February 20, 1998;
Transgene expression in Xenopus rods.
The photoreceptors of the vertebrate retina
express a large number of proteins that are involved in the process of light transduction. These genes appear to be coordinately regulated at the level of transcription, with rod- and cone-specific isoforms (J. Hurley (1992) J. Bioenerg. Biomembr. 24, 219-226). The mechanisms that regulate gene expression in a rod/cone-specific fashion have been difficult to address using traditional approaches and remain unknown. Regulation of the phototransduction proteins is medically important, since mutations in several of them cause retinal degeneration (P. Rosenfeld and T. Dryja (1995) in: Molecular Genetics of Ocular Disease (J.L. Wiggs, Ed.), pp. 99-126, Wiley-Liss Inc.). An experimental system for rapidly producing retinas expressing a desired mutant would greatly facilitate investigations of retinal degeneration. We report here that transgenic frog embryos (K. Kroll and E. Amaya (1996) Development 122, 3173-3183) can be used to study cell-specific expression in the retina
. We have used a 5.5 kb 5'' upstream fragment from the Xenopus principal rod opsin gene (S. Batni et al. (1996) J. Biol. Chem. 271, 3179-3186) controlling a reporter gene, green fluorescent protein (GFP), to produce numerous independent transgenic Xenopus. We find that this construct drives expression only in the retina
and pineal, which is apparent by 4 days post-nuclear injection. These are the first results using transgenic Xenopus for retinal promoter analysis and the potential for the expression in rod photoreceptors of proteins with dominant phenotypes.
[+] show captions
Integration of rhodopsin promoter‐GFP construct into the Xenopus genome. A: Map of the transgene construct pXOP(−5500/+41)GFP. On the mapline, the 5.5 kb Xenopus principal rod opsin promoter is shown as a solid box and the GFP coding region as an open box. Below the line are shown the probes for Southern analysis. The size marks are in kb and enzyme sites are shown: X, XhoI; B, BamHI; P, PstI; N, NotI; E, EcoRI. B: BamHI‐digested DNA from 2 month old tadpoles expressing GFP in the eye (X1, X2), control adult (A) and control tadpole (T) were analyzed by Southern blot using XOP (left) or GFP (right) probes. Arrow indicates the position of the endogenous 5.5 kb BamHI XOP fragment, arrowheads indicate major bands specific to the GFP‐expressing tadpoles and asterisks indicate non‐specific hybridizing species found only in tadpole DNA. The size markers were λ HindIII digest in kb: 23.1, 9.4, 6.6, 4.4, 2.3 and 2.0.
Figure 2 . Lateral view of a transgenic tadpole [Xla.Tg(rho:Eco.nfsA)] (stage 51) under bright field illumination (a), blue light (b) or both (c, the tungsten lamp was dimmed compared to a). The bright green fluorescence from the transgene is seen in the eye. d: Dorsal view of a transgenic tadpole (stage 48) under bright field illumination. The position of the eyes (e), optic nerve (on), olfactory organ (o), cerebral hemispheres (c) and pineal (p) are indicated. A higher magnification image of the pineal under bright field (e) and blue light (f). GFP‐specific fluorescence is seen as a punctate pattern in pineal. g: The anterior portion of a transgenic juvenile frog illuminated with both tungsten lamp and blue light.
An enucleated eye from a transgenic tadpole illuminated under bright field (a) or blue light (b). The position of the lens (L), iris (I), choroid (C) and embryonic fissure (F) which is not closed at this stage are indicated. A piece of whole mounted retina from the same eye is shown under bright field (c) and blue light (d). The rod outer (OS) and inner (IS) segments are indicated.
Figure 4 . Confocal images of a retina from a transgenic tadpole [Xla.Tg(rho:Eco.nfsA)Zuber] produced using pXOP(−5500/+41)GFP. Two separate sections are shown (A and B, C and D). The images under bright field (A, C) were obtained with both bright field and blue light, so the GFP fluorescence (green) is visible in these images. Sections were also viewed for fluorescence from GFP (green) and Cy3 which revealed B630N rod opsin staining (blue) (B, D). Cones are indicated, as well as opsin‐ and GFP‐positive rods.