Generation of <i>Tnnt2</i>^+/⁻ mice.

<p>A. A targeting construct containing a neomycin resistance gene (<i>neo/zeo</i>) between <i>lox</i>P sites (triangles) was introduced into the murine <i>Tnnt2</i> locus by homologous recombination in murine ES cells, ablating the 3′ segment of the gene, including exon 14. ES cells were microinjected into mouse blastocysts to generate chimeras, which were bred with wildtype mice for germline transmission of the <i>Tnnt2</i> ablation. The neomycin resistance gene was excised using Cre-mediated excision by mating with EIIa-Cre recombinase mice. Horizontal arrows indicate PCR primers (F1, F2, R1, R2, R3) used for genotyping as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002642#s2" target="_blank">Materials and Methods</a>.” B. Genomic DNA from mice prior to Cre-mediated excision of the neomycin resistance gene was digested with <i>Hin</i>d III and Southern blotted with the probe indicated in panel A to demonstrate homologous recombination. A <i>Hin</i>d III restriction site in the neomycin resistance gene produced a smaller restriction product in the presence of homologous recombination. WT, wildtype; TK, thymidine kinase gene.