EXPRESSION OF NONMUSCLE MYOSIN ISOFORMS IN EUKARYOTIC CELLS

两种非肌球蛋白重链 （NMHCII-A和NMHCII-B）已在本实验室克隆 并被证明是两种不同基因的产物。最近的工作 在NMHCII-B缺陷小鼠上（Tullio等，Proc. Natl. Acad. Sci. USA 94，12407，1997）已经阐明了心脏中的缺陷， 导致胚胎死亡。心脏的缺陷 包括心脏肥大、室间隔缺损， 血管错位。这种缺陷类似于人类 法洛四联症和右心室双出口。 脑缺陷的特征是脑积水。的 实验室还对一种NMHCII-B小鼠进行了表征， 不能将选择性外显子拼接到肌球蛋白重链中， ATP结合区域，并且还具有降低的表达， 未插入的NMHCII-B。目前的项目旨在建立一个 NMHCII-A缺陷小鼠。两种不同的结构， 为了中断NMHCII-A基因，并将NMHCII-A基因置于 新霉素抗性基因的起始外显子之一。于一体的 构建体中，新霉素抗性基因侧翼为loxP位点 这将允许其在交配后在小鼠胚胎中切除 用带有Cre重组酶的小鼠。所需的杂合子 胚胎干细胞已经产生用于胚细胞注射。

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.