Consequences of generalized lack of GHRH

描述（申请人提供）：虽然GHRH受体突变导致GH缺乏症（GHD），但GHRH基因突变从未报道过，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CNS区域，胎盘，胰腺，心脏，肝脏，肾脏和睾丸中表达，并且循环GHRH被认为主要是下丘脑外起源的。GHRH在睡眠调节中也有作用。最后，GHRH和GHRHR同种型在许多癌细胞中表达，其中GHRH可以自分泌/旁分泌方式起作用以调节癌症生长。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，我们使用质粒（“GHRHKO 1”），其中新霉素抗性盒（Neor）替代GHRH基因的基本部分。已经鉴定了25只GHRHKO 1杂合小鼠，它们都源自单个ES克隆。为了产生另外的KO系，我们随后产生了第二质粒（“GHRHKO 2”），其中Neor侧翼为IoxP位点。 规格目的1：分析靶向破坏GHRH基因对GHRHKO 1动物中小鼠表型的影响。将饲养F1 GHRHKO 1小鼠以产生纯合KO小鼠，以分析表型、大小、生长、活力、内脏外观和行为。如果纯合KO是致死性的，则将分析来自妊娠杂合雌性的胚胎和胎盘，以确定缺乏GHRH对胎儿发育的影响。如果该表型仅限于GHD，则由于缺乏下丘脑GHRH，该小鼠将是一种有用的GHD模型，并且将用于研究内源性GHRH对癌症发展和生长的影响。 具体目标2：创建无Neor盒的GHRH KO小鼠（GHRHKO 2）。将产生新的杂合KO系，其中Neor侧翼为loxP位点。通过重组酶技术，我们将产生不含Neor的KO系，避免与远距离或邻近基因的任何干扰。此外，在GHRHKO 1系中获得的结果将被证实或反驳。

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.