Green Fluorescent Protein Fusion Gene Research Articles

Three-dimensional visualization of mitochondria in conidia of Pyricularia oryzae using green fluorescent protein (GFP) fused with citrate synthase (CitA)

The mitochondrion is the target organelle for QoI (quinone outside inhibitor) antifungal drugs, but mitochondrial behavior in Pyricularia oryzae remains unclear. Conidial mitochondria in fungal isolate transformed with citrate synthase (CitA)-GFP fusion gene, were observed in situ using laser-scanning confocal microscopy and computer analysis. After mycelium was harvested at 36 h and aerial mycelia removed, conidia with 1, 2, or 3 cells were observed over time. The 3D visualization of mitochondria in these conidia showed that mitochondria were initially shaped like dots and developed into a network as the conidia aged.

Structure and Location Studies on Key Enzymes in Saponins Biosynthesis of Panax notoginseng.

Panax notoginseng is one of the most widely used traditional herbs for the treatment of various diseases, in which saponins were the main active components. At present, the research of P. notoginseng mainly focused on the discovery of new compounds and pharmacology. However, there were few studies on the molecular mechanism of the synthesis of secondary metabolites of P. notoginseng. In our study, four coding sequences (CDS) encoding the key enzymes involved in saponin biosynthesis were cloned, namely farnesyl diphosphate synthase (FPS), squalene synthase (SS), squalene epoxidase (SE), and dammarenediol-II synthase (DS), which contained open reading frame (ORF) of 1029 bp, 1248 bp, 1614 bp, and 2310 bp, and coded 342, 415, 537, and 769 amino acids, respectively. At the same time, their domains, secondary structures, three-dimensional structures, and phylogenetics trees were analyzed by kinds of bioinformatics tools. Their phylogenetics relationships were also analyzed. In addition, GFP (Green fluorescent protein) fusion genes were constructed by the plasmid transformation system to determine the subcellular localization. The results of subcellular localization showed that FPS, SE, and DS were mainly located in cytomembrane and its surrounding, while SS was located both in cytoplasm and cytomembrane. Our findings provided data demonstrating the expression patterns of genes involved in saponin biosynthesis and would facilitate efforts to further elucidate the biosynthesis of the bioactive components in P. notoginseng.

Excitatory GABAergic Action and Increased Vasopressin Synthesis in Hypothalamic Magnocellular Neurosecretory Cells Underlie the High Plasma Level of Vasopressin in Diabetic Rats.

Diabetes mellitus (DM) is associated with increased plasma levels of arginine-vasopressin (AVP), which may aggravate hyperglycemia and nephropathy. However, the mechanisms by which DM may cause the increased AVP levels are not known. Electrophysiological recordings in supraoptic nucleus (SON) slices from streptozotocin (STZ)-induced DM rats and vehicle-treated control rats revealed that γ-aminobutyric acid (GABA) functions generally as an excitatory neurotransmitter in the AVP neurons of STZ rats, whereas it usually evokes inhibitory responses in the cells of control animals. Furthermore, Western blotting analyses of Cl- transporters in the SON tissues indicated that Na+-K+-2Cl- cotransporter isotype 1 (a Cl- importer) was upregulated and K+-Cl- cotransporter isotype 2 (KCC2; a Cl- extruder) was downregulated in STZ rats. Treatment with CLP290 (a KCC2 activator) significantly lowered blood AVP and glucose levels in STZ rats. Last, investigation that used rats expressing an AVP-enhanced green fluorescent protein fusion gene showed that AVP synthesis in AVP neurons was much more intense in STZ rats than in control rats. We conclude that altered Cl- homeostasis that makes GABA excitatory and enhanced AVP synthesis are important changes in AVP neurons that would increase AVP secretion in DM. Our data suggest that Cl- transporters in AVP neurons are potential targets of antidiabetes treatments.

Construction and expression of a lentivirus expression vector carrying the VEGF165-EGFP fusion gene in breast cancer MCF-7 cells.

Vascular endothelial growth factor (VEGF)165 is one of the most abundant and potent angiogenic factors in both physiological and pathological conditions. However, the function and mechanism of VEGF165 in tumors and their environment remain to be elucidated. In the present study, a lentivirus vector (LV) that contained the VEGF165-enhanced green fluorescent protein (EGFP) fusion gene was constructed and transfected into the human breast cancer cell line MCF-7. Following transfection, the expression of VEGF165 in MCF-7 cells was detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blotting. Further cellular localization of VEGF165 was observed through fluorescence microscopy. The titer of the recombinant lentivirus was 5.44×107 TU/ml in the LV-VEGF165-EGFP group and 5.00×108 TU/ml in the LV-EGFP negative control group. RT-qPCR and western blotting demonstrated that the expression of VEGF165 was significantly increased in the LV-VEGF165-EGFP group compared with the control group. The present study lays the foundation for in vitro and in vivo studies on tumor cell derived-VEGF165. Furthermore, the present fusion gene expression vector may provide a potential approach for gene therapy treatment of cancer and other diseases that require regulation of angiogenesis.

Transgenic approach to express the channelrhodopsin 2 gene in arginine vasopressin neurons of rats

Optogenetics provides a powerful tool to regulate neuronal activity by light-sensitive ion channels such as channelrhodopsin 2 (ChR2). Arginine vasopressin (AVP; also known as the anti-diuretic hormone) is a multifunctional hormone which is synthesized in the magnocellular neurosecretory cells (MNCs) of the hypothalamus. Here, we have generated a transgenic rat that expresses an AVP-ChR2-enhanced green fluorescent protein (eGFP) fusion gene in the MNCs of the hypothalamus. The eGFP fluorescence that indicates the expression of ChR2-eGFP was observed in the supraoptic nucleus (SON) and in the magnocellular division of the paraventricular nucleus (PVN) that is known to contain AVP-secreting neurons. The eGFP fluorescence intensities in those nuclei and posterior pituitary were markedly increased after chronic salt loading (2% NaCl in drinking water for 5days). ChR2-eGFP was localized mainly in the membrane of AVP-positive MNCs. Whole-cell patch-clamp recordings were performed from single MNCs isolated from the SON of the transgenic rats, and blue light evoked repetitive action potentials. Our work provides for the first time an optogenetic approach to selectively activate AVP neurons in the rat.

Use of αv Integrin Linked to GFP to Image Molecular Dynamics in Trafficking Cancer-Cell Emboli.

Human osteosarcoma cells with an αv integrin green fluorescent protein (GFP) fusion gene were previously established and imaged in vitro and in vivo. In the present study, we imaged the interaction of αv integrin-GFP in osteosarcoma cells and collagen fibers in vascular-trafficking osteosarcoma emboli in nude mice. Human 143B osteosarcoma cells, expressing αv integrin-GFP, were injected by a vascular route in an abdominal skin flap in nude mice. Osteosarcoma cells were fluorescently imaged in the epigastric cranialis vein in the abdominal skin flap by confocal microscopy. Collagen fibers were imaged in reflectance mode. At early stages of tumor embolus-formation, cancer cells adhered firmly to each other, diffusely expressing αv integrin-GFP. Two weeks after injection, collagen fiber scaffolds were visualized at the margins of tumor emboli or within them. Four weeks after injection, cancer cells invading from emboli were strongly expressing αv integrin-GFP, and were aligned along collagen fibers. The results suggest αv integrin and collagen fiber scaffolds are important for tumor embolus formation, which are potential seeds of metastasis. J. Cell. Biochem. 118: 26-30, 2017. © 2016 Wiley Periodicals, Inc.

Germinal responses in murine Peyer’s Patches are regulated by a subpopulation of microbiota-induced IL-21high Tfh cells

Abstract IL-21 is the signature cytokine produced by T follicular helper (Tfh) cells and is vital in driving both the germinal centre (GC) reaction and formation of high affinity antibodies. However, the degree of Tfh cell heterogeneity and function is not fully understood. By utilising a novel IL-21eGFP reporter mouse carrying a diphtheria toxin receptor (DTR)-enhanced green fluorescent protein (eGFP) fusion gene, we identified a subpopulation of highly differentiated Tfh cells within Peyer’s Patches (PPs). This subpopulation demonstrated highest expression of the key Tfh molecules - Bcl6, ICOS and IL-21, and was found within germinal centres and surrounding B cell areas. TCRb repertoire analysis of GFP+Tfh cells revealed that these were polyclonal and closely related to GFP− Tfh cells, indicating selection by common antigens. In vitro stimulation of IL-21eGFP CD4+ cells in the presence of IL-6, TGFb and retinoic acid led to the induction of GFP+ cells that did not co-express IL-17 or Foxp3. Treatment of IL-21eGFP mice with antibiotics led to an ablation of PP GFP+CD4+ cells meaning that the presence of these cells relies on an intact bacterial microbiota. Furthermore, both polyclonal CD4+ T cell and B cell activation is necessary to support the differentiation of GFP+Tfh cells. Finally, GFP+ cell depletion resulted in a reduced frequency of PP GC B cells and IgG1+ cells and small but significant shifts in gut microbiome composition. Therefore, using a new IL-21-reporter mouse we have identified a subpopulation of Tfh cells induced by the gut microbiota and necessary for optimal PP GC and IgG1 responses.

Antitumor Effect of IP-10 by Using Two Different Approaches: Live Delivery System and Gene Therapy.

PurposeImmunotherapy is one of the treatment strategies for breast cancer, the most common cancer in women worldwide. In this approach, the patient's immune system is stimulated to attack microscopic tumors and control metastasis. Here, we used interferon γ-induced protein 10 (IP-10), which induces and strengthens antitumor immunity, as an immunotherapeutic agent. We employed Leishmania tarentolae, a nonpathogenic lizard parasite that lacks the ability to persist in mammalian macrophages, was used as a live delivery system for carrying the immunotherapeutic agent. It has been already shown that arginase activity, and consequently, polyamine production, are associated with tumor progression.MethodsA live delivery system was constructed by stable transfection of pLEXSY plasmid containing the IP-10-enhanced green fluorescent protein (IP-10-egfp) fusion gene into L. tarentolae. Then, the presence of the IP-10-egfp gene and the accurate integration location into the parasite genome were confirmed. The therapeutic efficacy of IP-10 delivered via L. tarentolae and recombinant pcDNA-(IP-10-egfp) plasmid was compared by determining the arginase activity in a mouse 4T1 breast cancer model.ResultsThe pcDNA-(IP-10-egfp) group showed a significant reduction in tumor weight and growth. Histological evaluation also revealed that only this group demonstrated inhibition of metastasis to the lung tissue. The arginase activity in the tissue of the pcDNA-(IP-10-egfp) mice significantly decreased in comparison with that in normal mice. No significant difference was observed in arginase activity in the sera of mice receiving other therapeutic strategies.ConclusionOur data indicates that IP-10 immunotherapy is a promising strategy for breast cancer treatment, as shown in the 4T1-implanted BALB/c mouse model. However, the L. tarentolae-(IP-10-EGFP) live delivery system requires dose modifications to achieve efficacy in the applied regimen (six injections in 3 weeks). Our results indicate that the arginase assay could be a good biomarker to differentiate tumoral tissues from the normal ones.

Synthesis of radial mesoporous bioactive glass particles to deliver osteoactivin gene.

Mesoporous bioactive glasses (MBGs) can be used as carriers for biomolecule delivery with improved functions. Although there are a great number of studies on drug delivery by MBGs, until now little work has been done to investigate the DNA gene transfection effect of MBGs. In this study, radial mesoporous bioactive glasses (rMBGs) were prepared by sol-gel process combined with a micro-emulsion method. The surface was further modified by amino groups in order to improve its affinity for DNA. Our study showed that rMBGs have good apatite-forming ability and cellular biocompatibility. In addition, rMBGs can enter cells in a time- and dose-dependent manner, and mainly localize in the cytoplasm. Agarose gel electrophoresis demonstrated that pOA-EGFP (containing the osteoactivin and the green fluorescent protein fusion gene) can be completely absorbed and protected from DNase I degradation by the aminated rMBGs. Additionally, the plasmid can be successfully expressed in cells transfected by rMBGs.

Sonic Hedgehog Signaling Pathway Supports Cancer Cell Growth during Cancer Radiotherapy

Tumor growth after radiotherapy is a commonly recognized cause of therapeutic failure. In this way, we examined tumor cell growth after radiotherapy by establishing a cancer cell growth model in vitro. We accomplished this model by seeding non-irradiated firefly luciferase2 and green fluorescent protein fusion gene (Fluc) labeled living cancer reporter cells onto a feeder layer of irradiated cancer cells. The living tumor cell growth was monitored by bioluminescence imaging. The living reporter cells grew faster when seeded onto lethally irradiated feeder cells than when seeded onto non-irradiated feeder cells or when seeded in the absence of feeder cells. We found that the expression levels of the Shh and Gli1 proteins, both of which are critical proteins in Sonic hedgehog (SHH) signaling, were increased after irradiation and that this expression was positively correlated with reporter cell growth. Moreover, the dying cell stimulation of living tumor cell growth was enhanced by the addition of SHH signaling agonists and inhibited by SHH signaling antagonists. SHH agonists also enhanced reporter cell growth in the absence of irradiated feeder cells, suggesting this mechanism plays a role in feeder cell growth stimulation. Given these results, we conclude that SHH signaling activation plays an important role during tumor repopulation after radiotherapy.

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

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.

Characterization of a Novel NBS-LRR Gene Involved in Bacterial Blight Resistance in Rice

Nucleotide-binding site leucine-rich repeat (NB-LRR) genes play important roles in plant disease resistance. Due to its similarity to Rp1 in maize (Zea mays), a novel NB-LRR gene was isolated from rice and designated as OsRP1L1 (Oryza sativa Rp1-like 1). Analysis of expression of a super green fluorescent protein (sGFP) fusion gene revealed that the OsRP1L1 protein localizes to the nucleolus. Expression patterns suggested that OsRP1L1 was involved in responses to several plant growth regulators (PGRs) and environmental stresses. To explore its function in bacterial blight (BB) resistance, OsRP1L1 was cloned and overexpressed in a susceptible japonica rice cultivar. Overexpression of OsRP1L1 moderately elevated the resistance of plants to Xanthomonas oryzae pv. oryzae strains PXO86 and PXO341. Transcriptome analysis showed that plants overexpressing OsRP1L1 had small changes in their transcriptome, with 26 genes showing statistically significant changes of more than two-fold. The most up-regulated transcripts—a DIN1-like gene and a terpene synthase (TPS) gene—were up-regulated 7.9- and 7.6-fold, respectively. It was also noted that seven of the up-regulated genes have also reportedly been induced by overexpression of OsbZIP46, which has been identified recently as a player in ABA sensing. All these findings suggest that OsRP1L1 plays an important role in rice BB resistance, and may be useful as a genetic resource for engineering disease resistance in plants.

Expression changes of tumor metastasis-related genes after overexpression of KAI1 in retinoblastoma Y79 cells

To investigate the expression changes of tumor metastasis-related genes after overexpression of KAI1 in retinoblastoma Y79 cells. Experimental study. Y79 cells were transfected with a lentivirus vector containing KAI1 and enhanced green fluorescent protein (EGFP) fusion gene, or a control lentivirus vector containing EGFP. Positive transfectants stably expressing high levels of KAI1 were named Y79-KAI1 and control transfectants were named Y79-KAI1/zero. These transfectants were selected by puromycin resistance and analysis with fluorescent microscopy. The expression of KAI1 mRNA and its protein among Y79, Y79-KAI1 and Y79-KAI1/zero were detected by fluorescent quantitative RT-PCR and Western blot. Differential expression of tumor metastasis-related genes in Y79-KAI1 and Y79-KAI1/zero was analyzed with human tumor metastasis PCR array. One-way ANOVA was used to analyze the differences of KAI1 mRNA and protein expression among the three groups. The stably transfected cell lines of Y79-KAI1 and Y79-KAI1/zero were established. The result of fluorescent quantitative real-time PCR showed that the relative quantification of mRNA level of KAI1 gene in the three kinds of cells above was 183.67 ± 21.20, 1.42 ± 0.55, 1.00 ± 0.00, respectively. And the expression level of KAI1 mRNA in Y79-KAI1 cells was significantly higher than those in Y79-KAI1/zero and Y79 cells (F = 108.74, P = 0.000). The results of Western blot showed that the expression level of the KAI1 protein in Y79-KAI1 cells was significantly higher than those in Y79-KAI1/zero and Y79 cells (F = 34.36, P = 0.001). Immunofluorescent staining showed that Y79 and Y79-KAI1/zero cells had no detectable KAI1 expression, while Y79-KAI1 cells expressed KAI1 in the cytoplasm surrounding the nuclei. Among the 84 tumor metastasis-related genes examined, 7 genes were up-regulated more than 2 folds and 6 genes were down-regulated over 50%. Over-expression of KAI1 may result in differential expression of tumor metastasis-related genes in Y79 cells, which may be related to the inhibitory effect on the tumor metastasis of retinoblastoma.

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

2530 Background: GL-ONC1 is a genetically engineered vaccinia 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 F14.5L, J2R (thymidine kinase) and A56R (hemaglutinin) loci. A phase I clinical trial of iv GL-ONC1 was pursued to evaluate safety, tolerability, tumour delivery, neutralizing antibody development and anti-tumour activity. Methods: GL-ONC1 was administered to patients with advanced solid tumours at escalating doses (1×105, 1×106, 1×107, 1×108, 1×109, 3×109 plaque-forming units (pfu) on day 1; 1.667×107 and 1.667×108 pfu on day 1-3 of a 28-day cycle) using a 3+3 dose escalation design. Green fluorescent protein (GFP) imaging was performed on skin rash and mucosal tumour lesions at baseline and after each cycle. Optional tumour biopsies were obtained for pharmacodynamic and viral delivery evaluation. Results: 27 patients (21 males, median age 60 years) were treated. One of six patients at the 1x109 pfu dose level developed a dose-limiting, grade 3 rise in aspartate transaminase levels after a single infusion. Other reported adverse events (grade 1/2) included pyrexia (21), musculoskeletal pain (9), fatigue (8), nausea (7), and vomiting (4). Two patients developed skin rash during the first week of treatment, which appeared green by GFP and were positive to viral plaque assay (VPA). VPA of blood, urine, stool and sputum were negative for viral shedding in all but one patient who had positive shedding for 11 days. Increased neutralizing antibody titres were detected in all tested patients apart from one. Best response by RECIST was stable disease at 48 weeks (1), 24 weeks (3) and 8-12 weeks (5). A patient with squamous cell carcinoma of the tongue had a biopsy after 4 cycles which showed positive IHC staining of vaccinia virus. This is the first time that a virus is shown to be delivered to solid tumour after iv delivery. Conclusions: GL-ONC1 administered iv is well tolerated, with documented colonisation in patient tumour, and preliminary evidence of anti-tumour activity.

遺伝子改変動物を用いた中枢神経系における侵害受容反応の可視化

Physiological response to acute and chronic nociceptive stimulation are important for living organisms. In our laboratory, we generated transgenic rats expressing the arginine vasopressin (AVP) and enhanced green fluorescent protein (eGFP) fusion gene, and the c-fos and monomeric red fluorescent protein 1 (mRFP1) fusion gene in the central nervous system. We made it possible to visualize the pain response in the living cells. Using these transgenic rats, the aim of our studies is the elucidation of the physiological role of AVP after nociceptive stimulation and the pathophysiology of work-related pain. We describe the previous findings of nociceptive response, using these transgenic animals.

Upregulation of arginine vasopressin synthesis in the rat hypothalamus after kainic acid-induced seizures

We examined the effects of kainic acid (KA)-induced seizures on arginine vasopressin (AVP) gene expression in the paraventricular (PVN) and the supraoptic nuclei (SON) of normal rats using in situ hybridization histochemistry. We also investigated the expression of the AVP-enhanced green fluorescent protein (eGFP) fusion gene after KA-induced seizures in transgenic rats. AVP heteronuclear (hn) RNA levels in the PVN and the SON were significantly increased at 3 h and 24 h after subcutaneous (s.c.) administration of KA in normal rats. AVP mRNA levels in the PVN and the SON did not change significantly at 3 h, 24 h and 1 week after s.c. administration of KA in normal rats. In KA-administered transgenic rats, AVP-eGFP fluorescence in the magnocellular and parvocellular divisions of the PVN and the SON were significantly stronger compared to vehicle-administered transgenic rats. By pretreatment with MK-801 (nonselective N-methyl-D-aspartate, NMDA, receptor antagonist), AVP-eGFP transgenic rats after administration of KA did not show preconvulsive symptoms or convulsions and AVP-eGFP fluorescence in the magnocellular and parvocellular divisions of the PVN and the SON of these rats was significantly reduced. These results suggested that KA-induced increases in AVP transcripts and AVP were prevented by MK801 because seizure activity was prevented or reduced.

A phase I clinical trial of a genetically modified and imageable oncolytic vaccinia virus GL-ONC1 with clinical green fluorescent protein (GFP) imaging.

2577 Background: GL-ONC1 is a genetically engineered vaccinia 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 F14.5L, J2R (thymidine kinase) and A56R (hemagglutinin) loci, respectively. A Phase I trial of intravenous GL-ONC1 was pursued to evaluate safety, tolerability, tumour delivery, neutralizing antibody development and anti tumour activity. Methods: GL-ONC1 was to be administered at escalating doses (1×105, 1×106, 1×107, 1×108, 1×109, 3×109 plaque-forming units (pfu) on day 1 only; 1.667×107 and 1.667×108 pfu on day 1-3; 1×109 pfu on day 1-5) using a 3+3 dose escalation design and a q28 days schedule. GFP imaging was performed at baseline and after each cycle on superficial and mucosal lesions. Results: To date, 21 patients (males 15, median age 57 years) have been treated without dose limiting toxicities. Adverse events reported were generally mild (grade 1/2) and included pyrexia (n:8), fatigue (n:4), myalgia (n:3), pustular rash (n:2), hypotension (n:2), nausea (n:2), tachycardia (n:2), anorexia (n:1), diarrhoea (n:1), hyperhidrosis (n:1), oedema (n:1), rhinorrhea (n:1), rigor (n:1), seborrhoea (n:1), thrombocytosis (n:1), flank pain (n:1), feet plantar tenderness (n:1), back pain (n:1) calf pain (n:1) and vomiting (n:1). One patient suffered a left common femoral artery embolism of uncertain causality (grade 3). Two patients had a pustular rash, 1 asymptomatic (grade 1) and 1 symptomatic with itching (grade 2). Both appeared during the first week of treatment with green fluorescence on GFP imaging and positivity by viral plaque assay (VPA) and resolved spontaneously by the end of cycle 1. VPA of blood, urine, stool and sputum were negative for viral shedding in all but 1 patient who had positive shedding for 11 days. Increased neutralizing antibody titres were seen in all tested patients except 1. Best response was RECIST stable disease > 6 months (n:1) and 3-6 months (n:5). Conclusions: GL-ONC1 can be administered intravenously, is well tolerated and its delivery can be imaged in cancer patients.

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

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.

Abstract 2051: Hypoxia-induced triple suicide gene TK/CD/UPRT expression enhances cytotoxicity to GCV and 5-FC and radiosensitization in human colorectal cancer cells

Abstract The hypoxic microenvironment, an important feature of human solid tumors but absent in normal tissues, may provide an opportunity for cancer-specific gene theapy. We constructed two vectors, both under the control of nine copies of hypoxia responsive element (HRE): (1) a herpes simplex virus type 1 thymidine kinase (TK) and enhanced green fluorescent protein (eGFP) fusion gene, and (2) a triple-fusion-gene, cytosine deaminase (CD), uracil phosphoribosyltransferase (UPRT) and monomeric DsRed (mDsRed). Human colorectal cancer cells (HCT8) were transfected with these plasmids, singly or in combination. Stable HCT8-HRE cells were established that contained and expressed the hypoxia-inducible vectors (i.e. HRE-TK/eGFP, HRE-CD/UPRT/mDsRed, or both HRE-TK/eGFP and HRE-CD/UPRT/mDsRed), as assessed by RT-PCR, Western blots and fluorescence microscopy analysis. In in vitro studies, we showed that hypoxia-induced TK/eGFP and CD/UPRT/mDsRed expression not only increased cytotoxicity to GCV and 5-FC, but also led to significant radiosensitization. In HCT8-HRE tumors transplanted in athymic mice, the distribution of TK/eGFP and CD/UPRT/mDsRed co-localized with that of the hypoxia marker pimonidazole. In addition, the administration of GCV and 5-FC in combination with local irradiation resulted in significant tumor regression, greater than that observed with drug or radiation treatment alone. Our data suggest that the hypoxia-inducible TK/GCV+CDUPRT/5-FC triple gene therapy has the ability to specifically target hypoxic cancer cells and significantly improve tumor control in combination with radiotherapy. This novel model is a valuable tool for studying tumor hypoxia and hypoxia-targeted radio-gene therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2051. doi:10.1158/1538-7445.AM2011-2051

Correction of Frameshift Mutations with Tailed Duplex DNAs

Tailed duplex (TD) DNAs, prepared by annealing an oligonucleotide to a several-hundred-base single-stranded (ss) DNA fragment, correct a base-substitution mutation with high efficiency. In the present study, the abilities of TD fragments to correct single-base insertion and deletion mutations were examined, using hygromycin-resistance and enhanced green fluorescent protein fusion (Hyg-EGFP) genes inactivated by +G and -C frameshift mutations. The 5'-TD and 3'-TD DNA fragments were co-transfected with plasmid DNA containing the inactivated Hyg-EGFP gene into CHO-K1 cells, and the gene correction efficiencies were determined by introducing the plasmid DNA recovered from the transfected cells into Escherichia coli cells. In contrast to their efficiencies for the substitution mutation, the gene correction abilities of the TD fragments were relatively low. The correction efficiencies by the TD fragments were apparently higher than that by a ss DNA fragment, one of the DNA fragments employed for gene correction. These results suggest that the TD fragments have the potential to correct frameshift mutations, although further improvement is required.