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.

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.

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 Strand Bias in Targeted Gene Repair Is Influenced by Transcriptional Activity

2002 ◽  
Vol 22 (11) ◽  
pp. 3852-3863 ◽  
Author(s):  
Li Liu ◽  
Michael C. Rice ◽  
Miya Drury ◽  
Shuqiu Cheng ◽  
Howard Gamper ◽  
...  
Keyword(s):  
Transcriptional Activity ◽  
Fluorescent Protein ◽  
Fusion Gene ◽  
Strand Bias ◽  
Nucleotide Exchange ◽  
Gene Repair ◽  
Protein Fusion ◽  
Template Strand ◽  
Targeted Gene Repair ◽  
Targeting Vector

ABSTRACT Modified single-stranded DNA oligonucleotides can direct nucleotide exchange in Saccharomyces cerevisiae. Point and frameshift mutations are corrected in a reaction catalyzed by cellular enzymes involved in various DNA repair processes. The present model centers on the annealing of the vector to one strand of the helix, followed by the correction of the designated base. The choice of which strand to target is a reaction parameter that can be controlled, so here we investigate the properties of strand bias in targeted gene repair. An in vivo system has been established in which a plasmid containing an actively transcribed, but mutated, hygromycin-enhanced green fluorescent protein fusion gene is targeted for repair and upon conversion will confer hygromycin resistance on the cell. Overall transcriptional activity has a positive influence on the reaction, elevating the frequency. If the targeting vector is synthesized so that it directs nucleotide repair on the nontranscribed strand, the level of gene repair is higher than if the template strand is targeted. We provide data showing that the targeting vector can be displaced from the template strand by an active T7 phage RNA polymerase. The strand bias is not influenced by which strand serves as the leading or lagging strand during DNA synthesis. These results may provide an explanation for the enhancement of gene repair observed when the nontemplate strand is targeted.

scholarly journals Spermine analogue-regulated expression of spermidine/spermine N1-acetyltransferase and its effects on depletion of intracellular polyamine pools in mouse fetal fibroblasts

2009 ◽  
Vol 422 (1) ◽  
pp. 101-109 ◽  
Author(s):  
Anne Uimari ◽  
Tuomo A. Keinänen ◽  
Anne Karppinen ◽  
Patrick Woster ◽  
Pekka Uimari ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fusion Gene ◽  
Wild Type Mouse ◽  
Egfp Expression ◽  
Wild Type ◽  
Protein Fusion ◽  
Reverse Transcription Pcr ◽  
Fetal Fibroblasts ◽  
Spermine Analogues ◽  
Ssat Activity

SSAT (Spermidine/spermine N1-acetyltransferase, also known as SAT1), the key enzyme in the catabolism of polyamines, is turned over rapidly and there is only a low amount present in the cell. In the present study, the regulation of SSAT by spermine analogues, the inducers of the enzyme, was studied in wild-type mouse fetal fibroblasts, expressing endogenous SSAT, and in the SSAT-deficient mouse fetal fibroblasts transiently expressing an SSAT–EGFP (enhanced green fluorescent protein) fusion gene. In both cell lines treatments with DENSpm (N1,N11-diethylnorspermine), CPENSpm (N1-ethyl-N11-[(cyclopropyl)-methy]-4,8-diazaundecane) and CHENSpm (N1-ethyl-N11-[(cycloheptyl)methy]-4,8-diazaundecane) led to high, moderate or low induction of SSAT activity respectively. The level of activity detected correlated with the presence of SSAT and SSAT–EGFP proteins, the latter localizing both in the cytoplasm and nucleus. RT–PCR (reverse transcription–PCR) results suggested that the analogue-affected regulation of SSAT–EGFP expression occurred, mainly, after transcription. In wild-type cells, DENSpm increased the amount of SSAT mRNA, and both DENSpm and CHENSpm affected splicing of the SSAT pre-mRNA. Depleted intracellular spermidine and spermine levels inversely correlated with detected SSAT activity. Interestingly, the analogues also reduced polyamine levels in the SSAT-deficient cells expressing the EGFP control. The results from the present study show that the distinct SSAT regulation by different analogues involves regulatory actions at multiple levels, and that the spermine analogues, in addition to inducing SSAT, lower intracellular polyamine pools by SSAT-independent mechanisms.

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.

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