Selective expression of Cre recombinase in skeletal muscle fibers

It is often difficult to study the function of genes that are widely expressed, because their critical function in one tissue can complicate or preclude study of their role in other tissues. Several lines of evidence support the idea that neuregulin (NRG) may be a synaptic signal that activates expression of certain genes in muscle nuclei positioned near the synaptic site (Burden, 1998). Since mice lacking NRG, or its receptors (ErbB2 or ErbB4) die, owing to a failure of cardiac differentiation (Lee et al., 1995; Gasmann et al., 1995; Meyer and Birchmeier, 1995), ;four days prior to neuromuscular synapse formation, it remains unclear whether NRG-mediated signaling is indeed required for synapse-specific gene expression. To determine the role of NRG-mediated signaling in neuromuscular synapse formation and to circumvent the problems associated with the early requirement for NRG-mediated signaling in early development, we sought to inactivate erbB genes selectively in skeletal muscle cells, using the Cre/loxP recombination system (Gu et al., 1994). These experiments require producing mice that selectively express Cre recombinase in skeletal muscle cells and crossing these mice with mice that carry an erbB target gene containing loxP sites flanking a critical exon. Although transgenic mice expressing Cre in heart and skeletal muscle cells have been described (Bruning et al., 1998), we are unaware of mice that express Cre selectively in skeletal muscle cells. Because the myosin light chain 1f (mlc1f) gene is expressed strongly and selectively in skeletal muscle cells, beginning early during muscle development (Lyons et al., 1990), we chose to introduce cre into the mlc1f locus by homologous recombination. Two mouse lines were generated using the targeting strategy illustrated in Fig. 1. mlcis identical to mlcexcept that mlc carries an extrinsic polyA signal, which we reasoned might alter Cre expression. We used Southern blots to show that cre or creA had been introduced into the mlc1f locus (Fig. 1B). The targeted mouse lines, mlcand mlc, were crossed to C57BL/6 mice and subsequently maintained on a C57BL/6 background. Homozygous mlcand mlcmice are viable and healthy. The specificity of Cre activity in mlc and mlc mice was determined by measuring excision of a loxPflanked (floxed) sequence in a target gene. mlcand mlcmice were crossed with mice carrying a floxed erbB4 allele (erbB4; R. Klein, personal communication), and the excision of the floxed DNA was determined by Southern hybridization (Fig. 2). Since muscle tissue contains endothelial cells associated with blood vessels, Schwann cells associated with axons, satellite cells, adipose cells and fibroblasts, in addition to skeletal muscle fibers, not all nuclei in skeletal muscle tissue are contained within muscle cells. Indeed, histological studies indicate that ;45% of the nuclei within skeletal muscle tissue are contained within muscle fibers (Schmalbruch and Hellhammer, 1977). Therefore, we reasoned that selective and efficient deletion of floxed erbB4 sequences in nuclei from skeletal muscle cells would result in ;45% deletion of floxed erbB4 sequences in DNA isolated from skeletal muscle tissue. We found that floxed erbB4 sequences were deleted selectively in skeletal muscle tissue, including gastrocnemius, tibialis anterior and biceps femoris muscles, and not in