Nonmuscle myosin is an ubiquitous protein present in all eukaryotic cells. It plays a role in cell motility, in cytokinesis and in capping. This laboratory has isolated two different cDNAs from human and chicken tissues encoding nonmuscle myosin heavy chains (MHCs). The two different isoforms are called MHC-A and MHC-B and, in humans, are located on two different chromosomes. In order to understand the in vivo function of the MHC-B isoform, we carried out experiments to produce transgenic mice in which the gene encoding nonmuscle MHC-B would be incapable of producing the gene product. We used a targeting vector containing 6.7 kb of the nonmuscle MHC-B fragment that was cloned for a mouse genomic lambda phage library. 2 kb of a neomycin gene was inserted into exon 2. The linearized vector was electroporated into J1 embryonic stem cells and Southern blot analysis of 352 selected clones revealed homologous recombination in 6 clones. All of these clones were used for microinjection of a 3.5 day-old C57Bj6 blastocysts as well as aggregation of 8 cell embryos. We observed germline transmission in the agouti progeny. The heterozygous mice looked healthy and were fertile. They were mated to produce homozygous mice. At the present moment, of the 32 offspring examined (including 10-15 day embryos), we have found only 1 homozygous mouse which was dead following birth. Considering the small size of our litters (average of 5 pups) and the fact that we have not observed a live homozygous mouse, we hypothesize that lethality is occurring during mouse embryonic development. To address this question, we are examining the embryos at a different stage of development. In addition to checking the phenotype and genotype, we are also checking the expressed protein using Western blot analysis with specific antibodies.
