NULL MUTATIONS OF VERTEBRATE NONMUSCLE MYOSIN HEAVY CHAINS

非肌肉肌球蛋白是一种普遍存在于所有真核生物中的蛋白质， 细胞 它参与细胞的运动和细胞内 胞质分裂和加帽。 这个实验室分离出了两种不同的 编码非肌球蛋白重链的人和鸡组织cDNA 链（NMMHC）。 这两种亚型称为NMMHC-A和-B， 位于两个不同的人类染色体上 为了解 在体内功能的两个异构体，我们正试图执行 使用同源性消除NMMHC基因的实验 在胚胎干细胞和转基因小鼠中的重组。 我们构建了一个14.5kb的含有6.7kb NMMHC-B片段的构建体 从小鼠基因组λ噬菌体文库克隆。 A新霉素基因 在外显子2中引入一个2kb的序列以中断转录 并在同源后提供新霉素抗性 重组 此外，单纯疱疹病毒胸苷激酶（TK）基因 在我们构建体末端的同源区域之外引入。 TK基因仅在随机整合的情况下整合，但 如果发生同源重组就不会。 通过限制性酶切图谱和部分测序检查构建体。 将突变的基因引入小鼠胚胎干细胞（ESC）（J-2005）。 1)通过电穿孔并在遗传霉素（300 μ g/ml）和FIAU（0.2 μ M）。 使用一种筛选方法筛选所选的克隆。 基因组Southern印迹以区分同源重组和随机重组 事件 我们也在鉴定来自肌动蛋白的小鼠NMMHC-B片段， 从相同的小鼠基因组λ噬菌体文库克隆的结合区 并用不同的探针进行筛选。 我们将利用这个基因区域 敲除NMMHC-B亚型，我们还将构建一个新的结构， 含有lox/cre序列的小鼠， 在肌动蛋白结合附近存在的mRNA的特定插入序列 脑NMMHC-B区。 同时，NMMHC-A的克隆 正在表征同种型，以制备类似于 NMMHC-B型

Nonmuscle myosin is an ubiquitous protein present in all eukaryotic cells. It is involved in the motility of cells and in cellular cytokinesis and capping. This laboratory has isolated two different cDNAs from human and chicken tissues encoding nonmuscle myosin heavy chains (NMMHCs). These two isoforms are called NMMHC-A and -B and are located on two different human chromosomes. In order to understand the in vivo function of the two isoforms, we are trying to perform experiments to eliminate the genes for NMMHC(s) using homologous recombination in embryonic stem cells and in transgenic mice. We generated a construct of 14.5 kb containing a 6.7 kb NMMHC-B fragment cloned from a mouse genomic lambda phage library. A neomycin gene sequence (2 kb) was introduced into exon 2 to interrupt the transcription of the gene and provide neomycin resistance after homologous recombination. Also, the herpes simplex virus thymidine kinase (TK) gene was introduced outside the homologous region at the end of our construct. The TK gene will be integrated only in cases of random integration, but not if homologous recombination occurs. The construct was checked by restriction maps and partial sequencing. The mutated gene was introduced into mouse embryonic stem cells (ESC) (J- 1) by electroporation and selected in the presence of Geneticin (300 ug/ml) and FIAU (0.2 uM). The selected clones were screened using a genomic Southern blot to distinguish homologous from random recombination events. We are also characterizing fragments of mouse NMMHC-B from the actin binding region cloned from the same mouse genomic lambda phage library and screened with a different probe. We will use this region of the gene to knock out the NMMHC-B isoform and we will also make a new construct containing a lox/cre sequence to try to generate a mouse lacking a particular inserted sequence of mRNA present near the actin binding region of brain NMMHC-B. At the same time, a clone for the NMMHC-A isoform is being characterized in order to make a construct similar to the NMMHC-B one.