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.
