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Generation of transgenic rats expressing enhanced green fluorescent protein in gonadotropin-releasing hormone neurons.

Hypothalamic gonadotropin-releasing hormone (GnRH) neurons govern reproductive function by controlling the release of gonadotropins from the pituitary. To facilitate identification of living GnRH neurons, here we attempted to generate transgenic rats that express enhanced green fluorescent protein (EGFP) in GnRH neurons. About 3 kb of rat GnRH promoter region was inserted into the EGFP reporter cassette, and the expression of EGFP fluorescence was confirmed in several cell lines following transient transfection. Then we successfully generated a transgenic rat by injecting linearized GnRH-EGFP transgene into the pronuclei of fertilized oocytes. The GnRH-EGFP transgenic rats expressed EGFP in the brain, but not in the ovary, testis or thymus. Immunohistochemical examination revealed that detectable EGFP fluorescence was confined to the cell body of GnRH-immunoreactive neurons in the septum and preoptic area, while no EGFP signal was discernible in the median eminence where abundant GnRH-immunoreactive fibers were observed. The mean percentage of EGFP-positive cells in the GnRH-positive cells was 76.3%. The GnRH-EGFP transgenic rats generated in the present study will enable characterization of properties of individual GnRH neurons.