NULL MUTATIONS OF VERTEBRATE NONMUSCLE MYOSIN HEAVY CHAINS

非肌肉肌球蛋白是真核生物中普遍存在的一种蛋白质。 细胞。它在细胞运动、胞质分裂和封顶过程中发挥作用。 本实验室从人和鸡中分离出两种不同的cdna。 编码非肌肉肌球蛋白重链(MHC)的组织。两个人 不同的亚型被称为MHC-A和MHC-B，在人类中是 位于两条不同的染色体上。为了了解In MHC-B亚型的体内功能，我们进行了实验 生产转基因小鼠，在其中编码非肌肉MHC-B的基因将 不能产生基因产物。我们使用了目标载体 包含6.7 kb的非肌肉MHC-B片段，该片段是为 小鼠基因组lambda噬菌体文库。2kb的新霉素基因 插入到外显子2中。线性化的载体电穿孔到J1 352个精选克隆的胚胎干细胞及Southern杂交分析 在6个克隆中发现同源重组。所有这些克隆人都是 用于3.5天龄C57Bj6囊胚的显微注射以及 8个细胞胚胎聚集。我们观察到生殖系在 刺鼠后代。杂合子小鼠看起来很健康，而且还能生育。 他们交配后产生了纯合的小鼠。此时此刻， 我们检查了32个后代(包括10-15天的胚胎)，我们发现 只有1只纯合子小鼠出生后死亡。考虑到 我们的窝很小(平均5只)，而且我们有 没有观察到活的纯合子小鼠，我们假设致命性是 发生在小鼠胚胎发育期间。为了解决这个问题， 我们正在检查处于不同发育阶段的胚胎。在……里面 除了检查表型和基因型外，我们还在检查 Western印迹分析表达蛋白的特异性 抗体。

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.