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. 2016 Jul 7;11(7):e0158597. doi: 10.1371/journal.pone.0158597

Fig 7. The effect of mouse GnRH-E1 RNA over-expression on Gnrh1 gene transcriptional activity in GN11 cells.

Fig 7

(A) A schematic diagram of the expression plasmid carrying the -3560 bp/-1128 bp segment from upstream of the mouse Gnrh1 TSS, which contains the full-length mouse GnRH-E1 RNA that is integrated in the forward orientation. Expression of the sense GnRH-E1 RNA is driven by the CMV promoter (CMVp) and terminated by a plasmid-specific 3’ polyadenylation element (BGH-PolyA). B-C) GN11 neurons were transiently co-transfected with either empty pcDNA2.1 vector (white bars) or expression plasmid carrying the forward mouse GnRH-E1 RNA (black bars) for over-expression and the luciferase reporter plasmids as indicated. (B) GN11 cells were co-transfected with luciferase reporter plasmids containing -5000 bp, -4199 bp, -3175 bp, or -2168 bp of the rat Gnrh1 regulatory region. Each luciferase reporter plasmid contains the indicated Gnrh1 enhancers (E1, E2 and E3) and Gnrh1 promoter (P). (C) GN11 cells were co-transfected with luciferase plasmids carrying the rat Gnrh1 enhancer 1 and promoter (GnRH-E1/GnRH-P), RSV enhancer and the rat Gnrh1 promoter (RSVe/GnRH-P), the rat Gnrh1 enhancer 1 and RSV promoter (GnRH-E1/RSVp), or the RSV enhancer and promoter (RSVe/RSVp) control (inset). For reporter plasmids carrying GnRH-E1, the rat Gnrh1 enhancer is integrated in the reverse orientation in the plasmid. (D) A schematic diagram of the antisense GnRH-E1 RNA expression plasmid carrying the full-length mouse GnRH-E1 RNA that is integrated in the reverse orientation, driven by CMVp and terminated by BGH-PolyA. (E) The antisense GnRH-E1 RNA expression plasmid (black bars) or empty pcDNA2.1 expression plasmid (white bars) were transiently co-transfected with a reporter plasmid carrying the -5 kb rat Gnrh1 gene regulatory region, GnRH-E1/GnRH-P, GnRH-E1/RSVp, or RSVe/RSVp control (inset). Since the RSVe/RSVp is a very strong reporter, relative luciferase/β-galactosidase values for the RSVe/RSVp reporter are graphed separately for clarity in C and D. Luciferase/β-galactosidase values were normalized to pGL3. Data are displayed as the mean ± SD. Asterisks indicate statistical significance determined using Student’s T-test comparison between pcDNA2.1-transfected and GnRH-E1 RNA-transfected cells, where p<0.05.