In these studies we are using a genetic approach to define the potential roles of luteinizing hormone releasing hormone (LHRH) and its receptors in brain. LHRH is the hypothalamic neuropeptide that controls sexual development and mature reproductive function. The first phase of the research plan is now complete. We have obtained two hemizygous transgenic mice (one male and one female) of the 671-Bx/Gal Nac (T8) strains carrying the Cre recombinase gene under the control of the neural cell-specific synapsin I promoter (Syn-Cre). They have been crossed to produce a first generation (F1) of mice carrying the Syn-Cre transgene. Seventy-five percent of the F1 pups (6 of 8) were positive for Syn-Cre, indicating that the transgene is transmitted in a Mendelian fashion. Two of these mice (one male and one female) were tentatively identified as homozygotes by semiquantitative PCR, and confirmed as such by backcrossing to wildtype mice. We then crossed a F1 Syn-Cre pos itive mal e with F1 Syn-Cre positive females to produce additional homozygous animals. As before, we used semiquantitative PCR and back crossing to identify homozygous mice of the F2 generation. The brains of two of these mice were fixed by transcardiac perfusion and subjected to in situ hybridization. The results of this analysis demonstrated the presence of Cre mRNA in neurons containing LHRH receptor mRNA. This colocalization was especially evident in the hippocampus, a region of the brain involved in learning and memory. We are currently using three pairs of young adult hemizygous animals to maintain this transgenic line. The second, and more complex phase of the proposal is well under way. We have prepared the targeting construct to Aflox@ exon 1 and proximal promoter of the LHRH receptor gene, and worked out a screening strategy to identify, first those ES clones carrying a homologously recombined targeting construct, and secondly, those clones in which Cre-mediated recombination was effective in generating the intended recombination events. ES cells have now been transfected with the targeting construct and grown under neomycin selection. A total of 112 clones were selected; they are currently being analyzed by PCR/ Southern blot analysis to identify those carrying an homologously integrated targeting construct. FUNDING NIH HD35958 PUBLICATIONS None
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