Usually, for overexpression, mass culture should work fine. Cloning the cells after selection is necessary when the cells "do not like" protein you overexpress, for example it is toxic or reduces proliferation (this would cause a negative selection for cells that overexpress your protein and positive selection for cells that lost expression so after a few passages you would loose expression even if you keep adding the antibiotic). Cloning would guarantee homogenicity in the plate but you introduce new biases: site of insertion in the genome, clonal defects/mutations, convenience (this clone gives the result you like while a diferent clone with similar overexpression levels does not). So, if you clone you have to confirm results in at least three different clones.

I suggest you freeze a lot of cells  as soon as possible after the transduction and selection (e.g. 10-15 days after starting selection with puromycin the resistant cells should grow well and have good expression), this way you can test your mass culture for overexpression and signalling and iif you notice incoherent results or unexpected variations in expression after a few passages you can go back to the frosen cells and clone.

Good luck. 

Das Beste wäre zusammen mit dem modifizierten Protein ein flankierendes Fluoro-Protein (möglichst im selben Konstrukt) zu transfizieren, dann kann man

1. Die Transfektionseffizienz anhand der Fluoreszenz überprüfen und

2. bei geringer Transfektionseffizienz ziemlich leicht die farblich fluoreszierenden Zellen heraussortieren

Viele Grüße nach Würzburg (wo ich auch gearbeitet habe)

Fred Lühder

I agree with Konstantinos. I would do a few rounds of puromycin selection in your cells before performing the experiments on collected transfected cells, in order to reduce bias (sequence insertion, level of expression, etc). Otherwise you will have to validate with several clones which may or may not give the same results. We store  transfected cells in liquid nitrogen that were sequentially frozen using a control rate freezer following several rounds of puromycin treatment. (We work in primary human skin cells, so it was important to ensure the cells would come back after taking them out of storage!) Before you freeze tons of cells, make sure to validate that your protein is being overexpressed above the baseline. I do not know if you are using a fluorescent tag (like Fred suggests) but validation by Western blot and immunofluorescence will help answer this question. I have had issues where my cells are puromycin resistant but when I try to validate K/O, there were no relative changes in protein levels.

Thanks for all your helpful tips!

I have both - i created a Fusionprotein once with GFP (for localization information) and once with V5 tag on the N-terminal side. I need both because my protein I'm interested in is very small and i have to exclude that GFP has an influence to the function of my protein. So, I planned to compare both in western blot analysis for their down stream signaling. Of course the GFP-tagged Protein i can sort with FACS, but not the V5-tagged? I think the idea of Konstantinos is good to freeze a lot of cells, of course test the expression of the bulk like Anne said, and if i will need clones  i can go back to my frozen cells, what do you think?

Yes, that is the idea, Susann. In all cases, anyway, it is important to be able to go back to an early passage frosen vial. As Anne said, you can check expression of the bulk culture. It is a good idea to test by immunofluorescence that the v5-tagged protein localizes similar to the Gfp-tagged one. This way you can also check how variable is the expression from cell to cell (population homogeneity). If the bulk culture does not give you good results you can always go on cloning.

Good luck