cre-loxP: how to check if the gene is expressed - (Oct/31/2010 )
Hi,
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.
You would usually transfect your targeted ES cell clone with a Cre recombinase, and compare GFP expression to untransfected cells. I would then also do a western or RT-PCR for your gene of interest (hoping that it has a tag).
rsm
Thanks a lot for your answer, but is there any way to check it before I transfect ES cells, i.e. for example I cotransfect HeLa cell line with my plasmid and a cre expressing plasmid and, after recombination occurs, observe both the gene and gfp expression. Can it be done this way?
since my plasmid has to be shipped to another lab for ES transfection, I would REALLY like to check if it really works and I dont't know how.
G.
if you want to do such an experiment, I would suggest that you use a mouse cell line similar to your target tissue.
gangut on Mon Nov 1 21:29:02 2010 said:
Thanks a lot for your answer, but is there any way to check it before I transfect ES cells, i.e. for example I cotransfect HeLa cell line with my plasmid and a cre expressing plasmid and, after recombination occurs, observe both the gene and gfp expression. Can it be done this way?
since my plasmid has to be shipped to another lab for ES transfection, I would REALLY like to check if it really works and I dont't know how.
G.
I guess it would work. Keep in mind that R26 is a rather big plasmid, so transfection efficiency may be low. Also, R26 is a weak promoter, you might not be able to see GFP expression with a microscope (I'd recommend FACS).
Good luck,
rsm
In the vectors we use for targeting the ROSA26 locus, there is no promoter present in the plasmid. After recombination into the genome, the endogenous ROSA26 promoter is used to drive transcription. If we would perform a transient transfection with our plasmid + Cre, we will not see GFP, as the chance of homologous recombination is quite low in HeLa cells. Of course, this depends whether or not the ROSA26 promoter is in your construct or not.
Great, thaks a lot to all. I've transfected cell lines and will let you know if it worked. Human cell lines, unfortunately as I don't have any mouse material. The plasmid is big indeed, > 17 kb, but transfection efficiency isn't very low. As for promoter, I have no information on the plasmid apart from its sequence, but it seems that the plasmid has CMV promoter. It was not detected by the online sequence analysis tools, but I performed blast with the CMV prom sequence and it showed it was there.
I know that in case of Rosa plasmids, transcription is normally driven from the endogenous promoter, but in this case, CMV is definetely in the plasmid.
So it seems you've never checked your plasmids this way?
G.
rsm
sorry, the plasmid was named Rosa26 (not R26-CMV) so I just pasted its name, at that time not even knowing it had CMV prom.
Rsm on Thu Nov 4 12:49:50 2010 said:
rsm