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Biochem Biophys Res Commun
2006 Jun 23;3451:140-7. doi: 10.1016/j.cub.2006.04.029.
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Stable knock-down of vomeronasal receptor genes in transgenic Xenopus tadpoles.
Kashiwagi A
,
Kashiwagi K
,
Saito S
,
Date-Ito A
,
Ichikawa M
,
Mori Y
,
Hagino-Yamagishi K
.
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Xenopus V2R (xV2R), a family of G-protein-coupled receptors with seven transmembrane domains, is expressed in the Xenopus vomeronasal organ (VNO). There are six subgroups of xV2R, one of which, xV2RE, is predominantly expressed in the VNO. To understand the function of xV2R during VNO development, we developed a new method to achieve stable siRNA-suppression of the V2RE genes by introducing siRNA expression transgenes into the genomes of unfertilized eggs. We found that some of the derived transgenic tadpoles lacked VNOs and that their olfactory epithelium was fused. With the exception of one tadpole, expression of xV2RE was not detected in morphologically abnormal mutant tadpoles, although the olfactory marker protein and the olfactory receptors were expressed. These results suggest that we successfully produced transgenic tadpoles in which xV2RE expression was stably suppressed by siRNA, and that xV2RE plays a role in the morphogenesis of olfactory organs.
Fig. 1.
Expression of xV2RE in Xenopus VNOs and the silencing effect of siRNA-expressing plasmids on the expression of xV2R1 in 293T cells. Cross-sections of adult (A) and tadpole VNOs at stage 50 (B) were hybridized with digoxigenin-labeled V2RE-specific antisense probe as described previously [17]. A schematic illustration of the siRNA-expressing plasmid (C). Arrows in (A,B) indicate the sites of xV2R1 expression. Chimeric rhodopsin-xV2R1 plasmid was transfected into HEK 293T cells with pA (E), pB (F), pA′ (G) or without siRNA plasmid (D), and expression of xV2R1 was detected by anti-rhodopsin antibody as described in the Materials and methods. Black spots on the periphery of the OE and VNO in cross-sections (A,B) are melanocyte aggregates. Bars indicate 50 μm.
Fig. 2.
Morphological features of olfactory organs of transgenic tadpoles. Photographs of pA′/E-derived EGFP-positive tadpole with morphologically normal nostrils (A) and pA/E- (D) and pB/E (G) -derived EGFP-positive tadpoles with fused nostrils at stage 50–51 (white arrows indicate nostrils). EGFP expression in tadpoles of A (B), D (E), and G (H) was monitored using a fluorescent dissecting microscope (white arrows indicate OE). Paraffin-embedded sections (15 μm) of pA′/E-derived EGFP-positive tadpoles with normal nostrils (C) and pA/E- (F), pB/E (I) -derived EGFP-positive tadpoles with fused nostrils were stained with hematoxylin/eosin. The VNO (arrows), OE (arrowheads), fused OE (asterisks), and Jacobson’s glands (double arrowhead) are shown. Bars indicate 50 μm.
Fig. 3.
Expression of the xV2RE genes in the olfactory organs. Cross-sections of pA′/E-derived EGFP-positive tadpoles with normal nostrils (A) and pA/E- (B,C), pB/E (D,E) -derived EGFP-positive tadpoles with fused nostrils. The sections were hybridized with digoxigenin-labeled V2RE-specific antisense probe as described in the Materials and methods. The VNO (black arrow), OE (arrowhead), and fused OE (asterisks) are shown. Black spots on the periphery of the OE and VNO in cross-sections (A–E) are melanocyte aggregates. Bars indicate 100 μm.
Fig. 4.
Expression of xOR1 in the olfactory organs. Cross-sections of normal adult principal cavity (PC) (A), middle cavity (MC) (B), VNO (C), the normal OE in the tadpole at stage 50–51 (D), pA/E- (E), and pB/E (F) -derived EGFP-positive tadpoles with fused nostrils. The sections were hybridized with digoxigenin-labeled xOR1-specific antisense probe as described in the Materials and methods. The fused OE (asterisks) and the expression sites of xOR1 (arrows) are shown. Bars indicate 50 μm.
Fig. 5.
Expression of xOMP-2 in the olfactory organs. Cross-sections of normal adults PC (A), MC (B), VNO (C), the normal olfactory organs in the tadpole at stage 50–51 (D), pA/E- (E), and pB/E (F) -derived EGFP-positive tadpoles with fused nostrils. The sections were hybridized with digoxigenin-labeled xOMP-2-specific antisense probe as described in the Materials and methods. The fused OE (asterisks) and the expression sites of xOMP-2 (arrows) are shown. Bars indicate 50 μm.
