Fig 4. GnRH-E1 RNA is localized in the GT1-7 neuron nucleus.

(A) Schematic diagram of the conserved regulatory elements upstream of the mouse Gnrh1 TSS, which contains enhancers 1, 2, and 3 (E3, E2, E1, respectively), the promoter (P), and the Gnrh1 gene with four exons (white boxes). Coordinates above the regulatory elements indicate positions with respect to the Gnrh1 TSS. RT-PCR primers used in B-D are indicated by arrows, and expected PCR products are represented by a connecting line. Positions of PCR primers are aligned to the mouse conserved regulatory region diagrammed above. Nuclear and cytoplasmic extracts from GT1-7 neurons were analyzed for GnRH-E1 RNA (B), Gnrh1 pre-mRNA (C), Gnrh1 mRNA (D), and H2afz mRNA control (E) by RT-PCR. RT-PCR analysis was performed on random hexamer-primed cDNA, where cDNA synthesized with (+) and without (-) reverse transcriptase were analyzed in parallel. PCR loading controls are plasmid containing the -3568/-1128 bp segment upstream of the Gnrh1 TSS and no-template control (NTC). The sizes of the PCR amplicons were marked by a 100 bp DNA ladder or a 1 kbp DNA ladder where indicated, that were resolved on the agarose gel in parallel.