A novel tetracycline-responsive transgenic mouse strain for skeletal muscle-specific gene expression

Fig. 1. A schematic of the HSA-rtTA transgene. The promoter and first exon (− 2,000 to + 239 relative to the transcription start site) of the human skeletal muscle α-actin (HSA) gene regulates expression of an optimized reverse tetracycline transactivator (rtTA) gene which has been reported to be sevenfold more active and 100-fold more doxycycline sensitive than the original Tet-On system [8]. The β-globin intron ΙΙ (BGI) and poly(A) tail were incorporated into the transgene to ensure proper splicing and transcript stability, respectively. The positions of the PCR primers used for genotyping are indicated by half-arrows

Fig. 2. Skeletal muscle-specific expression of rtTA. PCR analysis of rtTA mRNA expression of different tissues from the HSA-GFP transgenic mouse showed high levels of expression in skeletal muscle (gastrocnemius, soleus, plantaris, tibialis anterior (TA), and extensor digitorum longus (EDL)), modest expression in the diaphragm, very low expression in the heart, and not detectable in non-muscle tissue (brain, fat, lung, liver, stomach, spleen, and kidney)

Fig. 3. HSA-rtTA transgene drives robust myofiber expression of tetracycline-responsive H2B-GFP transgene. a Representative single fiber images of hind limb muscles taken from HSA-GFP mice (n = 4) treated with doxycycline. Single fiber images show robust myonuclear GFP expression in muscles composed of slow- and fast-twitch fibers. b Quantification of GFP+ myonuclei of single fibers from hind limb skeletal muscles (plantaris, gastrocnemius, soleus, tibialis anterior (TA), and extensor digitorum longus (EDL)) of HSA-GFP mice showed greater than 95% of all DAPI+ myonuclei within a fiber were GFP+. The gray bar represents the average percentage of GFP-positive myonuclei (n = 4) for each muscle

Fig. 4. GFP expression is specific to myonuclei in HSA-GFP mice. Representative muscle cross-section images of the plantaris and soleus muscles from HSA-GFP mice treated with doxycycline (n = 3). As indicated by white arrows, DAPI+/GFP+ myonuclei (green) did not show co-localization with DAPI+/Pax7+ satellite cells (pink). These results confirm that the HSA-rtTA transgene is able to drive myofiber-specific expression of a tetracycline-response gene

Fig. 5. Rapid GFP labeling of myonuclei. a Representative single fiber images of plantaris and diaphragm muscles taken from HSA-GFP mice (n = 2) treated with doxycycline for 24 h. b Quantification of GFP+ myonuclei showed ~ 90% of myonuclei were GFP-positive in myofibers from the plantaris with 60% of myonuclei GFP-positive in myofibers isolated from the diaphragm