pBK-CMV vs pcDNA3.1

Bernard Murray bpmurray*STUFFER* at socrates.ucsf.edu
Tue Jun 2 18:18:50 EST 1998


In article <6l0b97$rs8 at mserv1.dl.ac.uk>, Nico Dantuma
<nico.dantuma at mtc.ki.se> wrote:

> >I cloned an endocytic receptor in pBK-CMV and tried to monitor uptake of
> >fluorescently labeled ligands. The receptor was cloned in the MCS of
> >pBK-CMV. However, I didn't observe any uptake. I thought that the upstream
> >ATG of the beta-galactosidase was strongly inhibiting expression of the
> >receptor. Therefore I subcloned the insert in pcDNA3, which essentially
> >the same but lacks the beta-galactosidase. This time the staining of the
> >endocytic compartment by the fluorescent labeled ligand was very strong.
> >If you want a significant expression I would advise you to use pcDNA3 or
> >pBK-CMV from which the beta-gal 5' end is removed (Nhe1 site can be used
> >for this). Collegues used pBK-CMV for a signaling receptor (beta-gal ATG
> >was not removed) and could monitor signaling. Yet, in this case a limited
> >number of receptors at the cell surface can do the job. Because of my
> >experience I would discourage people to use a ZAP expression library
> >(Stratagene): in retrospect, I can tell that it would have been impossible
> >to pick up my receptor by screening our ZAP expression library for
> >endocytic uptake of the ligand.
> >Nico
> >
> >>Does anyone use pBK-CMV (Stratagene) to express your gene? Is there any
> >>difference from pcDNA3.1 (InVitrogen)? Please let me know.
> >>Jun-ichi Aikawa, Ph.D.

I had a similar problem to Nico in that I found it difficult to
insert a particular cDNA in pBK-CMV and transform bacteria
due to leaky expression (even in a lacIq strain in conditions
where lacP should have been suppressed).  When I removed lacP
I had no problem making the construct.  Stratagene acknowledge
this in the later versions of the pBK-CMV instructions and
suggest excising the NheI/SpeI fragment containing LacP
(this pair of enzymes gives cohesive ends - nice and easy).
     I think they've basically tried to cram far too much into
one vector.  The saving grace is that they use the bacterial
kan promoter to drive neo and so don't have to make the vector
even larger by adding an amp cassette.
     I have no experience with pcDNA3 or 3.1 but had poor results
with pcDNAI.  My favourite is Promega's pCIneo (in a side-by-side
comparison it was better than pBK-CMV) but YMMV, of course.
     Bottom line: try 'em and see...

     Bernard

[Disclaimer: No affiliation with Stratagene, Promega, Invitrogen etc.]
-- 
Bernard Murray, PhD
Dept. Cell. Mol. Pharmacol., UCSF, San Francisco, USA



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