In Vivo Function of Nonmuscle Myosin II-A

为了研究非肌球蛋白II亚型（NMII-A， II- b和II- c）的功能，在胚胎干细胞中使用同源重组来删除每个亚型，并产生空小鼠。NM II-A的缺失导致原肠胚形成前死亡（E6.5），胚胎组织紊乱，细胞粘附缺陷，不能形成柱状内脏内胚层。为了避免早期胚胎致死，我们创建了一个有条件的NMHC II-A null。在外显子3之前将一个新霉素耐药盒插入内含子，并且在新霉素盒和外显子的两侧插入loxP位点。在细胞或组织特异性启动子的控制下，这些小鼠与携带cre重组酶的小鼠交配，导致相应的NM II-A缺失。此外，三重转基因可以创建利用rtTA/tet操作系统赋予时间依赖于一个基因的删除。该系统使用摄入强力霉素在特定时间最终激活cre重组酶。众所周知，MYH9综合征患者在出血次数、听力丧失、白内障和肾小球性肾炎等方面存在缺陷，由于肾脏疾病的严重性，最初的研究主要集中在肾足细胞中NM II-A的缺失。足细胞是肾细胞，构成肾小球滤过屏障的一部分。将NM II-A小鼠与由试验操作者控制的cre重组酶和由podocin启动子控制的rtTA杂交。这些三重转基因允许肾足细胞中NM II-A的组织和时间依赖性缺失。通过测定尿液中葡萄糖/肌酐比率和肾脏病理，筛选三重转基因和对照幼崽（没有cre重组酶，没有rtTA或没有固定的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.