In Vivo Function of Nonmuscle Myosin II-A

非肌肉肌球蛋白 II-A 的体内功能

• 批准号: 7734986
• 负责人: ROBERT ADELSTEIN
• 金额: $ 37.61万
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7734986
• 关键词: A Mouse; Bleeding time procedure; Cataract; Cell-Cell Adhesion; Cells; Creatinine; Defect; Dependence; Development; Doxycycline; Embryo; Endoderm; Exons; Failure; Filtration; Genes; Glucose; Goals; Human; Ingestion; Introns; Kidney; Kidney Diseases; Knock-out; Knockout Mice; Mus; Mutation; Myosin Type II; NPHS2 protein; Neamin; Neomycin; Nephritis; Pathology; Protein Isoforms; Purpose; Research; Resistance; Severities; Site; Staging; Syndrome; System; Testing; Tetanus Helper Peptide; Time; Tissues; Transgenic Organisms; Urine; Visceral; embryonic stem cell; hearing impairment; homologous recombination; human disease; in vivo; kidney cell; mouse model; podocyte; promoter; recombinase

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.

为了研究非肌肉肌球蛋白II同工型（NMII-A，II-B和II-C）的功能，已经使用了同源重组来删除胚胎干细胞中的每种同工型，并且已经产生了无效的小鼠。柱状内脏内胚层。为了避免早期的胚胎杀伤力，已经创建了NMHC II-A的条件零。 在外显子3之前，已将新霉素的耐药盒插入内含子，而LOXP位点已插入Neomycin Cassette和Exon的两侧。这些小鼠在细胞或组织特异性启动子控制下携带CRE重组酶的小鼠的分子会导致NM II-A的相应缺失。此外，可以创建三重转基因，以利用RTTA/TET操作员系统来赋予对基因缺失的时间依赖性。该系统使用强力霉素的摄入来最终在特定时间激活CRE重组酶。 众所周知，患有MYH9综合征的人在出血时间，听力损失，白内障和肾小球肾炎中患有缺陷，由于肾脏疾病的严重程度，最初的研究着重于肾脏足细胞中NM II-A的缺失。 足细胞是肾小球构成肾小球中过滤屏障的一部分的肾细胞。 NM II-A小鼠已经用由测试操作员控制的CRE重组酶越过小鼠，也将小鼠的RTTA控制在Podocin启动子的控制下。这些三重转基因允许在肾脏足细胞中的NM II-A组织和时间依赖性缺失。三重转基因和对照同窝仔（如果没有CRE重组酶，无RTTA或没有Floxed NMHC II-A-A），通过测定尿液中的葡萄糖/肌酐比例来筛选。