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Dev Dyn
2005 Jun 01;2332:562-9. doi: 10.1002/dvdy.20350.
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Transgenic frogs expressing the highly fluorescent protein venus under the control of a strong mammalian promoter suitable for monitoring living cells.
Sakamaki K
,
Takagi C
,
Yoshino J
,
Yokota H
,
Nakamura S
,
Kominami K
,
Hyodo A
,
Takamune K
,
Yuge M
,
Ueno N
.
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To easily monitor living cells and organisms, we have created a transgenic Xenopus line expressing Venus, a brighter variant of yellow fluorescent protein, under the control of the CMV enhancer/chicken beta-actin (CAG) promoter. The established line exhibited high fluorescent intensity not only in most tissues of tadpoles to adult frogs but also in germ cells of both sexes, which enabled three-dimensional imaging of fluorescing organs from images of the serial slices of the transgenic animals. Furthermore, by using this transgenic line, we generated chimeric animals by brain implantation and importantly, we found that the brain grafts survived and expressed Venus in recipients after development, highlighting the boundary between fluorescent and nonfluorescent areas in live animals. Thus, Venus-expressing transgenic frogs, tadpoles, and embryos would facilitate their use in many applications, including the tracing of the fluorescent cells after tissue/organ transplantation.
Fig. 1. Generation of transgenic animals expressing Venus. A: Structure of the constructed transgene. The transgene CAG- Venus is composed of the fluorescence reporter gene Venus and the rabbit -globin polyA signal sequence under the control of the CAG promoter. Another transgene, XCar-GFP3, is composed of the fluorescence gene GFP3, the SV40 polyA signal sequence, and the Xenopus cardiac actin promoter (Mohun et al., 1986). B: Fluorescent analyses of the transgenic animals. Images of transgenic tadpoles carrying the CAG-Venus (upper animal in a) or the Xcar-GFP3 transgene (lower animal in a, and b) at stage 50 were captured as both brightfield (left panels) and fluorescence (right panels) under the microscope.
Fig. 2. Histological analysis of CAG-Venus transgenic tadpoles. A–F: Longitudinal (A–E) and transverse (F) sections prepared from transgenic tadpoles expressing Venus at stage 47 were taken as fluorescence images under the microscope. b, brain; d, digestive organs; e, eye; f, fin; g, gill; h, heart; m, muscle; n, notochord; s, spinal cord. Scale bars 200 m in A–F.
Fig. 4. (part 1) Morphological and histological analyses of Venus fluorescence in the tissues of CAG- Venus transgenic frogs. A,B: Observation of the fluorescence in the whole body; the dorsal (A) and ventral (B) sides of the wild-type (left side) and transgenic (right side) juvenile frogs were taken for the brightfield (left panels) and fluo- rescence (right panels) images under the micro- scope. C–L: Fluorescent analysis of Venus pro- teins in a variety of tissues dissected from transgenic animals; images of the brain (C), eye (D), hand (E), heart (F), kidneys (G), lobe of the liver (H), lung (I), thyroid gland (J), ovaries (continued in next figure.)
Fig. 5. Transplant engraftment with the brain from transgenic animals. A: Schematic diagram displaying the protocol for brain transplanta- tion. Wild-type tadpoles (n 15) at stages 55–58 were ectomized by removing the anterior one third of the forebrain, and a fragment of the whole brain from CAG-Venus transgenic tad- poles (stages 56–58) was implanted. After the transplantation, the recipient larvae were kept in water for 1, 3, and 4 weeks. B–E: Morpho- logical analysis of the implanted animals; heads of the implanted tadpoles (B,C) and frogs (D,E) were photographed in the light (left), fluorescent (middle), and magnified (right) fields under the microscope at 1 week (B,C), 3 weeks (D), and 4 weeks (E) after surgery. Arrows indicate an en- grafted area.
animals (data not shown). Thus, we embryo to adult stage. Consequently, are currently attempting to visualize we believe CAG-Venus transgenic
