Consequences of generalized lack of GHRH

描述（由申请人提供）：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，这可能表明GHRH缺乏可能导致比GHD更广泛的后果，甚至无法正常发育。事实上，除了下丘脑外，GHRH还在其他中枢神经系统区域表达，如胎盘、胰腺、心脏、肝脏、肾脏和睾丸，循环GHRH被认为主要来自下丘脑外。GHRH在睡眠调节中也有作用。最后，GHRH和GHRHR亚型在许多癌细胞中表达，其中GHRH可能以自分泌/旁分泌方式调节癌症生长。为了研究GHRH的功能，我们创造了一种普遍缺乏这种蛋白质的老鼠。最初，我们使用了一种质粒（“GHRHKO1”），其中新霉素耐药盒（Neor）替代了GHRH基因的重要部分，已经鉴定出25只GHRHKO1杂合小鼠，它们都起源于单个ES克隆。为了创建更多的KO系，我们随后创建了第二个质粒（“GHRHKO2”），其中Neor两侧是IoxP位点。

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.