cre-loxP: how to check if the gene is expressed - (Nov/01/2010 )
Hi, I've already posted this question in the Mol Biol section, but noone seems to know..
I have a Rosa26-loxP-stop-loxP-gfp construct that will be used for making mice overexpressing the gene of interest.
My question is whether I can check functionality of such a plasmid prior to using it in mice. Sequence is OK of course, but how will I know if the gene of interest will be expressed after final transfection and cre recombination?
Do you normally check your constructs by cotransfecting cell lines with the plasmid and a cre expressing plasmid, in order to see if gene expression occurs? or it doesn't work this way?
thanks a lot in advance
g.
gangut on Mon Nov 1 12:27:34 2010 said:
Hi, I've already posted this question in the Mol Biol section, but noone seems to know..
I have a Rosa26-loxP-stop-loxP-gfp construct that will be used for making mice overexpressing the gene of interest.
My question is whether I can check functionality of such a plasmid prior to using it in mice. Sequence is OK of course, but how will I know if the gene of interest will be expressed after final transfection and cre recombination?
Do you normally check your constructs by cotransfecting cell lines with the plasmid and a cre expressing plasmid, in order to see if gene expression occurs? or it doesn't work this way?
thanks a lot in advance
g.
when u clone ur gene in the vector there is no need to check the expression as it is well studied vector...usually u use a specific cre line where u want to express ur protein..eg..any organ..if u want to overexpress the protein only in muscle then u cud use MCK cre..hopefully i answered ur question...
best
V
PCR
Raise some mice, collect DNA samples (tail snips), and run a PCR on the samples 