Xenopus rod photoreceptor: model for expression of retinal genes.
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Animals↗
Biomedical subjects
C Schlueter
Publications and source records attributed to C Schlueter.
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.
Animals↗
Nuclear magnetic resonance (NMR) imaging of iron oxide-labeled neural transplants.
Nuclear magnetic resonance (NMR) imaging in vivo of adult rat brains was used to observe the fate of iron oxide-labeled intracerebral neural grafts. The host animals received grafts of fetal (E17-E18) rat tissue prepared as cell suspensions and labeled by incubation with reconstituted Sendai viral envelopes containing iron oxide particles. Control studies were performed in animals following surgical trauma alone or transplantation of unlabeled cell suspensions. In vivo NMR imaging (either two-dimensional Fourier transform or three-dimensional Fourier transform) was performed once on each animal between 5 and 60 days postsurgery. The NMR images of the host brains containing the labeled cells showed sufficient anatomical detail for the identification of the major brain structures. The graft sites were seen in the T2-weighted NMR images as dark regions (low-intensity signal) in the cortex. Histochemical staining for ferric iron (prussian blue stain) demonstrated the presence of numerous prussian blue-positive cells in tissue sections corresponding to the dark regions in the NMR images. Surviving prussian blue-positive cells with neuron-like morphology were relatively more numerous 10 days after transplantation than at 1-2 months postgrafting. The NMR images and immunohistochemical and histochemical staining of hosts containing labeled cells suggest cell migration of astrocytes and macrophages up to 2 mm away from the graft sites. The migrating cells were primarily along fiber tracts in the host white matter. Prussian blue deposits were also present within the brains of the control animals, but the NMR images from the same animals did not contain distinct dark regions like those in the images of brains which had received labeled cell transplants. These results demonstrate that NMR imaging can be used to study cell survival and migration following neural grafting procedures.
Animals↗
Immunobiochemical and molecular biologic characterization of the cell proliferation-associated nuclear antigen that is defined by monoclonal antibody Ki-67.
The monoclonal antibody Ki-67 detects a human nuclear antigen that is present in proliferating cells, but absent in quiescent cells. The aim of this study was to characterize the Ki-67 antigen by means of immunobiochemical and molecular biology techniques. Enzymatic digestion experiments showed that this antigen is highly susceptible to protease treatment, and the antigen cannot be extracted by 0.1 normal HCl, indicating that Ki-67 antigen is a nonhistone protein. Immunoblot analysis of cell lysates with Ki-67 showed a double band with apparent molecular weights of 395 kd and 345 kd, regardless of whether the gels were run under reducing or nonreducing conditions. It is noteworthy that these bands were exclusively detectable in lysates prepared from proliferating cells, whereas they were absent in lysates obtained from quiescent cells. These immunobiochemical data are further substantiated by our molecular cloning approaches. By means of immunocloning with Ki-67, the authors isolated and sequenced several cDNA fragments from lambda gt11 libraries. A 1095-bp fragment gave a strong hybridization signal at 7.5 to 9.5 kb in Northern blot analysis with RNA prepared from proliferating cells, whereas it was negative with RNA prepared from quiescent cells. This cDNA fragment could be bacterially expressed, and in subsequent immunoblot analysis Ki-67 reacted exclusively with those fusion proteins that were derived from bacteria containing the insert in the right reading frame.
Antibodies, Monoclonal↗