scholarly journals Robust Up-Regulation of Nuclear Red Fluorescent-Tagged Fos Marks Neuronal Activation in Green Fluorescent Vasopressin Neurons after Osmotic Stimulation in a Double-Transgenic Rat

Endocrinology ◽  
2009 ◽  
Vol 150 (12) ◽  
pp. 5633-5638 ◽  
Author(s):  
Hiroaki Fujihara ◽  
Yoichi Ueta ◽  
Hitoshi Suzuki ◽  
Akiko Katoh ◽  
Toyoaki Ohbuchi ◽  
...  
Keyword(s):  
Hypertonic Saline ◽  
Supraoptic Nucleus ◽  
Fluorescent Protein ◽  
Fusion Gene ◽  
Regulatory Sequences ◽  
Neuronal Activation ◽  
Protein Fusion ◽  
Transgenic Rats ◽  
Green Fluorescent ◽  
Vasopressin Neurons

Abstract The up-regulation in the expression of mRNA or protein encoded by the c-fos gene is widely used as a marker of neuronal activation elicited by various stimuli. To facilitate the detection of activated neurons, we generated transgenic rats expressing a fusion gene consisting of c-fos coding sequences in frame with monomeric red fluorescent protein 1 (mRFP1) under the control of c-fos gene regulatory sequences (c-fos-mRFP1 rats). In c-fos-mRFP1 transgenic rats, 90 min after hypertonic saline ip administration, nuclear mRFP1 fluorescence was observed abundantly in brain regions known to be osmosensitive, namely the median preoptic nucleus, organum vasculosum lamina terminalis, supraoptic nucleus, paraventricular nucleus, and subfornical organ. Immunohistochemistry for Fos protein confirmed that the distribution of Fos-like immunoreactivity in nontransgenic rats was similar to those of mRFP1 fluorescence after ip administration of hypertonic saline in the transgenic rats. Several double-transgenic rats were obtained from matings between transgenic rats expressing an arginine vasopressin-enhanced green fluorescent protein fusion gene (AVP-eGFP rats) and c-fos-mRFP1 rats. In these double-transgenic rats, almost all eGFP neurons in the supraoptic nucleus and PVN expressed nuclear mRFP1 fluorescence 90 min after hypertonic saline administration. The c-fos-mRFP1 rats are a powerful tool that enables the facile identification of activated neurons in the nervous system. Furthermore, when combined with transgenes expressing another fluorophore under the control of cell-specific regulatory sequences, activation of specific neuronal cell types in response to physiological cues can be readily detected.

scholarly journals Transgenic Expression of Enhanced Green Fluorescent Protein Enables Direct Visualization for Physiological Studies of Vasopressin Neurons and Isolated Nerve Terminals of the Rat

Endocrinology ◽  
2005 ◽  
Vol 146 (1) ◽  
pp. 406-413 ◽  
Author(s):  
Yoichi Ueta ◽  
Hiroaki Fujihara ◽  
Ryota Serino ◽  
Govindan Dayanithi ◽  
Hitoshi Ozawa ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Fusion Gene ◽  
Physiological Role ◽  
Transgenic Rats ◽  
Patch Clamp Technique ◽  
Axon Terminals ◽  
Green Fluorescent ◽  
Vasopressin Neurons

We have generated transgenic rats expressing an arginine vasopressin (AVP)-enhanced green fluorescent protein (eGFP) fusion gene. The expression of the eGFP gene and strong fluorescence were observed in the supraoptic nucleus (SON), the paraventricular nucleus (PVN), and the suprachiasmatic nucleus (SCN) in transgenic rats. The hypothalamo-neurohypophyseal tract, isolated SON neurons, and isolated axon terminals in the neurohypophysis also showed robust eGFP fluorescence. Water deprivation for 2 d increased the fluorescence of the eGFP in the SON and the PVN but not the SCN. The whole-cell patch-clamp technique was then used to record the electrical activities specifically identifying eGFP-expressing SON, PVN, and SCN AVP neurons in in vitro brain slice preparations. The AVP-eGFP transgenic rats are a unique new tool with which to study the physiological role of AVP-secreting neurons in the central nervous system and the dynamics of the regulation of AVP secretion in the living neurons and their axon terminals.

scholarly journals Specific expression of an oxytocin-enhanced cyan fluorescent protein fusion transgene in the rat hypothalamus and posterior pituitary

2009 ◽  
Vol 204 (3) ◽  
pp. 275-285 ◽  
Author(s):  
Akiko Katoh ◽  
Hiroaki Fujihara ◽  
Toyoaki Ohbuchi ◽  
Tatsushi Onaka ◽  
W Scott Young ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fusion Gene ◽  
Plasma Osmolality ◽  
Cyan Fluorescent Protein ◽  
Posterior Pituitary ◽  
Protein Fusion ◽  
Transgenic Rats ◽  
Salt Loading ◽  
Wide Range ◽  
Enhanced Cyan Fluorescent Protein

We have generated rats bearing an oxytocin (OXT)-enhanced cyan fluorescent protein (eCFP) fusion transgene designed from a murine construct previously shown to be faithfully expressed in transgenic mice. In situ hybridisation histochemistry revealed that the Oxt–eCfp fusion gene was expressed in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN) in these rats. The fluorescence emanating from eCFP was observed only in the SON, the PVN, the internal layer of the median eminence and the posterior pituitary (PP). In in vitro preparations, freshly dissociated cells from the SON and axon terminals showed clear eCFP fluorescence. Immunohistochemistry for OXT and arginine vasopressin (AVP) revealed that the eCFP fluorescence co-localises with OXT immunofluorescence, but not with AVP immunofluorescence in the SON and the PVN. Although the expression levels of the Oxt–eCfp fusion gene in the SON and the PVN showed a wide range of variations in transgenic rats, eCFP fluorescence was markedly increased in the SON and the PVN, but decreased in the PP after chronic salt loading. The expression of the Oxt gene was significantly increased in the SON and the PVN after chronic salt loading in both non-transgenic and transgenic rats. Compared with wild-type animals, euhydrated and salt-loaded male and female transgenic rats showed no significant differences in plasma osmolality, sodium concentration and OXT and AVP levels, suggesting that the fusion gene expression did not disturb any physiological processes. These results suggest that our new transgenic rats are a valuable new tool to identify OXT-producing neurones and their terminals.

Phase I clinical trial of a genetically modified and oncolytic vaccinia virus GL-ONC1 with green fluorescent protein imaging (NCT009794131).

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 3062-3062 ◽  
Author(s):  
Khurum Hayat Khan ◽  
Anna-Mary Young ◽  
Joaquin Mateo ◽  
Nina Tunariu ◽  
Timothy Anthony Yap ◽  
...  
Keyword(s):  
Clinical Trial ◽  
Green Fluorescent Protein ◽  
Phase I ◽  
Fluorescent Protein ◽  
Fusion Gene ◽  
Plaque Assay ◽  
Genetically Engineered ◽  
Renilla Luciferase ◽  
Protein Fusion ◽  
Green Fluorescent

3062 Background: GL-ONC is a genetically engineered virus attenuated by insertion of the ruc-gfp (Renilla luciferase and Aequorea green fluorescent protein fusion gene), beta-galactosidase (lacZ) and beta-glucuronidase (gusA) reporter genes into the FL14.5L, J2R (thymidine kinase) and A56R (hemagglutinin) loci, respectively. A phase I trial of intravenous (i.v) GL-ONC1 was pursued to evaluate safety, tolerability, tumour delivery, neutralising antibody development and antitumor activity. Methods: GL-ONC1 was administered at escalating doses (1x105, 1x106, 1x107, 1x108, 1x109, 3x109 plaque forming units (pfu) on day 1; 1.667x107 and 1.667x108, 1.667x109pfu on days 1-3) utilizing a 28-day cycle and a 3+3 dose escalation design. Paired biopsies before treatment and on day 8 for pharmacodynamic and viral delivery evaluation were obtained. Green flourescent protein (GFP) imaging was performed on skin rash and mucosal tumour lesions at baseline and after each cycle. Results: To date, 33 patients (pts) across 8 cohorts have been treated with 1 dose limiting toxicty reported of grade 3 transaminitis after a single infusion at 1x109pfu. Other reported adverse events (n) included pyrexia (26), musculoskeletal pain (10), fatigue (8), nausea and vomiting (4). 2 pts had transient transaminitis; both had liver metastases, which may have contributed to this. 2 pts developed minimally symptomatic poxvirus skin pustules, which appeared green by GFP and were positive to viral plaque assay (VPA). Overall, stable disease (SD) by RECIST was seen at >24 weeks (n=6) and 8-12 weeks (n=5). 2 out of 4 pts in cohort 8 (one with cholangiocarcinoma and another with non-small cell lung caner) achieved SD for median 5.5 months, with a drop in tumour markers at the time of infusions. Conclusions: GL-ONC1 is well tolerated; more frequent delivery of the virus (2 weekly, at the same dose) is planned in an attempt to increase agent exposure. Clinical trial information: NCT009794131.

scholarly journals Identification of Heterochronic Mutants in Caenorhabditis elegans: Temporal Misexpression of a Collagen::Green Fluorescent Protein Fusion Gene

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1335-1351
Author(s):  
Juan E Abrahante ◽  
Eric A Miller ◽  
Ann E Rougvie
Keyword(s):  
Green Fluorescent Protein ◽  
Caenorhabditis Elegans ◽  
Fluorescent Protein ◽  
Fusion Gene ◽  
Terminal Differentiation ◽  
Protein Fusion ◽  
Gene Pathway ◽  
Seam Cell ◽  
Heterochronic Gene ◽  
Green Fluorescent

Abstract The heterochronic genes lin-4, lin-14, lin-28, and lin-29 specify the timing of lateral hypodermal seam cell terminal differentiation in Caenorhabditis elegans. We devised a screen to identify additional genes involved in this developmental timing mechanism based on identification of mutants that exhibit temporal misexpression from the col-19 promoter, a downstream target of the heterochronic gene pathway. We fused the col-19 promoter to the green fluorescent protein gene (gfp) and demonstrated that hypodermal expression of the fusion gene is adult-specific in wild-type animals and temporally regulated by the heterochronic gene pathway. We generated a transgenic strain in which the col-19::gfp fusion construct is not expressed because of mutation of lin-4, which prevents seam cell terminal differentiation. We have identified and characterized 26 mutations that restore col-19::gfp expression in the lin-4 mutant background. Most of the mutations also restore other aspects of the seam cell terminal differentiation program that are defective in lin-4 mutant animals. Twelve mutations are alleles of three previously identified genes known to be required for proper timing of hypodermal terminal differentiation. Among these are four new alleles of lin-42, a heterochronic gene for which a single allele had been described previously. Two mutations define a new gene, lin-58. When separated from lin-4, the lin-58 mutations cause precocious seam cell terminal differentiation and thus define a new member of the heterochronic gene pathway.

scholarly journals Highly Visible Expression of an Oxytocin-Monomeric Red Fluorescent Protein 1 Fusion Gene in the Hypothalamus and Posterior Pituitary of Transgenic Rats

Endocrinology ◽  
2011 ◽  
Vol 152 (7) ◽  
pp. 2768-2774 ◽  
Author(s):  
Akiko Katoh ◽  
Hiroaki Fujihara ◽  
Toyoaki Ohbuchi ◽  
Tatsushi Onaka ◽  
Takashi Hashimoto ◽  
...  
Keyword(s):  
Median Eminence ◽  
Fluorescent Protein ◽  
Fusion Gene ◽  
Female Rats ◽  
Posterior Pituitary ◽  
Red Fluorescent Protein ◽  
Protein Fusion ◽  
Transgenic Rats ◽  
Salt Loading ◽  
Male And Female

We have generated rats bearing an oxytocin (OXT)-monomeric red fluorescent protein 1 (mRFP1) fusion transgene. The mRFP1 fluorescence was highly visible in ventral part of the supraoptic nucleus (SON) and the posterior pituitary in a whole mount. mRFP1 fluorescence in hypothalamic sections was also observed in the SON, the paraventricular nucleus (PVN), and the internal layer of the median eminence. Salt loading for 5 d caused a marked increase in mRFP1 fluorescence in the SON, the PVN, the median eminence, and the posterior pituitary. In situ hybridization histochemistry revealed that the expression of the mRNA encoding the OXT-mRFP1 fusion gene was observed in the SON and the PVN of euhydrated rats and increased dramatically after chronic salt loading. The expression of the endogenous OXT and the arginine vasopressin (AVP) genes were significantly increased in the SON and the PVN after chronic salt loading in both nontransgenic and transgenic rats. These responses were not different between male and female rats. Compared with nontransgenic rats, euhydrated and salt-loaded male and female transgenic rats showed no significant differences in plasma osmolality, sodium concentration, OXT, and AVP levels. Finally, we succeeded in generating a double-transgenic rat that expresses both the OXT-mRFP1 fusion gene and the AVP-enhanced green fluorescent protein fusion gene. Our new transgenic rats are valuable new tools to study the physiology of the hypothalamo-neurohypophysial system.

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