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Mouse Genetic Models Core

Director

David Ornitz
Developmental Biology
314-362-3908
dornitz@wustl.edu

Associate Director

Fanxin Long
Orthopaedic Surgery
314-454-8795
longf@wudosis.wustl.edu

Mouse Model Database

Debabrata Patra
314-454-8823
patrad@wudosis.wustl.edu

Billing Administrator

Kami McGhee
Orthopaedic Surgery
314-747-5993
314-454-5900 - fax
mcgheek@wudosis.wustl.edu

Mouse line: Runx2-rtTA knockin mouse line

(to target osteoblast lineage)

A critical barrier to progress in bone research is the lack of tools that allow for gene manipulations in osteoprogenitors specifically in postnatal animals. To overcome this problem, we have developed a doxycycline-dependent system targeting osteoprogenitors. Specifically, we created a mouse strain (termed Runx2-rtTA) that expresses the reverse tetracycline-controlled transactivator (rtTA) from the Runx2 genomic locus; this mouse, when combined with the well-characterized tetO-Cre strain (Perl et al., 2002), directs Cre expression selectively in Runx2-positive cells in response to the tetracycline analog doxycycline (Dox). To generate the Runx2-rtTA mouse, we first modified a BAC (bacterial artificial chromosome) clone that spans the Runx2 locus (Children’s Hospital Oakland Research Institute) by employing the BAC recombination methods to replace the first exon of Runx2 with rtTA cDNA (Lee et al., 2001); the engineered BAC was then injected into the pro-nucleus of fertilized eggs to generate transgenic mice. The rtTA cDNA we used encodes an optimized rtTA (rtTA2S-M2) that exhibits minimal leakage or cell toxicity but maximal inducibility (Urlinger et al., 2000). Among 30 presumptive founder mice that were screened, one line was established to target Runx2-positive cells specifically in response to Dox.

To tested for specificity and inducibility of the Runx2-rtTA transgene, the Runx2-rtTA mouse was bred with the tetO-Cre and the R26-mT/mG mice to generate progenies harboring all three transgenes (Runx2-rtTA;tetO-Cre;R26-mT/mG) (Muzumdar et al., 2007) (Fig. 1A). The R26-mT/mG allele contains cDNA for membrane-tethered GFP (termed mG) proceeded by a loxP-flanked cDNA for membrane-tethered Tomato (mT), all within the Rosa26 genomic locus; this allele ubiquitously expresses Tomato (red) but switches to GFP in cells containing Cre activity. The triple transgenic mice were treated or not with Dox in the drinking water, either in utero through the pregnant dam or postnatally. To assay for Cre activity, frozen sections were prepared from the long bones, and examined for Tomato or GFP expression under a fluorescence microscope. No GFP was detected in any cells in the absence of Dox (Fig. 1C). With Dox, the triple transgenic mice elicited robust GFP specifically in cells known to express Runx2, following Dox treatment either in utero (Fig. 1B) or at one month of age (Fig.1D). Overall, we have established a Runx2-rtTA strain that can direct gene manipulations in Runx2-postitive cells in a Dox-dependent manner. We have successfully used the Runx2-rtTA mouse to induce Wnt7b expression in the postnatal skeleton. These mice exhibited excessive bone formation (Chen et al., 2014).


Citing the grant in publications:


“Washington University Musculoskeletal Research Center (NIH P30 AR057235)”