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Hygromycin selection


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#1 JdC09

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Posted 04 February 2009 - 08:48 AM

I co-transfected 293Flp cells with pOG44 (Flp recombinase) and a custom FRT vector. 24 hours later I split the cells into a 10cm dish.
I noticed lots of cell death and the cells that were attached looked unhealthy. I did not want to put the cells in hygromycin until the cell had a few days to recover. So I waited 72 hours after transfection to put these cells in hygromycin selection. Will I still be able to select stable integrants this far removed from transfection?

Thanks

#2 cotchy

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Posted 05 February 2009 - 06:24 AM

I co-transfected 293Flp cells with pOG44 (Flp recombinase) and a custom FRT vector. 24 hours later I split the cells into a 10cm dish.
I noticed lots of cell death and the cells that were attached looked unhealthy. I did not want to put the cells in hygromycin until the cell had a few days to recover. So I waited 72 hours after transfection to put these cells in hygromycin selection. Will I still be able to select stable integrants this far removed from transfection?

Thanks


hi jdc,

Does the plasmid you inserted stably transfect in the hygromycin into your cells?

If so you shoulde be able to select the cells of interest forever by culturing in the optimal amount of hygromycin (which you would have worked out on untransfected cells prior to transfection)

That is you should know what the lowest concentration of hygromycin is enough to kill your cells, thereby when exposing cells to it only ones with resistance will survive, ratherr than treating the cells with too litle hygromycin where most cells will survive regardless of wheter they have the resistance gene or not.

Supposing you have done this and the cells have been stably transfected with the hygromycin plasmid you should be able to select the cells by maintaining them in your normal media, supplemented with the optimal concentration of hygromycin.

If on the other hand the co-transfection used a transient hygromycin expression [plasmid 72 hours could be on the late ennd of the scale for selection, as i am thinking to when i use beta gal for co-transfection mpost suppliers recommned from 24-48 hours later to test for uptake.

Hope this explains it.

What i would do if i was in your situation is, as the cells still seem to be very sensitive which is proably due to the liposomes or whatever other transfection reagent/protocol you used is treat the cells with the lowest concentration you can find on papers for selecting for this marker 50 ug/ml seems to be the lowest i see on a quick internet search at (http://www.invivogen...mycin_03D03.pdf)

And see if the cells survive.

If you wanted to risk trypsinising again and running the experiment one extra day it could be possible to seed cells into a 96 well plate and expose to increasing concs of hygromyicn maybe 20 ug up to 400 ug and see if any cells survive in the higher concs, if they do they should still have the plasmid.

Hope this helps you out.

Cotchy.

#3 JdC09

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Posted 05 February 2009 - 07:18 AM

No the FRT plasmid does not confer hygromycin resistance. What happens is the pOG44 encodes flp recombinase which inserts my GOI as a single copy into the genome. This puts hygromycin in frame and gives the cells resistance.

#4 JdC09

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Posted 05 February 2009 - 07:20 AM

I also want to clear up that I never put the cells in hygromycin until 72 hours after the transfection.
thanks for any help.




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