An Inducible and Reversible Mouse Genetic Rescue System

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.

Transgenic Mice for Intersectional Targeting of Neural Sensors and Effectors with High Specificity and Performance

“…Previous work showed that TRE-driven genes targeted (via a retrovirus) to the mouse TIGRE locus, situated on Chromosome 9 between the AB124611 (HIDE1) and Carml loci, could be expressed in most tissues in the presence of tTA (Zeng et al, 2008). However, targeting of TRE driven transgenic cassette to the Rosa26 locus resulted in silencing or mosaicism of the transgene expression (Tasic et al, 2012).…”

“…To complement existing Cre driver lines, we focused on the intersection of Cre with another recombinase or with a transcriptional activator. In addition, we built and validated a new docking site in a permissive genomic locus, the TIGRE locus (Zeng et al, 2008), which supports repeated targeting. By introducing a tTA-based transcriptional amplification approach to the TIGRE locus, all reporter lines doubly regulated by Cre and tTA drove robust expression of sensors and effectors at levels substantially higher than those in comparable Rosa-CAG based reporters.…”

Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization

“…The BFP/mCherry FUCCI reporter (pEN435) was assembled by serial modification of the base vector vector pFNF (Addgene #22687) using Gibson assembly with the following templates: hGeminin and mCherry-Cdt1 were PCR amplified from pRetroX-S2G2M and pRetroX-G1-Red (Clonetech); tagBFP cDNA from pHR-Tet3G-2A-BFP (Kind gift from Stanley Qi); CAGGS promoter and puroR are of the same source as pEN113; homology arms to the Tigre locus (Zeng et al, 2008) were PCR amplified from E14Tg2A genomic DNA (1kb each).…”

Beyond vascular inflammation—recent advances in understanding atherosclerosis

“…This idea has partially been based on the observation that statins – potent anti-inflammatory drugs developed to lower cholesterol by blocking endogenous cholesterol synthesis – resulted in regression of atherosclerotic plaques in individuals with coronary heart disease 66 . Likewise, inducible reconstitution of ApoE in mice expressing a hydromorphic ApoE allele or that lack ApoE demonstrated diminished atherosclerotic plaques and foam cell content 67, 68 . On the other hand, aggravation of atherosclerosis is associated with increased accumulation of macrophages, which possibly lost their ability to leave the plaque.…”