Generation of mice carrying a <i>Mstn</i> point mutation rendering the propeptide resistant to cleavage by BMP-1/TLD proteases.

<p>(a) Gene targeting strategy. Black and stippled boxes represent coding exons for the propeptide and C-terminal domain, respectively. The location of the point mutation (D76A) is denoted by an asterisk, and LoxP sites are denoted by triangles. Removal of the neo cassette using EIIa-cre mice resulted in <i>Mstn</i> alleles containing a single LoxP site (in intron 1) either with (<i>Mstn^D76A</i>) or without (<i>Mstn^LoxP</i>) the point mutation. (b) Northern analysis of <i>Mstn</i> RNA expression in mutant mice. Muscle RNA isolated from <i>Mstn^−/−</i>, wild type, and <i>Mstn^D76A/D76A</i> mice was electrophoresed, blotted, and hybridized with a <i>Mstn</i> probe. The blots were re-hybridized with a probe for the S26 ribosomal protein to control for loading. (c) Analysis of myostatin protein in mutant mice. Hydroxylapatite-bound serum samples isolated from wild type, <i>Mstn^−/−</i>, and <i>Mstn^D76A/D76A</i> mice were electrophoresed, blotted, and probed with antiserum directed against either the C-terminal domain <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001628#pone.0001628-McPherron1" target="_blank">[2]</a> or the propeptide <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001628#pone.0001628-Lee3" target="_blank">[15]</a>. In each gel, the first lane contains purified myostatin latent complex isolated from Chinese hamster ovary cells <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001628#pone.0001628-Lee3" target="_blank">[15]</a>.