Conditional Ablation and Mutation of Nonmuscle Myosins

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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.