Zero background/Multifunctional reporter vectors

riyer at rascal.med.harvard.edu riyer at rascal.med.harvard.edu
Sat Nov 13 22:42:13 EST 1993


Hi netters,

pow
    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  
f
eature 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 wel
l
 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 bee
n
 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 r
e
sulted 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 5-pr
i
me 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.   

Ravi



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