Conditional Ablation and Mutation of Nonmuscle Myosins

非肌肉肌球蛋白的条件性消融和突变

• 批准号: 7594413
• 负责人: ROBERT ADELSTEIN
• 金额: $ 29.96万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7594413
• 关键词: Ablation; Adult; Amino Acids; Animal Model; Cardiac; Defect; Development; Dilated Cardiomyopathy; Embryo; Etiology; Exons; Genes; Genotype; Glomerulonephritis; Heart; Human; Introns; Molecular; Mus; Mutation; Myosin ATPase; Myosin Heavy Chains; Myosin Type II; Neomycin; Pathogenesis; Phenotype; Point Mutation; Protein Isoforms; Proteins; Purpose; Reporting; Screening procedure; Syndrome; Thrombocytopenia; day; deafness; design; embryonic stem cell; homologous recombination; human disease; insight; mouse model; research study; restriction enzyme

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.

到目前为止，在椎骨中已经鉴定出三种非肌肉肌球蛋白重链（NMHC）II蛋白的同工型：II-A，II-B和II-C。 据报道，MYH9基因Gencoding在NMHC II-A中的单个氨基酸突变会引起人类的血小板减少症，以及其他缺陷的不同组合，例如肾小球肾炎和耳聋。 人和小鼠非肌中肌球蛋白II-A重链之间98％的氨基酸的身份表明，验证基因型 - 表现型连接并了解缺陷的分子发病机理可能是合适的动物模型。 我们正在MyH9基因的外显子31中生成具有点突变D1424N的小鼠。 我们已经将新霉素盒放在外显子31和32之间的内含子内。最终构建体已用几种限制酶验证并进行了测序。 具有正确序列的构建体被转染到ES细胞中，我们正在筛选出显示同源重组的克隆。 我们还表征了纯合和杂合性心脏特异性的条件非肌球蛋白II-B消融小鼠相对于心脏异常。 其中一些小鼠似乎显示出成年后发育的扩张性心肌病。