Transgene expression in Xenopus rods

Department of Anatomy and Cell Biology, University of Kansas, Lawrence, Kansas, United States
FEBS Letters (Impact Factor: 3.17). 02/1998; 423(2):117-21. DOI: 10.1016/S0014-5793(98)00018-0
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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.

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Available from: Brooke M Steenhard, Oct 09, 2015
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    • "Furthermore, the 5′US enhancement of synergistic activation appears to be specific to Xenopus transcription factors. Qualitatively, the relative activity of the promoters containing these 5′US sequences (−503, -5361) is similar in transgenic Xenopus ([3,37] and M. Haeri, personal communication). However, position effects and copy number variation prevent a direct comparison between transgenics and in vitro experiments. "
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    ABSTRACT: In vertebrates, rod photoreceptor-specific gene expression is regulated by the large Maf and Pax-like transcription factors, Nrl/LNrl and Crx/Otx5. The ubiquitous occurrence of their target DNA binding sites throughout rod-specific gene promoters suggests that multiple transcription factor interactions within the promoter are functionally important. Cooperative action by these transcription factors activates rod-specific genes such as rhodopsin. However, a quantitative mechanistic explanation of transcriptional rate determinants is lacking. We investigated the contributions of various paired-like transcription factors and their cognate cis-elements to rhodopsin gene activation using cultured cells to quantify activity. The Xenopus rhodopsin promoter (XOP) has a bipartite structure, with ~200 bp proximal to the start site (RPP) coordinating cooperative activation by Nrl/LNrl-Crx/Otx5 and the adjacent 5300 bp upstream sequence increasing the overall expression level. The synergistic activation by Nrl/LNrl-Crx/Otx5 also occurred when XOP was stably integrated into the genome. We determined that Crx/Otx5 synergistically activated transcription independently and additively through the two Pax-like cis-elements, BAT1 and Ret4, but not through Ret1. Other Pax-like family members, Rax1 and Rax2, do not synergistically activate XOP transcription with Nrl/LNrl and/or Crx/Otx5; rather they act as co-activators via the Ret1 cis-element. We have provided a quantitative model of cooperative transcriptional activation of the rhodopsin promoter through interaction of Crx/Otx5 with Nrl/LNrl at two paired-like cis-elements proximal to the NRE and TATA binding site. Further, we have shown that Rax genes act in cooperation with Crx/Otx5 with Nrl/LNrl as co-activators of rhodopsin transcription.
    BMC Molecular Biology 02/2014; 15(1):4. DOI:10.1186/1471-2199-15-4 · 2.19 Impact Factor
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    • "The Gal4-VP16-GR module (consisting of 147 amino acids of S. cerevisiae Gal4 N-terminal transactivation domain, 59 amino acids of herpes viral protein VP16 and 266 amino acids of rat glucocorticoid receptor protein C terminal domain) was amplified from TOPtk-iGFP plasmid [25] and inserted into peGFP-N1 vector (Clonetech, Mountain View, CA) with poly-Gly linker (LEPLEGTGGGGG) to create the pCMV:G3 plasmid. The CMV promoter was replaced with XOP (-503/+41) promoter [33] to create the XOP:G3 construct. A fragment containing five copies of UAS immediately upstream of Hsp promoter was amplified from pUAS:GFP [34] and subcloned into pGL2 (Promega, Madison, WI) to create pUASLuc, pmCherry-N1 (Clonetech, Mountain View, CA) to create pUAS:mCherry and replacing XOP promoter in pRho-mCherry [5] to create pUAS:Rho-mCherry. "
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    ABSTRACT: We developed an inducible transgene expression system in Xenopus rod photoreceptors. Using a transgene containing mCherry fused to the carboxyl terminus of rhodopsin (Rho-mCherry), we characterized the displacement of rhodopsin (Rho) from the base to the tip of rod outer segment (OS) membranes. Quantitative confocal imaging of live rods showed very tight regulation of Rho-mCherry expression, with undetectable expression in the absence of dexamethasone (Dex) and an average of 16.5 µM of Rho-mCherry peak concentration after induction for several days (equivalent to >150-fold increase). Using repetitive inductions, we found the axial rate of disk displacement to be 1.0 µm/day for tadpoles at 20 °C in a 12 h dark /12 h light lighting cycle. The average distance to peak following Dex addition was 3.2 µm, which is equivalent to ~3 days. Rods treated for longer times showed more variable expression patterns, with most showing a reduction in Rho-mCherry concentration after 3 days. Using a simple model, we find that stochastic variation in transgene expression can account for the shape of the induction response.
    PLoS ONE 12/2013; 8(12):e82629. DOI:10.1371/journal.pone.0082629 · 3.23 Impact Factor
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    • "Plasmids for transgene expression in Xenopus were based upon the pEGFP-N1 (Stratagene) backbone as previously described [22]. DNA fragments from Xenopus opsin (545 or 5417 bp) [22], Xenopus arrestin (XAR7, 285 bp) [50] or Xenopus α-transducin upstream (4996 bp) sequences (unpublished data) were generated by PCR and subcloned into the pEGFP(-) vector [22] at the XhoI-BamHI site. Dual transgene expression constructs were made by assembling each transgene protein cassette separately by PCR and then sub-cloning both into a vector containing duplicate XOP(-504/+41) promoters [50] in the same transcription direction. "
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    ABSTRACT: The rod outer segment (OS), comprised of tightly stacked disk membranes packed with rhodopsin, is in a dynamic equilibrium governed by a diurnal rhythm with newly synthesized membrane inserted at the OS base balancing membrane loss from the distal tip via disk shedding. Using transgenic Xenopus and live cell confocal imaging, we found OS axial variation of fluorescence intensity in cells expressing a fluorescently tagged rhodopsin transgene. There was a light synchronized fluctuation in intensity, with higher intensity in disks formed at night and lower intensity for those formed during the day. This fluctuation was absent in constant light or dark conditions. There was also a slow modulation of the overall expression level that was not synchronized with the lighting cycle or between cells in the same retina. The axial variations of other membrane-associated fluorescent proteins, eGFP-containing two geranylgeranyl acceptor sites and eGFP fused to the transmembrane domain of syntaxin, were greatly reduced or not detectable, respectively. In acutely light-adapted rods, an arrestin-eGFP fusion protein also exhibited axial variation. Both the light-sensitive Rho-eGFP and arrestin-eGFP banding were in phase with the previously characterized birefringence banding (Kaplan, Invest. Ophthalmol. Vis. Sci. 21, 395-402 1981). In contrast, endogenous rhodopsin did not exhibit such axial variation. Thus, there is an axial inhomogeneity in membrane composition or structure, detectable by the rhodopsin transgene density distribution and regulated by the light cycle, implying a light-regulated step for disk assembly in the OS. The impact of these results on the use of chimeric proteins with rhodopsin fused to fluorescent proteins at the carboxyl terminus is discussed.
    PLoS ONE 11/2013; 8(11):e80059. DOI:10.1371/journal.pone.0080059 · 3.23 Impact Factor
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