Figure 1.

Development of GnRH-specific Kiss1r knockout mouse. A, Schematic diagrams of constructs used to generate GKirKO mice. Mice bearing LoxP sites flanking exon 2 of the Kiss1r were crossed with transgenic mice expressing Cre recombinase specifically in GnRH neurons. Primers used in PCR genotyping are labeled P1 (exon 1) and P3 (exon 3). X, XbaI; A, Asp718I; S, SpeI restriction enzyme sites. B, Genotyping by PCR analysis of the genomic DNA produced a band migrating at 2096 bp in the mice bearing a floxed allele and a band at 1882 bp in WT mice. These primers were designed to produce a 1120-bp band after excision of the sequence between the LoxP sites in DNA extracted from the hypothalamus (that is, tissue that express Cre recombinase). C, Southern blot analysis was performed by digesting the DNA with Asp718I and detected a band of 9.2 kb in the floxed allele and 12.2 kb in the WT allele with probe 1. D, qPCR analysis of Kiss1r mRNA extracted from male and female mouse tissues (P ≤ .001, n = 3). WT, wild-type; Kiss1r^WT/fl, heterozygous floxed; Kiss1r^fl/fl, homozygous floxed; ND, not detected.