EXPRESSION OF NONMUSCLE MYOSIN ISOFORMS IN EUKARYOTIC CELLS

非肌肉肌球蛋白重链的两种形式 (NMHCII-A和NMHCII-B)已在本实验室克隆 并被证明是两个不同基因的产物。近期工作 关于NMHCII-B缺陷小鼠(Tullio等人，Proc.娜塔莉。阿卡德。 SCI。美国94、12407、1997年)阐明了心脏的缺陷和 导致胚胎死亡的大脑。心脏的缺陷 包括心肌肥厚、室间隔缺损以及 血管错位。这样的缺陷类似于人类 法洛四联症和右室双出口疾病。 大脑缺陷的特征是脑积水。这个 实验室还鉴定了一种NMHCII-B小鼠 不能将替代外显子拼接到肌球蛋白重链附近 三磷酸腺苷结合区，也降低了 未插入的NMHCII-B。当前的项目旨在创建一个 NMHCII--一只缺陷小鼠。已经有两种不同的构造 以中断NMHCII-A基因并将 新霉素抗性基因的起始外显子之一。在其中一个 构建后，新霉素抗性基因的两侧是loxP位点。 这将允许在交配后的小鼠胚胎中切除它 携带Cre重组酶的小鼠。所需的杂合子 胚胎干细胞已经被培养出来，用于胚泡细胞注射。

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 defects in the heart include cardiac hypertrophy, a ventricular septal defect, and malposition of the vessels. Such defects resemble the human diseases Tetralogy of Fallot and double outlet right ventricle. Defects in the brain are characterized by hydrocephalus. 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. The required heterozygotic embryonic stem cells have been generated for blastocyte injection.