We have successfully expressed full length wild type and mutated NM II proteins using the Sf9-baculovirus system. We also expressed two chimeric NM II proteins and GFP-NM II fusion proteins. We find that: I) although full length NM II-A, II-B and II-C exhibit biochemical differences, the morphology of the filaments determined by negative-staining electron microscopy (EM) is essentially indistinguishable among the three paralogs. In the presence of ATP all three paralogs display a similar ability to adopt the 10S compact conformation. II) EM images of chimeric molecules show that the tail domains of the paralogs are interchangeable in terms of filament formation and formation of the 10S compact conformation;III) In contrast to a previous report, the presence of point mutations in full length NM IIA proteins (N93K, D1424N, E1841K) causing human diseases has little or no obvious effects on filament formation;IV) GFP fused to NM II allows us to directly analyze in vitro motility by TIRF microscopy. We have identified a gene targeting locus (6 kb), in the region of exon 2 of the Myh9 gene, that displays an extremely high and repeatable frequency of HR in mouse embryonic stem (ES) cells (95% in this case vs 1-10% in most cases). To our knowledge this is the highest rate that has been reported to date. Our initial investigations indicated no evidence for a specific DNA sequence that is responsible for this high targeting efficiency since a gradual shortening of the homologous arms results in a corresponding reduction of targeting frequency. Further studies using different cell lines including mouse ES cells, induced pluripotent stem (iPS) cells and mouse embryonic fibroblasts (MEFs) showed that the GT efficiency at the same targeted site exhibits an order of ES>iPS>MEF cells, and that GT frequency gradually decreases with the shift of the targeted sites 3 from exon 2 to the intron between exon 2 and 3 and then to exon 3 in mouse ES cells. These results imply the influence of chromosome structure and possible epigenetic modification on GT efficiency. Additionally, our findings have important applications as the Myh9 locus can provide a safe harbor for transgene insertions in the absence of the influence of the integration site, allowing multiple transgenic lines to be more accurately compared. In our current study we took advantage of the high frequency of HR at the Myh9 locus by: 1) generating genetic replacement mouse models to study the isoform and domain specificity of, for example, nonmuscle myosin IIs (Zhang Y et al., Blood, 2012, 119:238-50). So far, at least 5 mouse models have been produced for these purposes. 2) Creating Myh9 related disease (Myh9-RD) mouse models which successfully mimic the Myh9-RD phenotype found in humans. 3) Obtaining high purity cardiomyocytes derived from mouse embryonic stem cells. To this end, a cassette encoding Puromycin resistance controlled by a cardiac-specific promoter was integrated into the Myh9 locus. 4) Integrating a shRNA expression cassette into this site for gene-specific knockdown. In each case, the high HR frequency facilitated isolation of the desired ES cell clones.
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