Inducible and reversible regulation of gene expression is a powerful approach for uncovering gene function. We have established a general method to efficiently produce reversible and inducible gene knockout and rescue in mice. In this system, which we named iKO, the target gene can be turned on and off at will by treating the mice with doxycycline. This method combines two genetically modified mouse lines: a) a KO line with a tetracycline-dependent transactivator replacing the endogenous target gene, and b) a line with a tetracycline-inducible cDNA of the target gene inserted into a tightly regulated (TIGRE) genomic locus, which provides for low basal expression and high inducibility. Such a locus occurs infrequently in the genome and we have developed a method to easily introduce genes into the TIGRE site of mouse embryonic stem (ES) cells by recombinase-mediated insertion. Both KO and TIGRE lines have been engineered for high-throughput, large-scale and cost-effective production of iKO mice. As a proof of concept, we have created iKO mice in the apolipoprotein E (ApoE) gene, which allows for sensitive and quantitative phenotypic analyses. The results demonstrated reversible switching of ApoE transcription, plasma cholesterol levels, and atherosclerosis progression and regression. The iKO system shows stringent regulation and is a versatile genetic system that can easily incorporate other techniques and adapt to a wide range of applications.
We describe a technology for the creation of inducible and reversible gene inactivation in mice. It combines two genetically modified mouse lines: a knock-out line with a tetracycline transactivator replacing the endogenous target gene, and a line in which a tetracycline-inducible cDNA of the target gene has been inserted into a specific genomic locus. A critical component of this system is the unique chromosomal loci we have identified and engineered that offer a platform for easy insertion of any gene of interest for tightly controlled expression. Because of its simple binary nature, allowing independent modification of each of the two components and possibility of use in a high-throughput mode, we believe that our system will be useful for multiple applications, such as introducing mutant or humanized form of the target gene as well as functional manipulating tools. We have applied this technology to the Apolipoprotein E (ApoE) gene and have demonstrated that: a) the expression of ApoE is strictly dependent on the presence of doxycycline, a tetracycline group antibiotic, in the mouse diet, b) in the absence of doxycycline (ApoE repressed) atherosclerotic plaques are formed, confirming the importance of ApoE in the process, and c) upon re-induction of ApoE in the animals with doxicyclin, atherosclerosis regressed.