Establishment of an autocatalytic conditional mammalian system for expression of stringently regulated genes

Analysis of the biological roles and regulation of isolated genes in mammalian cells often requires inducible expression systems, particularly when the studied gene product is essential for viability and/or cytotoxic upon overexpression. Notable among these systems is the LAP267-regulated expression...

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Bibliographic Details
Published in:Nucleic acids research 1995-01, Vol.23 (2), p.307-309
Main Authors: Dotan, Iris, Kopatz, Idit, Naiman, Tova, Perelman, Boris, Dafni, Naomi, Canaani, Dan
Format: Article
Language:English
Subjects:
3T3 Cells
Actins - genetics
Animals
Catalysis
Chloramphenicol O-Acetyltransferase - genetics
Gene Expression Regulation
herpes simplex virus
Kinetics
Lac Operon
Mice
Promoter Regions, Genetic
Repressor Proteins - genetics
RNA, Messenger - metabolism
Simian virus 40 - genetics
Trans-Activators - biosynthesis
Trans-Activators - genetics
Transfection
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Summary:Analysis of the biological roles and regulation of isolated genes in mammalian cells often requires inducible expression systems, particularly when the studied gene product is essential for viability and/or cytotoxic upon overexpression. Notable among these systems is the LAP267-regulated expression system developed by the groups of A. Levine and T. Shenk. It is based on converting the lacI repressor into a mammalian transcriptional activator protein (LAP267) by inserting the herpes simplex virus VP16 transactivator domain after amino acid 267 of the lac repressor protein, and including a nuclear localization signal from SV40 large tumor antigen. The insertion generates a temperature-sensitive transactivator which induces expression from an enhancerless simian virus 40 early promoter linked to lac operators at 32 degree C, but not at 37 or 39.5 degree C. LAP267 activity can be rescued at the nonpermissive temperatures by IPTG addition. We report the generation and characterization of mouse NIH 3T3 cell lines expressing the LAP267 transcriptional activator conditionally from the IPTG inducible LAP267-responsive L7 promoter (ILAP267). We compare the conditional system to the former constitutive one (CLAP267) and demonstrate that the autocatalytic conditional expression system is superior to the former in its absolute level of activity, as well as its faster kinetics of induction.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/23.2.307