4 replies to this topic

[Bomber]

Hi all,

I hope somebody can share some experiences.
I generated clones stably expressing my gene of interest using cloning cylinders and picking single colonies after selection with puromycin.
The plasmid is designed in such a way that the gene of interest and the selection cassette is driven by the same promoter.
I started to check the cells with confocal microscopy and realized that the level of expression of my gene of interest is quite heterogenous (though all cells express it compared to the control) within cells derived from one colonie.

I wondered if I should 'select' my cells again with a higher puromycin concentration to obtain a culture that shows a uniform and highest expression of my gene?

Thanks in advance for any help.
Best regards

[Dr Teeth]

You could try selecting in higher concentrations of puromycin as you said, if high levels of expression are what you want.  Otherwise, you could harvest the cells and plate them onto 96 well plates at a concentration of 1 cell/well.  Then select individual clones from that population.  In this manner, you can select populations with differing levels of your target gene, which may be desirable in future studies.

[stardust]

Hi,

did you check if your promoter is influenced by the cell cycle of the cells? Which promoter are you using? Maybe it would help to synchronise the cells for 48 h by serum starvation, then culturing in normal medium for 24 h and redoing your check for expression?

Stardust

[Bomber]

Hi,

thanks for your input.
The gene and the selection cassette are driven by the CMV promoter.
Currently, I am just growing the cells with half of the puromycin concentration that I used for selection to obtain enough cells
to freeze etc... . (The mock cells were completely dead after 2 days).
I will try both approaches with one of the clones and see if it makes a differende regarding the cell cycle and the increase of puromycin.
Thanks again and best regards!

[stardust]

Hi again,

In my cells (CHO-K1) expression of a transgene from a CMV promoter was cell cycle dependent. Western Blot after starvation followed by growth (important, otherwise your cells don't produce protein) gave pretty nice and consistent results.

Stardust