Plasmid Construction .. Will this work?

jmh at canctr.mc.duke.edu jmh at canctr.mc.duke.edu
Wed Jul 21 16:27:08 EST 1993


In article <1993Jul14.164924.5520 at midway.uchicago.edu>, sjchmura at kimbark.uchicago.edu (steven joseph chmura) writes:
>I am trying to contruct a single plasmid from 2.
>I have a CMV-rabbit globin-Gene#1 plasmid
>My second plasmid is a CMV-B-gal report gene.
>
>There is a cut site between the CMV-Rabbit which has the same overhangs
>as the cut site for B-gal.
>
>Can I simply remove the B-gal gene(assuming it has no intrinsic stop loops -
>but then agin should that matter since it is eucariotic machinery?)an d
>insert it between the CMV promoter and the rabbit exon-intron-exon 
>sequence?
>
>Will the message still be transctribed all the way down to Gene#1?
>
>Thanks.....
>
>-- 
>________________________________________________________________________________Steven Chmura				University of Chicago Medical School(M1)
>  "Given enough time, the impossible becomes probable, and the probable
>	inevitable.."  -George Wald, "On the Origins of Life"

Steve:

If transcription in a eukaryotic cell is all you are interested in then your
construction should result in a bicsitonic message (i.e. a message that encodes
two proteins).  However, if co-translation is the goal you're in for a heap of
trouble.  Eukaryotic ribosomes will not initiate internally within a message,
so if Bgal is the 3' cDNA it will not be translated (only globin will be).  If
you need to co-express both cDNAs you have two alternatives:

	1. Construct two independent vectors that each carry a single cDNA and
co-transfect them into cells (usually one would use two distinct promoters so
that you don't run into promoter competition).

	2. You could clone two independent cDNAs into the same plasmid with
each under the transcriptional control of a distinct promoter (again for the
reason outlined above).

Hope this has been helpful.
Good luck.
********************************************
Jon Horowitz 
Section of Cell Growth, Regulation and Oncogenesis
Department of Microbiology
Duke University Medical Center
jmh at canctr.mc.duke.edu
********************************************



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