construct! We used the Mr03987581_mr primer-probe set from Life Technologies and saw no LacZ mRNA expression in our tissue of interest, agreeing with the LacZ staining. We used the Rosa-LacZ mouse (http://jaxmice.jax.org/strain/003309.html) combined with a Cre transgenic expressing Cre in our tissue of interest as a positive control. That produces robust LacZ staining and robust LacZ mRNA signals. Our working hypothesis is that the spice acceptor site in the KOMP construct is doing a poor job of splicing the LacZ from the intron into our mRNA. Instead the cell simply splices out the LacZ in the intron and makes the normal transcript. Please email me at [email protected] to discuss further. I can tell you more about the LacZ qRT-PCR and I'm curious about the KOMP constructs you're using. Maybe they are similarities among all of the constructs giving no LacZ signal.
Adam
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