Muscle Fiber Type-Predominant Promoter Activity in Lentiviral-Mediated Transgenic Mouse

Abstract

Variations in gene promoter/enhancer activity in different muscle fiber types after gene transduction was noticed previously, but poorly analyzed. The murine stem cell virus (MSCV) promoter drives strong, stable gene expression in hematopoietic stem cells and several other cells, including cerebellar Purkinje cells, but it has not been studied in muscle. We injected a lentiviral vector carrying an MSCV-EGFP cassette (LvMSCV-EGFP) into tibialis anterior muscles and observed strong EGFP expression in muscle fibers, primary cultured myoblasts, and myotubes isolated from injected muscles. We also generated lentiviral-mediated transgenic mice carrying the MSCV-EGFP cassette and detected transgene expression in striated muscles. LvMSCV-EGFP transgenic mice showed fiber type-dependent variations in expression: highest in types I and IIA, intermediate in type IID/X, and lowest in type IIB fibers. The soleus and diaphragm muscles, consisting mainly of types I and IIA, are most severely affected in the mdx mouse model of muscular dystrophy. Further analysis of this promoter may have the potential to achieve certain gene expression in severely affected muscles of mdx mice. The Lv-mediated transgenic mouse may prove a useful tool for assessing the enhancer/promoter activities of a variety of different regulatory cassettes.

Defective excitation-contraction coupling is partially responsible for impaired contractility in hindlimb muscles of Stac3 knockout mice

“…Cross-sections of 8 μm thick were cut and mounted on Fisher Superfrost Plus slides (Thermo Fisher Scientific). Myosin-ATPase staining was performed as described 37 . Briefly, muscle sections were pre-incubated for 15 min in sodium barbital buffer containing 20 mM sodium barbital and 36 mM CaCl 2 at pH 10.2 and then incubated for 45 min in sodium barbital buffer containing 3.5 mM ATP, 20 mM sodium barbital, and 18 mM CaCl 2 at pH 9.4.…”

“…The NADH-TR staining was performed as previously described 37 . Briefly, muscle sections were incubated at 37 °C for 10 min and then with 1 mg/ml nitroblue tetrazolium (Sigma-Aldrich) and 0.4 mg/ml β-NADH (Sigma-Aldrich) in 50 mM Tris-HCl buffer, pH 7.3, at 37 °C for 30 min.…”

Supplementation with a selective amino acid formula ameliorates muscular dystrophy in mdx mice

“…Myofibrillar ATPase staining with pre-incubation at pH 4.3 was used to identify fibre types IIa, IIx and IIb in TA and VM muscles. ATPase (pH 4.3) staining was performed as described in recent publications 77 – 79 . After staining, the sections were washed with several changes of tap water, dehydrated with ethanol, cleared in xylene, and mounted in glycerol-gelatin aqueous slide mounting medium (Sigma-Aldrich, USA).…”

The SH3 and cysteine-rich domain 3 (Stac3) gene is important to growth, fiber composition, and calcium release from the sarcoplasmic reticulum in postnatal skeletal muscle

“…Myosin-ATPase and NADH-TR staining of the EDL and SOL sections prepared above were conducted as described previously [ 12 ]. Briefly, for myosin-ATPase staining, muscle sections were pre-incubated for 5 min in a sodium barbital buffer containing 20 mM sodium barbital and 36 mM CaCl 2 at pH 4.21 and then incubated for 55 min in a sodium barbital buffer containing 3.5 mM ATP, 20 mM sodium barbital, and 18 mM CaCl 2 at pH 9.45.…”