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。NM II-A的缺失导致原肠胚形成（E6.5）之前的致死性，并且胚胎因细胞-细胞粘附的缺陷和未能形成柱状内脏内胚层而紊乱。为了避免早期胚胎致死性，已经创建了NMHC II-A的条件无效。 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。此外，可以产生三重转基因，其利用rtTA/泰特操纵子系统赋予基因缺失的时间依赖性。该系统利用强力霉素的摄入最终在特定时间激活cre重组酶。 已知患有MYH 9综合征的人在出血时间、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脏足细胞中NM II-A的缺失上。 足细胞是形成肾小球中的过滤屏障的一部分的肾细胞。将NM II-A小鼠与具有由测试操作员控制的cre重组酶的小鼠以及在podocin启动子控制下的rtTA的小鼠杂交。这些三重转基因允许肾脏足细胞中NM II-A的组织和时间依赖性缺失。通过测定尿液中的葡萄糖/肌酐比值和肾脏病理学筛选三重转基因和对照同窝仔（不含cre重组酶、不含rtTA或不含floxed NMHC II-A）。

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 NM 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 conditional null of NMHC II-A 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. Additionally, triple transgenics can be created which take advantage of the rtTA/tet operator system to confer time dependence on the deletion of a gene. This system uses ingestion of doxycycline to ultimately activate cre recombinase at a specific time. It is known that humans with MYH9 syndrome 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. Podocytes are the kidney cells which form part of the filtration barrier in the glomerulus. The NM II-A mice have been crossed to mice with cre recombinase controlled by the test operator and also mice the rtTA under control of the podocin promoter. These triple transgenics permit tissue and time dependent deletion of NM II-A in the kidney podocytes. Triple transgenics and control littermates (either without the cre recombinase, without rtTA or without the floxed NMHC II-A) are being screened by determination of glucose/creatinine ratios in urine and by kidney pathology.