In Vivo Function of Nonmuscle Myosin II-A

为了研究非肌肉肌球蛋白II同种型（匪II-A、II-B和II-C）的功能，已使用同源重组来删除胚胎干细胞中的每种同种型，并产生了无效小鼠。非肌肉肌球蛋白重链（NMHC）II-A的缺失导致原肠胚形成（E6.5）之前的致死性，胚胎因细胞-细胞粘附缺陷和未能形成柱状内脏内胚层而紊乱。为了避免早期胚胎死亡，已经创建了NMHC II-A floxed小鼠。 新霉素抗性盒已经插入到外显子3之前的内含子中，并且loxP位点已经插入到新霉素盒和外显子的侧翼。 这些小鼠与在细胞或组织特异性启动子控制下携带cre重组酶的小鼠交配，导致NM II-A的相应缺失。已知MYH 9突变的人在出血时间、听力损失、白内障和肾小球肾炎方面存在缺陷。由于肾脏疾病的严重性，最初的研究集中在肾足细胞中NM II-A的缺失，肾足细胞是形成肾小球中滤过屏障的一部分的肾细胞。在MYH 9 RD患者的肾活检中观察到足细胞消失。由于目前尚不清楚该疾病是由突变型肌球蛋白与野生型NMIIA的干扰还是由单倍不足引起的，因此这些缺失可能提供关于NMIIA在足细胞功能中的作用的重要信息。NM II-A小鼠已经与具有由足细胞标志物podocin控制的cre重组酶的小鼠杂交，podocin已经成功地用于产生足细胞特异性缺失。 缺失NMIIA的纯合子小鼠（Apod/Apod）在6月龄时尿样中白蛋白浓度增加，表明肾小球滤过减少。

In order to study the function of the nonmuscle myosin II isoforms (NMII-A, II-B, and II-C), homologous recombination has been used to delete each isoform in embryonic stem cells and null mice have been generated. Deletion of nonmuscle myosin heavy chain (NMHC) II-A causes lethality prior to gastrulation (E6.5) and the embryos are disorganized with defects in cell-cell adhesion and a failure to form a columnar visceral endoderm. In order to avoid the early embryonic lethality, a NMHC II-A floxed mouse has been created. A neomycin-resistance cassette has been inserted into the intron prior to exon 3 and loxP sites have been inserted flanking both the neomycin cassette and the exon. Matings of these mice to mice bearing cre recombinase under the control of a cell or tissue specific promoters causes the corresponding deletion of NM II-A. It is known that humans with mutations in MYH9 have defects in bleeding times, hearing loss, cataracts, and glomerular nephritis. Because of the severity of the kidney disorder, initial studies focus on deletion of NM II-A in kidney podocytes which are the kidney cells which form part of the filtration barrier in the glomerulus. Effacement of podocytes is seen in kidney biopsies from MYH9RD patients. Since it is currently not clear whether the disease results from interference of the mutant myosin with the wild type NMIIA or from haploinsufficiency these deletions may provide important information about the role of NMIIA in podocyte function. The NM II-A mice have been crossed to mice with cre recombinase controlled by the podocyte marker, podocin which has been successfully used to generate podocyte-specific deletions. Homozygous mice for deletion of NMIIA (Apod/Apod) have increased concentrations of albumin in urine samples by 6 months of age indicating a decrease in glomerular filtration.