Abstract
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.
Highlights
In the post-genome era, a major challenge is deciphering the function of thousands of newly identified genes
As a proof of concept, we report the characterization of an iKO of the apolipoprotein E gene (ApoE iKO)
On examination of aortic atherosclerosis in the ApoE iKO mice we found that Dox treatment before the onset of atherosclerotic lesions completely prevented lesion formation and Dox treatment after extensive lesions had already formed resulted in regression of the lesions
Summary
In the post-genome era, a major challenge is deciphering the function of thousands of newly identified genes. A common problem with these methods stems from the fact that the gene of interest is usually mutated throughout the animal’s life. Conditional knockout and gene expression technologies, such as the Cre/loxmediated tissue-specific knockout [1] and the tetracycline (Tet) regulated transcriptional activation system [2], can regulate gene expression in a more spatially and temporally controlled fashion. These technologies are often laborious to establish and the results are frequently variable
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