Transfections...?

John Ladasky jladasky at cmgm.Stanford.EDU
Wed Jan 14 21:31:39 EST 1998


In article <34BCEE5A.4A5B at umich.edu>, Eric Tsai  <ericbt at umich.edu> wrote:
>Hi, everyone!
>
>I have a question about subsequent transfections.  I am using,
>primarily, the neomycin gene (G418).  I would like to follow this with a
>secondary transfection using the psv2gpt gene (mycophenolic acid, etc). 
>As far as selection medium following the first transfection, I would
>ordinarily maintain my transfectants in low dose G418.  If I am doing a
>second transfection, would my selection medium contain both G418 and the
>resistance reagents for psv2gpt and would my final maintenance medium
>also contain reagents for both resistances? ( I am not even totally
>convinced that the maintenance of stable expression depends on low dose
>G418).  Any help would be appreciated!

Hello, Eric,

	The need to include drugs in your medium indefinitely depends on
your expression vector.  Does it integrate?  Vectors that do not integrate,
that are maintained episomally, *must* be maintained under drug selection.
But drugs *can* be eliminated from cultures that have been transfected 
with an integrating vector -- and if you're planning to co-culture these
cells with others, eliminating the drug is probably a good idea.
 
	Now, as for doing subsequent transfections, do you have a means for
purifying your successful transfectants from the first round?  Drug resist-
ance, alas, does not guarantee the co-expression of your gene of interest.
In our lab's experience, with the integrating vector pBJ1-neo and with 
class I MHC and beta-2 microglobulin inserts, the number of cells that 
express our inserted gene can be anywhere from zero to 100% of the drug-
resistant cells, with 70% being typical.  But 10% expression is not un-
common.  Fortunately, we are able to identify and purify the positive 
cells using monoclonal antibodies and flow cytometry.  Can you do some-
thing similar?

	Here's another idea.  Can you insert both of your gene products of
interest into a single vector?  It would save you a transfection step.
There are plasmids available with two cloning sites separated by an IRES
element, and that might make your job easier.

	Good luck!

-- 
Rainforest laid low.
"Wake up and smell the ozone,"
Says man with chainsaw.					- John Ladasky



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