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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 40008967

~I~HE COUNCIL 1~O1~ TOBACCO I~ESEARCH - U.S.A., IN0.~.. S~PPOP.~INO BXO~DI~.t. |~V~STIOATIO~ 900 THIRD ~V.~NUE (212) 421-83~5 GBORGE A. HASHIM~ PH.D. P~~Y ~PLICA~ON BV~UA~ON ":" Date: FEB. I~, 1995 Reviewer 1: DR. ~EN~.N Reviewer 2: DR. JOKLIK RE: Prelimin~--y Application Number: 5582 Applicant's Name and Degree(s): Institu.tion: Title: DAN CANAANI, PH.D. TEL AVIV UNIVERSITY A NATURAL OCCURING FUMAN #~I'ISENSE RNA- A REPRESSOR OF CELLULAR P~IORESISTANCE 1. Please review this application and indicate your scoring below. -. A return envelope is provided for your convenience. 2. We expect the top 30% of Preliminary Applications will be encouraged to submit Full Applications. Top 10% I~1 Top 40% Top 20% I~ Top 50%. Top 30% ~l B~Iow ~0% Reviewer's comments: Copi~to: Drs. Eisenbcrg, Ford, pa-vev2.doe r~v07.11.94 Signature: 40008988