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do you have any experience with using the Flp In system to create stable expressing cell lines? We use CHOs and up to now we used the classical vectors (pcDNA3.1) to create single and double expressing clones (adenosine and dopamine receptors). We spent two years to create about 5 good clones with correct expressions of the receptors and good affinities but the work was quite frustrating (welcome to science
) and some clones were not perfect anyways.We saw following problems
- high number of false positive clones that survive in the antibiotic (hygromycin, geneticine)
- - from 1:2, 1:5, even 1:20 for the A2a receptor in Hygro
- too high expression of the receptors, so they had bad affinities
- mixed clones or clones that lost their expression
So we think to try the Flp In cells. I have this doubts:
- We can express only receptor or express a fusionated cDNA of the two receptors
- Even if the cDNA integrates into the FRT target, cannot it get integrated in any other places too as before?
- This is less frequent because the FLP recombinases cuts all the vector because its over abundant?
- All the clones that survive the selection have the same expression? I read someone commented it was no so true
Thank you a lot for you help and opinions
