I have a plasmid to use to generate new transgenic mice, but I would like to try to see my protein expression before starting the transfection of ES cells.
The plasmid of interest has the promoter silenced by a stop cassette flanked by loxP sequence, that I was thinking to remove by co-transfection with a cre-Plasmid.
I have 293T cells ready to use to test it, but I wonder whether Cre-excision requires the loxP sequences to be in the genome, because in the 293T cells the plasmid will not be integrated.
Thanks in advance for the help!
I believe as long as the Cre recombinase has access to the DNA, it doesn't require the plasmid to be integrated into the genome.
No, you don't need the loxP sites integrate in the genome. We have co-transformed target plasmids (containing loxP sites) and the Cre-expressing plasmids into bacteria for transient assay to see whether or not both constructs are ok. This also apply to other Site-specific system such as phiC31-att and Bxb1-att etc uni-directional SSR systems.
We do this type of testing by co-transfection of Cre and Cre-dependent plasmids in HEK cells all the time for routine testing. If your targeting vector is large, just be sure you isolate enough endotoxin free high quality DNA for the transfections. If it is smaller than 10Kb you shouldn't worry about it much. You also need to be sure the promoter in your plasmid(s) will mediate expression in HEK cells, and I can't tell you for sure since you didn't mention which one. EF1a, CMV, etc work great in HEK lines. One other thing that tripped me up previously is make sure the targeting vector does not have any selection markers that might cause toxicity on cell lines. If so, you need to remove that portion by restriction digestion and purification before transfection, and though less efficient, the linearized DNA can also work for transfection and Cre dependent expression in HEK cells.
Ok!!! Thanks a lot for the useful information. I hope it's gonna work. Otherwhise I will try to question all the possibilities. My vector is quite big (more than 10 Kb), but it's should be clean enough. The promoter is CAG, so it should be fine. I don't think I have any selection markers that could cause toxicity, but I will double check just to be 100% sure!
In that case, perhaps you can just use a bit more DNA in micrograms to compensate for the large construct size. I usually just use a smaller amount of Cre construct for co-transfection...a little bit is sufficient. For example, maybe something like 2ug of your very large targeting construct and 0.25-0.5ug of your Cre construct per well of a 24 well plate. You could scale that for larger well sizes.
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