Session: SAT 88-108-GHRH, GH & IGF Biology & Signaling
Poster Board SAT-107
Methods and Results: In order to construct the transgene, a 1458 bp mouse GHRH promoter fragment was cloned upstream of a 1353 bp Cre-recombinase gene obtained from the ACN cassette (a gift from Dr. Mario Capecchi) and sequenced to ensure that the genes were in the correct orientation and in-frame. The GHRH-CRE transgene was linearized for pronuclear injection by the Johns Hopkins Transgenic Core. In brief, 1-cell stage embryos were obtained from superovulated B6SJLF1 females and the DNA was injected into one pronucleus of each embryo. Following injection, surviving embryos were surgically transferred to oviducts of psuedopregnant ICR females (25 embryos/female). Genomic DNA from a total of 117 pups was screened for the GHRH Cre transgene of which 16 were Cre positive. These mice were then mated with normal mice and pups that were Cre positive were sacrificed. RNA was obtained from cortex, hypothalamus, cerebellum, pituitary, heart, lung, liver, stomach, pancreas, spleen, kidney, fat, muscle, uterus and gonads. GHRH-Cre was expressed in the hypothalamus, but not in the pituitary using Quantitative Real Time PCR with primers specific for targeting Cre recombinase in cDNA.
Conclusion: We describe the development of a transgenic GHRH-Cre mouse that can be used to selectively ablate genes in the GHRH neuron to further study and characterize the development and functional regulation of these cells. In addition, this model system will provide insight into the role of hypothalamic GHRH on regulation of mammalian growth.
Disclosure: SR: Ad Hoc Consultant, CVS/Caremark, Speaker, Novo Nordisk. Nothing to Disclose: CJR, EA, VC, BF, RS, AMW
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