Reporter gene vectors

Cornelius Krasel krasel at alf.biochem.mpg.de
Tue Jan 11 06:58:10 EST 1994


riyer at rascal.med.harvard.edu wrote:
: I recd. so many requests from people who did not get my earlier post that I
: decided to save time by re-posting it. At the outset I would like to state
: that I am only a satisfied user of the above vectors and am not associated
: with the company that markets them.

And now here comes a readable version (with <80 chars/line) :-)

: Hi netters,  I would like to report the availability of a set of
: reporter gene vectors that have some distinct advantages over those
: currently in use for the purpose of analysis of eukaryotic promoters
: & enhancers. The vectors , SV40-Syncat, Syncat I, Syncat II,
: SV40-pFlash, pFlash I and pFlash II, are as their names suggest
: CAT & Luciferase (Luc) reporter gene vectors. What distinguishes
: them from other vectors in this category is that these vectors produce
: near-zero background. This feature is due to the fact, that they use a
: modified SV40-t-intron as the donor for the poly-adenylation signal.
: The wild type SV40-t-intron is the generic donor for this function
: and is widely used in a variety of reporter gene vectors, eg. pCAT-
: basic, pCAT-promoter, pGL-basic and pGL-promoter (Promega).
: However one little known fact about the wild-type-t-intron is that it
: contains cryptic enhancer sequences that produce significant
: background activity, thereby raising the threshold of sensitivity.
: Until now this feature was not a serious limitation since scientist
: were analysing strong promoters/enhancers that produced high
: signal to noise ratios. However the focus in the past couple of
: years has shifted to the analysis of weak promoters, or promoters
: that have very low basal transcription rates but are specifically
: induced to high levels by cytokines, or promoters that function only
: in cells that are very poorly transfectable. Analysis of such
: regulatory elements requires systems of high sensitivity as well as
: specificity. The Syncat & pFlash series of vectors provide precisely
: these capabilities, since they contain a t-intron polyA signal that has
: been modified to be devoid of cryptic enhancer activity.
: The 2nd. feature of these vectors is the choice of the
: heterologous promoter in Syncat II and pFlash II. These vectors use
: the well characterized HSV-tk TATA box containing minimal
: promoter situated immediately upstream of the reporter gene. The
: HSV-tk is a widely used basal promoter that has been documented
: to produce no spurious interactions with enhancers of interest. By
: contrast vectors like pCAT-promoter or pGL-promoter use the SV40
: promoter. The SV40 promoter contains an atypical TATA-box and
: more seriously has been documented to spuriously repress certain
: cytokine inducible enhancers (Benech, et.al.. J.Exp.Med. 1992,
: Vol. 176: 1115-1123). I should know, because I am one of the
: authors of that paper and this defect in the pCAT-promoter vector
: cost me 6 months of time and nearly resulted in our being scooped
: by a competing lab.  Other features of these vectors include a very
: versatile multiple cloning site upstream and downstream of the
: reporter gene cassette, flanked by T3 & T7 promoter sequences for
: direct sequencing and creation of unidirectionally deleted mutant
: libraries as well as f1 origin of replication for ssDNA recovery and
: site-directed mutagenesis. The combination of these features enabled
: me to perform all my DNA manipulations from cloning of a 5 kb
: genomic 5U flanking region upstream of the reporter gene ---to---
: creation of nested deletion mutant libraries followed by sequence
: verification ---to--- ssDNA-site-directed mutagenesis and
: transfection of each construct --- ALL IN THE SAME REPORTER
: GENE VECTOR I STARTED WITH. The time savings alone were
: upto 50%.  These vectors are available from SynapSys Corp. To
: get more info about them send E-mail to :- #
: #
: #
: #
: synapsys at world.std.com. #

Just wanted to note that I am not affiliated with this company either.
In fact, I have never worked with reporter genes :-)

--Cornelius.

--
/* Cornelius Krasel, Abt. Lohse, Genzentrum, D-82152 Martinsried, Germany */
/* email: krasel at alf.biochem.mpg.de                 fax: +49 89 8578 3975 */
/* "People are DNA's way of making more DNA." (R. Dawkins/anonymous)      */



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