Conditional Ablation and Mutation of Nonmuscle Myosins

到目前为止，已在脊椎动物中鉴定出三种非肌肉肌球蛋白重链（NMHC）II蛋白亚型：II-A、II-B和II-C。 据报道，编码NMHC II-A的MYH 9基因中的单个氨基酸突变可导致人类血小板减少症，沿着其他缺陷的不同组合，如肾小球肾炎和耳聋。 人类和小鼠非肌肉肌球蛋白II-A重链之间98%的氨基酸的同一性表明，它可能是一个合适的动物模型，以验证基因型-表型连接，并了解缺陷的分子发病机制。 我们正在产生MYH 9基因外显子31中具有点突变D1424 N的小鼠。 我们将新霉素盒置于外显子31和32之间的内含子内。 用几种限制性酶验证最终构建体并测序。 将具有正确序列的构建体转染到ES细胞中，我们正在筛选显示同源重组的克隆。 我们还对纯合子和杂合子心脏特异性条件性非肌肉肌球蛋白II-B消融小鼠的心脏异常进行了表征。 这些小鼠中的一些似乎表现出扩张型心肌病，它们在成年后发展。

Until now, three isoforms of nonmuscle myosin heavy chain (NMHC) II protein have been identified in vertebrares: II-A, II-B, and II-C. Single amino acid mutations in the MYH9 gene gencoding for NMHC II-A have been reported to cause thrombocytopenia in humans along with different combinations of other defects such as glomerulonephritis and deafness. The identity of 98% of the amino acids between the human and mouse nonmuscle myosin II-A heavy chain suggested that it could be an appropriate animal model to verify a genotype-phenotype connection and to understand the molecular pathogenesis of the defects. We are generating mice with a point mutation, D1424N, in exon 31 of the MYH9 gene. We have placed the neomycin cassette inside the intron between exons 31 and 32. The final construct was verified with several restriction enzymes and sequenced. The construct with the correct sequence was transfected into ES cells and we are screening for clone(s) showing homologous recombination. We are also characterizing homozygous and heterozygous cardiac-specific conditional nonmuscle myosin II-B ablated mice with respect to abnormalities in the heart. Some of these mice appear to show a dilated cardiomyopathy that they develop as adults.