Two forms of the nonmuscle myosin heavy chain (NMHCII-A and NMHCII-B) have been cloned in this laboratory and shown to be the products of two different genes. Recent work on the NMHCII-B deficient mouse (Tullio et al., Proc. Natl. Acad. Sci. USA 94, 12407, 1997) has elucidated defects in the heart and brain which lead to embryonic lethality. The laboratory is also characterizing an NMHCII-B mouse which cannot splice an alternative exon into the myosin heavy chain near the ATP-binding area and also has decreased expression of noninserted NMHCII-B. The current project is aimed at creating a NMHCII-A deficient mouse. Two different constructs have been generated in order to interrupt the NMHCII-A gene and place the neomycin resistance gene in one of the initial exons. In one of the constructs, the neomycin resistance gene is flanked by loxP sites which would permit its excision in the mouse embryo after mating with a Cre recombinase bearing mouse. Blastocyst injection and production of chimeric mice have been carried out. Chimeras have been mated to C57Bl/6 and 219SVJ mice to produce mice on mixed and uniform genetic backgrounds respectively. The resulting heterozygous mice are fertile but have not yet produced homozygous offspring. We are presently analyzing embryos in order to determine the possible date of embryonic lethality. - nonmuscle myosin II-A; embryonic stem cells; loxP; Cre recombinase