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Consequences of generalized lack of GHRH

普遍缺乏 GHRH 的后果

基本信息

批准号：
6896781
负责人：
Roberto Salvatori
金额：
$ 8.18万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-07-01 至 2006-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/6896781
关键词：
biological signal transduction gene expression genetically modified animals growth /development growth hormone releasing hormone hormone receptor immunocytochemistry laboratory mouse phenotype pituitary dwarfism polymerase chain reaction protein deficiency recombinase southern blotting western blottings

项目摘要

DESCRIPTION (provided by applicant): While mutations in the GHRH receptor cause GH deficiency (GHD), mutations in the GHRH gene have never been reported, possibly indicating that lack of GHRH may cause broader consequences than GHD, or even not allow normal development. Indeed, in addition to the hypothalamus, GHRH is expressed in other CNS areas, in placenta, pancreas, heart, liver, kidney and testes, and circulating GHRH is believed to be mostly of extra-hypothalamic origin. GHRH has also a role in sleep regulation. Finally, GHRH and GHRHR isoforms are expressed in numerous cancer cells, where GHRH may act in autocrine/paracrine fashion to regulate cancer growth. To investigate the functions of GHRH, we worked on creating a mouse that ubiquitously lacks this protein. Initially, we used a plasmid ("GHRHKO1") in which the Neomycin resistance cassette (Neor) substitutes an essential part of the GHRH gene, Twenty-five GHRHKO1 heterozygous mice, all originating form a single ES clone, have been identified. In order to create additional KO lines, we have subsequently created a second plasmid ("GHRHKO2"), in which the Neor is flanked by IoxP sites. Spec. aim 1 : Analysis of the effect of targeted disruption of the GHRH gene on mouse phenotype in GHRHKO1 animals. F1 GHRHKO1 mice will be bred to generate homozygous KO mice, to be analyzed for phenotype, size, growth, viability, appearance of internal organs, and behavior. If homozygous KO is lethal, embryos and placentas from pregnant heterozygous females will be analyzed to determine the effect of lack of GHRH on fetal development. If the phenotype is limited to GHD, this mouse will be a useful model of GHD due to lack of hypothalamic GHRH, and will be useful to study the effect of endogenous GHRH on cancer development and growth. Specific aim 2: Creation of a GHRH KO mouse without Neor cassette (GHRHKO2). New heterozygous KO lines will be created in which the Neor is flanked by loxP sites. By recombinase technique, we will generate KO lines with no Neor, avoiding any interference with distant or neighboring genes. In addition, results obtained in the GHRHKO1 line will be confirmed or disproved.

描述(申请人提供)：虽然GHRH受体的突变会导致GH缺乏症(GHD)，但GHRH基因的突变从未被报道过，可能表明GHRH缺乏可能导致比GHD更广泛的后果，甚至不允许正常发育。事实上，除了下丘脑，GHRH在其他中枢神经系统区域也有表达，如胎盘、胰腺、心脏、肝脏、肾脏和睾丸，循环中的GHRH被认为主要来自下丘脑以外。GHRH也在睡眠调节中发挥作用。最后，GHRH和GHRHR亚型在许多癌细胞中表达，GHRH可能以自分泌/旁分泌的方式调节肿瘤的生长。为了研究GHRH的功能，我们致力于创造一种普遍缺乏这种蛋白质的小鼠。最初，我们使用了一个新霉素耐药盒(Neor)替换了GHRH基因的一个重要部分的质粒(“GHRHKO1”)，已经鉴定了25只GHRHKO1杂合子小鼠，它们都来自一个ES克隆。为了创造更多的KO系，我们随后创造了第二个质粒(“GHRHKO2”)，其中Neor的两侧是IoxP位点。规格。目的：分析GHRH基因靶向阻断对GHRHKO1小鼠表型的影响。F1GHRHKO1小鼠将被培育成纯合子KO小鼠，以进行表型、大小、生长、活力、内脏外观和行为分析。如果纯合子KO是致命的，将分析来自杂合子怀孕女性的胚胎和胎盘，以确定缺乏GHRH对胎儿发育的影响。如果表型仅限于GHD，由于下丘脑GHRH缺乏，该小鼠将是一个有用的GHD模型，并将有助于研究内源性GHRH对肿瘤发生和生长的影响。具体目标2：建立一只无Neor盒的GHRH KO小鼠(GHRHKO2)。新的杂合KO系将被创造，其中Neor的侧翼是loxP位点。通过重组酶技术，我们将产生没有NeOR的KO系，避免了与远近基因的任何干扰。此外，在GHRHKO1品系中获得的结果将得到确认或反驳。