This is common when generating stable cell lines.  The location of the insertion can have a big effect on the expression of the gene.  As the plasmids integrate randomly, you will get a wide range of expression levels.  

We routinely select a number of clones (~20) and screen for the ones that have the desired properties (e.g. highest expression levels) before expansion.

Good luck,

J

Dear Jonathan,

Thank you for your quick response! In order to "avoid" picking so many clones we are using the FlpIn System. The cells harvest a single Flp-recombination site and by co-transfecting the vector with the GOI together with the Flp-recombinase (pOG44) the recombination event should only take place in one specific locus. The recombination event switches the cells from Zeonzin to Hygromycin resistance by bringing the Hygromycin resistance gene into frame. We never got a single clone when we were omitting the Flp-recombinase. Of course we cant exclude random integration completely but chances are very low I guess.

Anybody familiar with the FlpIn System and their caveates?

Gernot

Hi Gernot

I've made and worked with a few FlpIn (HeLa) cell lines now and have seen a similar variance in expression. It's almost certainly down to the integration locus being controlled by the cell in one way or another. It could be as simple as the region switching between hetero- and eu-chromatin. I'm afraid the hardest part of using the FlpIn system is establishing the best parent cell line you can, with the most steady rate of expression from its Flp locus.

Good luck with your experiments!

Please, take into consideration that a DNA vector, a transfection reagent, expression of an antibiotic resistance (trans)gene, expression of a reporter (trans)gene, and selection by acute/chronic antibiotic treatment may evoke cellular responses that affect the biochemical processes under investigation. In the following review, we demonstrate that an assumption that empty vector-transfected cells preserve the cytogenetic and phenotypic characteristics, and represent the adequate control in transfection experiments is not universally valid. We exemplify a number of studies, which reported obvious genomic, transcriptomic and phenotypic changes of tumor cells after transient/stable transfection of an empty vector. Finally, we conceptualize that the diverse experimental manipulations (e.g., transgene overexpression, gene knock out/down, chemical treatments, acute changes in culture conditions, etc.) may act as a system stress, promoting intensive genome-level alterations (chromosomal instability, CIN), epigenetic and phenotypic alterations, which are beyond the function of manipulated genes.

Please see the following manuscript for details:

Stepanenko AA, Heng HH. Transient and stable vector transfection: Pitfalls, off-target effects, artifacts. Mutat Res. 2017 Jul;773:91-103. doi: 10.1016/j.mrrev.2017.05.002.