Inducible Cre transgenic mouse strain for skeletal muscle-specific gene targeting

Abstract

Background: The use of the Cre/loxP system for gene targeting has been proven to be a powerful tool for understanding gene function. The purpose of this study was to create and characterize an inducible, skeletal muscle-specific Cre transgenic mouse strain.

Methods: To achieve skeletal muscle-specific expression, the human α-skeletal actin promoter was used to drive expression of a chimeric Cre recombinase containing two mutated estrogen receptor ligand-binding domains.

Results: Western blot analysis, PCR and β-galactosidase staining confirmed that Cre-mediated recombination was restricted to limb and craniofacial skeletal muscles only after tamoxifen administration.

Conclusions: A transgenic mouse was created that allows inducible, gene targeting of floxed genes in adult skeletal muscle of different developmental origins. This new mouse will be of great utility to the skeletal muscle community.

Figure 1

A schematic of the HSA-MCM transgene. The promoter and first exon (−2,000 to +239 relative to the transcription start site) of the human α-skeletal actin (HSA) gene drives expression of the MerCreMer (MCM) gene which harbors a mutated estrogen receptor (Mer) ligand-binding domain on each terminus of the Cre recombinase gene. 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.

Figure 2

Skeletal muscle-specific expression of MerCreMer (MCM) protein. Western blot analysis of different muscle and nonmuscle tissues from the human α-skeletal actin (HSA)-MCM transgenic strain detected the MCM protein (112 kDa) only in skeletal muscle (Esop, esophagus; Gstn, gastrocnemius; Pln, plantaris; Sol, soleus; EDL, extensor digitorum longus; EOM, extraocular muscle; Mastr, masseter; Tong, tongue; Quad, quadriceps; TA, tibialis anterior; Diaph, diaphragm) and not in the heart (Hrt), samples containing smooth muscle (Stom, stomach; S. Int, small intestine) or nonmuscle tissue (Lung; Panc, pancreas; Liver; Brain; Fat; Spln, spleen; Kdny, kidney). Both glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and tubulin, γ1 (TUBG1) were used as loading controls.

Figure 3

PCR analysis of inducible, skeletal muscle-specific, Cre-mediated recombination. (A) Schematic of the lacZ reporter gene showing the floxed (flanking loxP sites, solid arrowheads) STOP cassette prevents expression of the downstream lacZ cDNA. Following tamoxifen administration, a Cre-mediated recombination event resulted in deletion of the STOP cassette, thereby allowing expression of the lacZ cDNA. (B) Qualitative PCR analysis of genomic DNA shows Cre-mediated recombination (deleted, 0.4-kb band) occurred only in skeletal muscle samples (Gstn, gastrocnemius; Pln, plantaris; Sol, soleus; TA, tibialis anterior; Diaph, diaphragm) and not in the heart (Hrt) following tamoxifen (t) administration, with no recombination (floxed, 3.2-kb band) in vehicle-treated (v) samples. The positions of the PCR primers are indicated by half-arrows.

Figure 4

Skeletal muscle-specific recombination as assessed by β-galactosidase activity. The HSA-MCM strain was bred to a lacZ reporter mouse to visually assess tamoxifen-induced recombination. (A) consistent with the Western blot analysis and PCR results, strong β-galactosidase expression (blue precipitate) was observed only in skeletal muscle (Gstn, gastrocnemius; Pln, plantaris; Sol, soleus; TA, tibialis anterior; EDL, extensor digitorum longus; Diaph, diaphragm; EOM, extraocular muscle) and not in the heart (Hrt) or liver following tamoxifen administration. (B) When we used a second lacZ reporter mouse (that contained a nuclear localization signal), β-galactosidase-positive, “blue” myonuclei were observed in Gstn skeletal muscle after tamoxifen treatment, but not in vehicle-treated Gstn. Inset shows enlarged image of labeled nuclei that reside within the muscle fiber.