Establishment of an autocatalyfic conditional.mammalian-system for " exPression stringently regula ted genes -Iris Dotan, Idit Kopa~z, Tova Naiman; Boris Perelman, Naomi Dafni and Dan Canaani* Dep~xtment of Bio¢hemlstry, Tel A~dv University, Rama't Avlv 6997g, Israel
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• NAR nat 23/214l~409 rob 05/01/95 Page
Establishment of an autocatalyfic
conditional.mammalian-system for "
exPression stringently regula ted genes
-Iris Dotan, Idit Kopa~z, Tova Naiman; Boris Perelman, Naomi
Dafni and Dan Canaani*
Dep~xtment of Bio¢hemlstry, Tel A~dv University, Rama't Avlv 6997g, Israel
Received November 3, 1994; Acccptc~l D~mb~r 2, 1994
=To whom correspondence should be add.tossed
Analysis of the biological roles and regulation of isolated genes in mammalian cells o.Rca
requires inducible expression syste~ns, particularly when the sty. died gene product is essential
.for vlabiliW and/or cytotoxi~ upon overexpression. :Notable among these systems i~
LAP26~-reEulated expression system developed by the groups ofA. Levlne and T~ Shenk
(I). :It is baaed on converting the la'c.I repr~ssor into a mammalian "transcriptiona/activator
protein (LA_P267) by irtsertRng the herpe~ simplex -¢-;_ms VP16 trans, ctivator domaha .~fter
amino acid 267 of the lac repressor protein, a_~d iricluding a nuclear l~calizatioa si_~tal from
S~,~40 large tum~)r antigen. The insertio~a generates a temperature-sensitive transac'tb,'ator
which induces express- ion from an enhaneertess simian ~dr:us 40 eaa-ly promoter .lhaked to
operators at 32°C, but not at 37¢C or 39.5°C. LA_P267 acti,,'i~" ca,a be rescued at the
nonpermissive temperatut:~s by IPT(~ atidition.
Previousb), the LAP267 gene ~,as transcribed offthe c.~n. rdmtb,'e hu .manJ3-a .c'dn. or
%aomegalovirus :lie promoter (1). The constiarl%'e production of the ~ransac~i~or might
represent a drawback. In ~he initial phase of the generation ofs~able transfect~.,m~s, as well as
in transient transfeetion, there is a massive ent-fy ofDNA moL~-ules into the fraction of
competent cells amohg the recipie~a~ population. Ix is possible "&erefore that transfection of a
to×ic.gerte under zb.e inducibl~ promoter imo con~kutlvcly LAP267-produciny, c,.*J, ls is
---.ccomp~aied, prior to inte_~.at:,ort, by an inducer-;-ndependent expression (le.~&) "-.v1"dch
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~I~HE COUNCIL 1~O1~ TOBACCO I~ESEARCH - U.S.A., IN0.~..
S~PPOP.~INO BXO~DI~.t. |~V~STIOATIO~
900 THIRD ~V.~NUE
GBORGE A. HASHIM~ PH.D.
P~~Y ~PLICA~ON BV~UA~ON
Date: FEB. I~, 1995
Reviewer 1: DR. ~EN~.N
RE: Prelimin~--y Application Number: 5582
Applicant's Name and Degree(s):
DAN CANAANI, PH.D.
TEL AVIV UNIVERSITY
A NATURAL OCCURING FUMAN #~I'ISENSE RNA-
A REPRESSOR OF CELLULAR P~IORESISTANCE
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