The lac operator-repressor system is functional in the mouse

Abstract

We report the successful transfer of a fully functional lac operator-repressor gene regulatory system to the mouse. The key component is a lac repressor transgene that resembles a typical mammalian gene both in codon usage and structure and expresses functional levels of repressor protein in the animal. We used the repressor to regulate the expression of a mammalian reporter gene consisting of the tyrosinase promoter embedded with three short lac operator sequences and the tyrosinase coding sequence. Pigmentation of the mouse was controlled by the interaction of the lac repressor with the regulatable Tyrosinase transgene in a manner that was fully reversible by the lactose analog IPTG. Direct control of mammalian promoters by the lac repressor provides tight, reversible regulation, predictable levels of de-repressed expression, and the promise of reversible control of the endogenous genome. The mouse is the most widely used experimental animal to model mammalian development and disease. In recent years, technological advances in genetic manipulation of the mouse genome have made it even more valuable as a research tool, and a great deal of information has been learned from conventional knockout and transgenic experiments. Despite this, it can be difficult to draw definite conclusions from these studies. Embryonic lethality might preclude the possibility of analyzing adult phenotype, or activation of compensatory systems confuse the analysis. Tight, reversible control of gene expression would greatly broaden the possible experimental questions that can be addressed. To gain such control, the ideal system would enable a target gene to be switched on and off repeatedly, without affecting the expression of nontargeted genes. With this ideal in mind, we have developed the lac repressor regulatory system for use in the mouse.The lac operon of Escherichia coli consists of a set of genes coordinately regulated by lactose (Jacob and Monod 1961). The regulatory components of the system are the lac repressor and its DNA-binding sequence, the lac operator. In the absence of lactose, lac repressor occupies the lac operators and prevents transcription. Lactose causes a conformational change in the repressor, and it vacates the operators, allowing RNA polymerase to gain access to the promoter and initiate transcription. Hu and Davidson (1987) were the first to use lac elements to control reporter-gene expression reversibly in mammalian cells. Their results were extended by Figge et al. (1988), who demonstrated that the lac repressor was able to gain access to the mammalian chromosome to regulate a stably integrated reporter gene.Our goal has been to adapt the lac regulatory system to control gene expression in the mouse. In an earlier paper, we reported that transgenes containing the bacterial-coding sequence for the lac repressor downstream of the ␤-actin promoter were heavily methylated and only transcribed in the testis of transgenic mice (Scrable and Stambrook 1997). Methylation and silencing in mice also was observed by W...

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A novel model to identify interaction partners of the PTEN tumor suppressor gene in human bladder cancer

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