pBK-CMV vs pcDNA3.1
nico.dantuma at mtc.ki.se
Tue Jun 2 02:57:27 EST 1998
>Date: Tue, 2 Jun 1998 08:59:26 +0100
>To: jaikawa at UCSD.EDU (Jun-Ichi Aikawa)
>From: Nico Dantuma <nico.dantuma at mtc.ki.se>
>Subject: Re: pBK-CMV vs pcDNA3.1
>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.
>>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.
>>University of California, San Diego
Microbiology and Tumorbiology Center
S-171 77 Stockholm
Phone: +46 8 7286280; fax: +46 8 331399
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