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 costeffective 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.

A collection of genetic mouse lines and related tools for inducible and reversible intersectional misexpression

“…Another problem that initially encumbered Tet systems was that the TRE DNA sequences can be inactivated through epigenetic mechanisms, rendering them unreliable unless de-repressed by additional genetic modules (Godecke et al, 2017). Fortunately, this drawback was overcome when a systematic screen identified a locus in the mouse genome that does not show this inactivation (Zeng et al, 2008). Indeed, Tigre (also known as Igs7) is an intergenic region on mouse chromosome 9 that provides a safe harbor from which TRE-dependent expression can be tightly regulated (hence the name).…”

Simultaneous mesoscopic Ca2+ imaging and fMRI: Neuroimaging spanning spatiotemporal scales

“…Calcium imaging was performed through the intact skull on transgenic mice expressing GCaMP6f in cortical excitatory neurons (Slc17a7-Cre/Camk2a-tTA/Ai93 mice, Methods). [19][20][21][22] However, it should be emphasized that the methods described here are suitable for any mouse line with a sufficiently bright optical signal (we have tested: Emx/Camk/TeTG6f; Slc17a7/Camk/TeTG6f; Slc17a7/Ai162; Vip/Ai162, and Som/Ai162 lines, data not shown).…”

“…We report data from a firstgeneration TIGRE (genomic locus) line crossed with reporter lines controlled by Cre recombinase (promoter) with a tetracycline-regulated transcriptional trans-activator (tTA) for amplification. [19][20][21][22] For detailed expression level data, refer to: http://connectivity.brainmap.org/transgenic;http://www.alleninstitute.org/what-we-do/brainscience/research/productstools/. 19 More specifically, we used Ai93 mice (or TIT2L-GCaMP6f, TIGRE -Insulators -TRE2 promoter -LoxPStop1LoxP -GFP CalModulin fusion Protein 6 fast) crossed to CaMK2a-tTA mice (CalModulin dependent protein Kinase 2 alpha) driven by Slc17a7 (or Vglut1) -IRES (internal ribosome entry site) 2 -Cre promotor mice, all purchased from Jackson Labs (JAX stock numbers: 024103, 003010 and 023517) and bred in-house.…”

What a Fish Can Learn from a Mouse: Principles and Strategies for Modeling Human Cancer in Mice

“…This application has been shown for animal models of atherosclerosis. 49 There are numerous methods by which this system could model mutations in cancer. For example, tumor suppressor genes could be repressed until the onset of a lesion and then reexpressed after tumor formation to determine their relative contribution to continued tumor progression, metastasis, or angiogenesis.…”