Help me, please!!!! (successive transfections)

Antoine VEKRIS vekris at u-bordeaux2.fr
Thu Nov 30 13:04:30 EST 1995


Hello Net transfecters,
I do not have any experience with puromycin as we do not have the
resistance fragment (it would be very kind of you if you can mail us some
plasmid) but I am using  multiple transfections using the G418 resistance.
It is clear that if you use a strong promoter (as CMV) there is no way to
differenciate double transfected cells. Using a SV40 promoter some
difference occur following long term culture (at least 2 weeks) and double
transfected clones may be isolated after about a month from the second
transfection.
In this case at least 1 mg/ml of  G418 have to be used and changed every
day! (does any body know something about G418's half life time?).

Well at the end point it seems more easy to us to use a single
transfection system; to allow multiple transgenes expression using a
single promoter it may be interesting to use IRES between coding
sequences. I am preparing a construction with beta-gal/GFP/CFTR to test
this system ( reported to work well as polycistrinic for expression of
IL-12; Zitvogel L. et al, Human Gene Therapy (1994) 5:1493-1506)

Good luck fellows


In article <v01510104ace24fee1e44@[205.149.179.34]>,
jack_owicki at MOLDEV.COM (Jack Owicki) wrote:

: If I understand correctly, you want to use puromycin in the second
: transfection as a selective agent on cells that have already acquired
: puromycin resistance in the first transfection.  I would have thought that
: you needed to use a different selective agent for the second transfection,
: both to obtain effective selection and to assure long-term maintenance of
: both genes that you want to express.  We've been wondering about such
: multiple transfections and would also appreciate comments from the net on
: the subject.  Of course, this should be distinguished from the usual
: (simultaneous) co-transfection procedure, which would be fine for some
: purposes, but not all.
: 
: Jack Owicki
: Molecular Devices
:
--------------------------------------------------------------------------------
: >Hi netters,
: >I'm trying to (super)-transfect BHK-cells with an expression plasmid (the
: >selection marker is
: >Puromycin), which is used in the same cells in other transfection
: >procedere before. So I'll
: >use sucessive the same expression vector for two different cDNA's.
: >Positive clones of the
: >first transfection are used as recipient cells for the second one. The
: >problem is the
: >puromycin concentration during the selection in the second transfection
: >step.Are there any
: >experiences with this kind of double transfections? The transfections are
: >compounded with
: >Lipofectin reagent.
: >
: >Sincerely
: >
: >Mathias

-- 
Antoine VEKRIS
University of Bordeaux II
Medical Biochemistry and Molecular Biology laboratory



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