Green Fluorescent Protein Transgene Research Articles

Organ Culture and Immunostaining of Mouse Embryonic Kidneys

The study of organogenesis in mammals allows investigation of a wide variety of basic cell biological processes in the context of the intact organ. This has become especially important in the age of genetics, as the consequences of gene deletion or mutation in the mouse can be directly linked to human congenital abnormalities. The ability to culture some organs ex vivo during development has emerged as an important tool to understand how tissues are constructed and the signaling pathways that regulate these processes. It has been especially useful in organs that grow via branching morphogenic mechanisms, such as the lung and kidney. Here we demonstrate isolation, ex vivo growth, and fluorescent immunostaining of mouse embryonic day 12.5 (E12.5) kidneys. To demonstrate nephron formation using live imaging, we have isolated and cultured kidneys from mice carrying a green fluorescent protein (GFP) transgene driven by the Hes 1 promoter, which is expressed early in the developing nephron. We also provide a protocol for robust imaging of multiple kidney structures in the whole-mount setting. These techniques serve as a basic platform for the analysis of branching morphogenesis and nephron formation in genetic mouse models or in response to exogenous factors, such as agonists or inhibitors, which can be directly added to the culture medium.

Gene targeting with zinc finger nucleases to produce cloned eGFP knockout pigs

We report using zinc finger nucleases (ZFNs) and somatic cell nuclear transfer (SCNT) to generate pigs with a knockout (KO) mutation of an enhanced green fluorescent protein (eGFP) transgene. ZFNs are synthetic modular proteins composed of a FokI endonuclease domain linked to a sequence-specific zinc finger DNA-binding domain. Pairs of ZFNs bind to the target region, allowing FokI dimerization and subsequent DNA cleavage. Mutation of an eGFP transgene by using ZFNs has been demonstrated in rats (Geurts et al., 2009); however, there are no reports of ZFN-mutated livestock offspring. Using ZFNs to increase the efficiency of gene modification may advance the production of agriculturally and clinically relevant animal models, particularly in species where modification is difficult. All animal procedures followed an approved IACUC protocol. Adult porcine ear fibroblasts hemizygous for the eGFP transgene (Whitworth et al., 2009), from a grandson of the founder animal, were cultured to 75% confluency, trypsinized, and cotransfected (Ross et al., 2010) with a pair of ZFN plasmids that bind to opposing strands at the eGFP target site (Sigma–Aldrich CompoZr®) and a red fluorescent CAG-tomato plasmid as a transient selectable fluorophore reporting transfection efficiency. Transfected fibroblasts were cultured for 96 h and selected for red fluorescence by automated cell sorting (FACS; Fig. 1A,B) and seeded into 96-well plates (100 cells/well) based on CAG-tomato expression (2% of total cells sorted). A second round of FACS using negative selection of eGFP fluorescence enriched for eGFP KO cells (~5% of sorted cells). PCR of genomic DNA from fibroblasts in wells containing predominantly non-green cells was used to amplify a fragment bracketing the ZFN targeting site. The PCR product was cloned into E. coli, and 16 colonies showed mutations by Sanger sequencing. These mutations at the ZFN cleavage site included a 6-bp deletion, a 333-bp deletion, and a 222-bp deletion replaced with a 113-bp inversion of the deleted sequence (Supplemental Information http://animalsciences.missouri.edu/faculty/prather/). Fibroblast colonies determined to carry ZFN-induced mutations were used as donor cells for SCNT, and embryo transfer (n = 3; Whitworth et al., 2009). One Day-12 embryo collection recovered 7 of 9 embryos that did not fluoresce. Quality sequence was obtained from four embryos, and confirmed that the two embryos that fluoresced had an intact eGFP and two embryos that did not fluoresce were mutated. One of seven piglets recovered via Cesarean section at term for the other two pregnancies fluoresced (Fig. 1C). DNA sequencing confirmed that this piglet had unaltered sequence at the predicted ZFN cut site, while the non-fluorescing piglets had various deletions and insertions. While a direct comparison to a 12% mutation rate in the rat made by microinjection (Geurts et al., 2009) cannot be made because of our FACS preselection and SCNT methods, six out of seven founders being mutated is consistent with their results. This study establishes ZFN-based gene modification in a large animal model. The methods used are anticipated to be useful in selective knockout of endogenous swine genes of agricultural and biomedical importance without introducing any transgenic sequence into the genome. Figure 1 A: Control porcine ear fibroblasts; eGFP and Hoechst DNA stain fluorescence. B: Porcine ear fibroblasts treated with eGFP-specific ZFNs; eGFP and Hoechst DNA stain fluorescence. GFP fluorescence is absent in a high proportion of the treated fibroblasts ...

Renal tubular Fas ligand mediates fratricide in cisplatin-induced acute kidney failure

Cisplatin, a standard chemotherapeutic agent for many tumors, has an unfortunately common toxicity where almost a third of patients develop renal dysfunction after a single dose. Acute kidney injury caused by cisplatin depends on Fas-mediated apoptosis driven by Fas ligand (FasL) expressed on tubular epithelial and infiltrating immune cells. Since the role of FasL in T cells is known, we investigated whether its presence in primary kidney cells is needed for its toxic effect. We found that all cisplatin-treated wild-type (wt) mice died within 6 days; however, severe combined immunodeficiency (SCID)/beige mice (B-, T-, and natural killer-cell-deficient) displayed a significant survival benefit, with only 55% mortality while exhibiting significant renal failure. Treating SCID/beige mice with MFL3, a FasL-blocking monoclonal antibody, completely restored survival after an otherwise lethal cisplatin dose, suggesting another source of FasL besides immune cells. Freshly isolated primary tubule segments from wt mice were co-incubated with thick ascending limb (TAL) segments freshly isolated from mice expressing the green fluorescent protein (GFP) transgene (same genetic background) to determine whether FasL-mediated killing of tubular cells is an autocrine or paracrine mechanism. Cisplatin-stimulated primary segments induced apoptosis in the GFP-tagged TAL cells, an effect blocked by MFL3. Thus, our study shows that cisplatin-induced nephropathy is mediated through FasL, functionally expressed on tubular cells that are capable of inducing death of cells of adjacent tubules.

Visualization of plant viral suppressor silencing activity in intact leaf lamina by quantitative fluorescent imaging

BackgroundTransient expression of proteins in plants has become a favoured method over the production of stably transformed plants because, in addition to enabling high protein yields, it is both fast and easy to apply. An enhancement of transient protein expression can be achieved by plant virus-encoded RNA silencing suppressor proteins. Since viral suppressor proteins differ in their efficiency to enhance transient protein expression in plants, we developed a whole-leaf green fluorescent protein (GFP)-based imaging assay to quantitatively assess suppressor protein activity.ResultsIn a transient GFP-expression assay using wild-type and GFP-transgenic N. benthamiana, addition of the plant viral suppressors Beet mild yellowing virus (BMYV-IPP) P0 or Plum pox virus (PPV) HC-Pro was shown to increase fluorescent protein expression 3-4-fold, 7 days post inoculation (dpi) when compared to control plants. In contrast, in agroinfiltrated patches without suppressor activity, near complete silencing of the GFP transgene was observed in the transgenic N. benthamiana at 21 dpi. Both co-infiltrated suppressors significantly enhanced GFP expression over time, with HC-Pro co-infiltrations leading to higher short term GFP fluorescence (at 7 dpi) and P0 giving higher long term GFP fluorescence (at 21 dpi). Additionally, in contrast to HC-Pro co-infiltrations, an area of complete GFP silencing was observed at the edge of P0 co-infiltrated areas.ConclusionsFluorescence imaging of whole intact leaves proved to be an easy and effective method for spatially and quantitatively observing viral suppressor efficiency in plants. This suppressor assay demonstrates that plant viral suppressors greatly enhanced transient GFP expression, with P0 showing a more prolonged suppressor activity over time than HC-Pro. Both suppressors could prove to be ideal candidates for enhancing target protein expression in plants.

Effects of Gene Methylation Reprogramming in Cloned Calves Derived from In Vitro-Transfected Somatic Cells

In vitro transfection of cultured cells combined with nuclear transfer currently is the most effective procedure to produce transgenic livestock. In the present study, bovine primary fetal fibroblasts were transfected with a green fluorescent protein (GFP) reporter transgene and used as nuclear donor cells in oocyte reconstructions. To examine the role of host cytoplasm on transgene expression and developmental outcome, GFP-expressing fibroblasts were fused to oocytes reconstructed either metaphase or telophase activation, and PCR technology was also employed. The results showed that GFP became detectable at the 8- to 16-cell stage, approximately 80h after reconstruction, and remained positive at all later stages. Embryonic development to the blastocyst stage was not significantly different among metaphase and telophase groups. Therefore, GFP transgene technology can be used to select embryoes derived from transgenic animals.

Fluorescent Leishmania species: Development of stable GFP expression and its application for in vitro and in vivo studies

Reporter genes have proved to be an excellent tool for studying disease progression. Recently, the green fluorescent protein (GFP) ability to quantitatively monitor gene expression has been demonstrated in different organisms. This report describes the use of Leishmania tarentolae (L. tarentolae) expression system (LEXSY) for high and stable levels of GFP production in different Leishmania species including L. tarentolae, L. major and L. infantum. The DNA expression cassette (pLEXSY-EGFP) was integrated into the chromosomal ssu locus of Leishmania strains through homologous recombination. Fluorescent microscopic image showed that GFP transgenes can be abundantly and stably expressed in promastigote and amastigote stages of parasites. Furthermore, flow cytometry analysis indicated a clear quantitative distinction between wild type and transgenic Leishmania strains at both promastigote and amastigote forms. Our data showed that the footpad lesions with GFP-transfected L. major are progressive over time by using fluorescence small-animal imaging system. Consequently, the utilization of stable GFP-transfected Leishmania species will be appropriate for in vitro and in vivo screening of anti-leishmanial drugs and vaccine development as well as understanding the biology of the host–parasite interactions at the cellular level.

Amino acids stimulate cholecystokinin release through the Ca2+-sensing receptor

Cholecystokinin (CCK) is produced by discrete endocrine cells in the proximal small intestine and is released following the ingestion of food. CCK is the primary hormone responsible for gallbladder contraction and has potent effects on pancreatic secretion, gastric emptying, and satiety. In addition to fats, digested proteins and aromatic amino acids are major stimulants of CCK release. However, the cellular mechanism by which amino acids affect CCK secretion is unknown. The Ca(2+)-sensing receptor (CaSR) that was originally identified on parathyroid cells is not only sensitive to extracellular Ca(2+) but is activated by extracellular aromatic amino acids. It has been postulated that this receptor may be involved in gastrointestinal hormone secretion. Using transgenic mice expressing a CCK promoter driven/enhanced green fluorescent protein (GFP) transgene, we have been able to identify and purify viable intestinal CCK cells. Intestinal mucosal CCK cells were enriched >200-fold by fluorescence-activated cell sorting. These cells were then used for real-time PCR identification of CaSR. Immunohistochemical staining with an antibody specific for CaSR confirmed colocalization of CaSR to CCK cells. In isolated CCK cells loaded with a Ca(2+)-sensitive dye, the amino acids phenylalanine and tryptophan, but not nonaromatic amino acids, caused an increase in intracellular Ca(2+) ([Ca(2+)](i)). The increase in [Ca(2+)](i) was blocked by the CaSR inhibitor Calhex 231. Phenylalanine and tryptophan stimulated CCK release from intestinal CCK cells, and this stimulation was also blocked by CaSR inhibition. Electrophysiological recordings from isolated CCK-GFP cells revealed these cells to possess a predominant outwardly rectifying potassium current. Administration of phenylalanine inhibited basal K(+) channel activity and caused CCK cell depolarization, consistent with changes necessary for hormone secretion. These findings indicate that amino acids have a direct effect on CCK cells to stimulate CCK release by activating CaSR and suggest that CaSR is the physiological mechanism through which amino acids regulate CCK secretion.

Model to study in vivo transdifferentiation of somatic cells into urothelium

The development of reconstructive therapy of the urinary tract using pluripotent and somatic stem cells, e.g., mesenchymal stem cells (MSC), is currently in the stage of experimental studies. These studies include the investigation of the main functions of MSC and the urothelium lining the organs of the urinary tract. An important role in the regulation of proliferation and differentiation of urothelium belongs to EGF and Wnt/β-catenin signaling pathways, the activity of which may be evaluated by the level of Her-4 and Tcf-3, 4, respectively. We found that MSC labeled by transgenic green fluorescence protein (GFP) did not pro-duce Her-4 and Tcf3, 4 in vitro, but activated their production after cell grafting into the cryoinjured bladders of the syngenic mice. In mice transplanted with these MSC GFP was detected by RT-PCR in the bladder. GFP colocalization with Her-4 or Tcf3, 4 in a few urothelial cells was detected by immunohistochemical staining with specific antibodies. These results suggest that MSC labeled with GFP an be used as a proper model to study the transdifferentiation of somatic cells into urothelium.

Analysis of 4D DIC Microscopic Data to Determine Cell Contacts in Caenorhabditis elegans Embryos

The Caenorhabditis elegans embryo is particularly amenable to microscopy and embryological studies because of its short developmental time, transparent shell, and nonpigmented cells. Identification of cell contacts is important for understanding how cells within the embryo can be polarized by their neighbors. This protocol describes a technique for identifying cell membranes and potential contacts between different blastomeres in the embryo and for following those contacts through development. This protocol involves manual segmentation of the membrane of each blastomere from four-dimensional (4D) differential interference contrast (DIC) data sets. Although this technique is described for use with C. elegans embryos, it will work with any 4D DIC data set. The recent development of a pleckstrin homology (PH)-domain tagged::GFP expressed in C. elegans embryos simplifies this analysis; however, many organisms lack appropriate green fluorescent protein (GFP) transgenes. The use of the GFP transgene improves on laser-mediated techniques for labeling embryonic cell membranes with fluorescent dye. Such methods require that a laser be used to carefully permeabilize the eggshell for entry of the dye into the embryo. The technique described in this protocol requires only easily obtainable 4D DIC data sets.

Development of a virus induced gene silencing vector from a legumes infecting tobamovirus

Medicago truncatula, the model plant of legumes, is well characterized, but there is only a little knowledge about it as a viral host. Viral vectors can be used for expressing foreign genes or for virus-induced gene silencing (VIGS), what is a fast and powerful tool to determine gene functions in plants. Viral vectors effective on Nicotiana benthamiana have been constructed from a number of viruses, however, only few of them were effective in other plants. A Tobamovirus, Sunnhemp mosaic virus (SHMV) systemically infects Medicago truncatula without causing severe symptoms. To set up a viral vector for Medicago truncatula, we prepared an infectious cDNA clone of SHMV. We constructed two VIGS vectors differing in the promoter element to drive foreign gene expression. The vectors were effective both in the expression and in the silencing of a transgene Green Fluorescent Protein (GFP) and in silencing of an endogenous gene Phytoene desaturase (PDS) on N. benthamiana. Still only one of the vectors was able to successfully silence the endogenous Chlorata 42 gene in M. truncatula.

Stochastic simulation of the spatio-temporal dynamics of reaction-diffusion systems: the case for the bicoid gradient.

Reaction-diffusion systems are mathematical models that describe how the concentrations of substances distributed in space change under the influence of local chemical reactions, and diffusion which causes the substances to spread out in space. The classical representation of a reaction-diffusion system is given by semi-linear parabolic partial differential equations, whose solution predicts how diffusion causes the concentration field to change with time. This change is proportional to the diffusion coefficient. If the solute moves in a homogeneous system in thermal equilibrium, the diffusion coefficients are constants that do not depend on the local concentration of solvent and solute. However, in nonhomogeneous and structured media the assumption of constant intracellular diffusion coefficient is not necessarily valid, and, consequently, the diffusion coefficient is a function of the local concentration of solvent and solutes. In this paper we propose a stochastic model of reaction-diffusion systems, in which the diffusion coefficients are function of the local concentration, viscosity and frictional forces. We then describe the software tool Redi (REaction-DIffusion simulator) which we have developed in order to implement this model into a Gillespie-like stochastic simulation algorithm. Finally, we show the ability of our model implemented in the Redi tool to reproduce the observed gradient of the bicoid protein in the Drosophila Melanogaster embryo. With Redi, we were able to simulate with an accuracy of 1% the experimental spatio-temporal dynamics of the bicoid protein, as recorded in time-lapse experiments obtained by direct measurements of transgenic bicoidenhanced green fluorescent protein.

Action of brain-derived neurotrophic factor on function and morphology of visual cortical neurons

Brain-derived neurotrophic factor (BDNF) is known to play a role in experience-dependent plasticity of the developing visual cortex. For example, BDNF acutely enhances long-term potentiation and blocks long-term depression in the visual cortex of young rats. Such acute actions of BDNF suggested to be mediated mainly through presynaptic mechanisms. A chronic application of BDNF to the visual cortex of kittens is known to expand ocular dominance columns in the cortex. With the method of direct intranuclear injection of plasmid cDNAs of BDNF tagged with green fluorescence protein (GFP) we have demonstrated that BDNF-GFP is transferred from presynaptic axon terminals to postsynaptic neurons in an activity-dependent manner, suggesting the possibility that BDNF also has chronic postsynaptic actions. In this study, however, it was not clear whether endogenous BDNF exerts such a postsynaptic action, because BDNF-GFP is a kind of artificial protein. In a subsequent study, therefore, we tested whether endogenous BDNF has any effect on dendritic morphology of postsynaptic neurons in igchimera culture of visual cortical neurons prepared from two types of transgenic mice, GFP mice and BDNF knockout mice. Neurons derived from the former mice have endogenous BDNF as well as GFP. We found that neurons derived from the latter mice, BDNF (-/-) neurons, have relatively poor dendrites if they were not contacted by GFP-positive terminals, whereas BDNF (-/-) neurons had complex dendritic morphology if they were directly contacted by GFP-positive terminals and thus supplied with endogenous BDNF. These results indicate that endogenous BDNF play a role in development of dendrites of visual cortical neurons in an activity-dependent manner.

Zebrafish: an exciting model for investigating the spatio-temporal pattern of enteric nervous system development

Recently, the zebrafish (Danio rerio) has been shown to be an excellent model for human paediatric research. Advantages over other models include its small size, externally visually accessible development and ease of experimental manipulation. The enteric nervous system (ENS) consists of neurons and enteric glia. Glial cells permit cell bodies and processes of neurons to be arranged and maintained in a proper spatial arrangement, and are essential in the maintenance of basic physiological functions of neurons. Glial fibrillary acidic protein (GFAP) is expressed in astrocytes, but also expressed outside of the central nervous system. The aim of this study was to investigate the spatio-temporal pattern of GFAP expression in developing zebrafish ENS from 24 h post-fertilization (hpf), using transgenic fish that express green fluorescent protein (GFP). Zebrafish embryos were collected from transgenic GFP Tg(GFAP:GFP)(mi2001) adult zebrafish from 24 to 120 hpf, fixed and processed for whole mount immunohistochemistry. Antibodies to Phox2b were used to identify enteric neurons. Specimens were mounted on slides and imaging was performed using a fluorescent laser confocal microscope. GFAP:GFP labelling outside the spinal cord was identified in embryos from 48 hpf. The patterning was intracellular and consisted of elongated profiles that appeared to migrate away from the spinal cord into the periphery. At 72 and 96 hpf, GFAP:GFP was expressed dorsally and ventrally to the intestinal tract. At 120 hpf, GFAP:GFP was expressed throughout the intestinal wall, and clusters of enteric neurons were identified using Phox2b immunofluorescence along the pathway of GFAP:GFP positive processes, indicative of a migratory pathway of ENS precursors from the spinal cord into the intestine. The pattern of migration of GFAP:GFP expressing cells outside the spinal cord suggests an organized, early developing migratory pathway to the ENS. This shows for the first time that Tg(GFAP:GFP)(mi2001) zebrafish model is an ideal one to study spatio-temporal patterning of early ENS development.

Use of human MAR elements to improve retroviral vector production

Retroviral vectors have many favorable properties for gene therapies, but their use remains limited by safety concerns and/or by relatively lower titers for some of the safer self-inactivating (SIN) derivatives. In this study, we evaluated whether increased production of SIN retroviral vectors can be achieved from the use of matrix attachment region (MAR) epigenetic regulators. Two MAR elements of human origin were found to increase and to stabilize the expression of the green fluorescent protein transgene in stably transfected HEK-293 packaging cells. Introduction of one of these MAR elements in retroviral vector-producing plasmids yielded higher expression of the viral vector RNA. Consistently, viral titers obtained from transient transfection of MAR-containing plasmids were increased up to sixfold as compared with the parental construct, when evaluated in different packaging cell systems and transfection conditions. Thus, use of MAR elements opens new perspectives for the efficient generation of gene therapy vectors.

OP07 Macrophage cell therapy causes the hepatic recruitment of host effector cells and improves structure and function in a murine model of chronic liver disease

IntroductionBone marrow (BM) cell populations have a number of roles in the development and resolution of chronic liver disease. Clinical trials of BM cell therapy have already begun. These have...

GFAP-Driven GFP Expression in Activated Mouse Müller Glial Cells Aligning Retinal Blood Vessels Following Intravitreal Injection of AAV2/6 Vectors

BackgroundMüller cell gliosis occurs in various retinal pathologies regardless of the underlying cellular defect. Because activated Müller glial cells span the entire retina and align areas of injury, they are ideal targets for therapeutic strategies, including gene therapy.Methodology/Principal FindingsWe used adeno-associated viral AAV2/6 vectors to transduce mouse retinas. The transduction pattern of AAV2/6 was investigated by studying expression of the green fluorescent protein (GFP) transgene using scanning-laser ophthalmoscopy and immuno-histochemistry. AAV2/6 vectors transduced mouse Müller glial cells aligning the retinal blood vessels. However, the transduction capacity was hindered by the inner limiting membrane (ILM) and besides Müller glial cells, several other inner retinal cell types were transduced. To obtain Müller glial cell-specific transgene expression, the cytomegalovirus (CMV) promoter was replaced by the glial fibrillary acidic protein (GFAP) promoter. Specificity and activation of the GFAP promoter was tested in a mouse model for retinal gliosis. Mice deficient for Crumbs homologue 1 (CRB1) develop gliosis after light exposure. Light exposure of Crb1−/− retinas transduced with AAV2/6-GFAP-GFP induced GFP expression restricted to activated Müller glial cells aligning retinal blood vessels.Conclusions/SignificanceOur experiments indicate that AAV2 vectors carrying the GFAP promoter are a promising tool for specific expression of transgenes in activated glial cells.

Transplantation of Nonhematopoietic Adult Bone Marrow Stem/Progenitor Cells Isolated by p75 Nerve Growth Factor Receptor Into the Penis Rescues Erectile Function in a Rat Model of Cavernous Nerve Injury

Radical prostatectomy for prostate cancer frequently results in erectile dysfunction and decreased quality of life. We investigated the effects of transplanting nonhematopoietic adult bone marrow stem/progenitor cells (multipotent stromal cells) into the corpus cavernosum in a rat model of bilateral cavernous nerve crush injury. Multipotent stromal cells were isolated from the bone marrow of transgenic green fluorescent protein rats by plastic adherence (rat multipotent stromal cells) or magnetic activated cell sorting using antibodies against p75 low affinity nerve growth factor receptor (p75 derived multipotent stromal cells). Bilateral cavernous nerve crush injury was induced in adult male Sprague-Dawley rats. Immediately after injury 8 rats each were injected intracavernously with phosphate buffered saline (vehicle control), fibroblasts (cell control), rat multipotent stromal cells (cell treatment) or p75 derived multipotent stromal cells (cell treatment). Another 8 rats underwent sham operation (phosphate buffered saline injection). Four weeks after the procedures we assessed erectile function by measuring the intracavernous-to-mean arterial pressure ratio and total intracavernous pressure during cavernous nerve stimulation. Intracavernous injection of p75 derived multipotent stromal cells after bilateral cavernous nerve crush injury resulted in a significantly higher mean intracavernous-to-mean arterial pressure ratio and total intracavernous pressure compared with all other groups except the sham operated group (p <0.05). Rats injected with typical multipotent stromal cells had partial erectile function rescue compared with animals that received p75 derived multipotent stromal cells. Fibroblast (cell control) and phosphate buffered saline (vehicle control) injection did not improve erectile function. Enzyme-linked immunosorbent assay suggested that basic fibroblast growth factor secreted by p75 derived multipotent stromal cells protected the cavernous nerve after bilateral cavernous nerve crush injury. Transplantation of adult stem/progenitor cells may provide an effective treatment for erectile dysfunction after radical prostatectomy.

Modulators of BK and SK channels alter electrical activity in vitro in single vasopressin neurons isolated from the rat supraoptic nucleus

Release of arginine vasopressin (AVP) from magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus (SON) is controlled by the electrical activities of the MNCs. Ca 2+-activated K + channels, such as the BK and SK channels, are K +-selective ion channels that are activated in response to increased intracellular calcium concentrations. Intrinsic affinities for Ca 2+ permit these channels to exert a negative feedback effect on cellular excitability. In the present study, we used the whole-cell patch-clamp technique to examine the effects of BK or SK channel modulators on neuronal activity in single isolated rat SON MNCs that express an AVP-enhanced green fluorescent protein (eGFP) transgene. Application of BK or SK channel activators abolished the action potentials and induced hyperpolarization. In contrast, the number of action potentials was significantly increased after application of BK or SK channel blockers. Our results suggest that BK and SK channels in AVP neurons may play a role in the regulatory mechanisms of neural activity.

Action Potential–Enhanced ATP Release From Taste Cells Through Hemichannels

Only some taste cells fire action potentials in response to sapid stimuli. Type II taste cells express many taste transduction molecules but lack well-elaborated synapses, bringing into question the functional significance of action potentials in these cells. We examined the dependence of adenosine triphosphate (ATP) transmitter release from taste cells on action potentials. To identify type II taste cells we used mice expressing a green fluorescence protein (GFP) transgene from the alpha-gustducin promoter. Action potentials were recorded by an electrode basolaterally attached to a single GFP-positive taste cell. We monitored ATP release from gustducin-expressing taste cells by collecting the electrode solution immediately after tastant-stimulated action potentials and using a luciferase assay to quantify ATP. Stimulation of gustducin-expressing taste cells with saccharin, quinine, or glutamate on the apical membrane increased ATP levels in the electrode solution; the amount of ATP depended on the firing rate. Increased spontaneous firing rates also induced ATP release from gustducin-expressing taste cells. ATP release from gustducin-expressing taste cells was depressed by tetrodotoxin and inhibited below the detection limit by carbenoxolone. Our data support the hypothesis that action potentials in taste cells responsive to sweet, bitter, or umami tastants enhance ATP release through pannexin 1, not connexin-based hemichannels.

Glioma stem cells involved in tumor tissue remodeling in a xenograft model

Although tissue remodeling plays a crucial role in the tumorigenesis and progression of human gliomas, its mechanisms remain largely uncertain. In the current study, the authors investigated the potential role of human glioma stem cells (hGSCs) in the tissue remodeling of gliomas. Transgenic nude mice with ubiquitous green fluorescent protein (GFP) expression were obtained by crossing nontransgenic NC athymic nude mice with the GFP transgenic C57BL/6J mice. As a result, GFP was expressed in essentially all tissues in the offspring. Human glioma stem cells were then orthotopically implanted into the GFP nude mice in an effort to assess the hGSC-host brain interactions and thereby elucidate the roles of tissue remodeling during tumorigenesis and progression of human gliomas. All of the essential tissues in the GFP transgenic nude mice, including the brain, fluoresced green under an excitation light; therefore, tumor remodeling by hGSCs can be unambiguously distinguished from a bright green background composed of adjacent host GFP-expressing components. This technique enabled the authors to address the following concerns: 1) hGSCs were involved in the invasiveness of gliomas and adjacent stroma degradation of the host. 2) An in vivo study demonstrated that cell fusion occurred between hGSCs and host cells. 3) Vasculogenic mimicry--the formation of patterned, tubular networks of vascular channels by transdifferentiated hGSCs--could be observed. 4) Differentiation mimicry--namely, the differentiation direction of hGSCs bearing multidifferentiation potentials--seemed to be decided by the local host cellular microenviroment. The results of this study indicated that the GFP transgenic nude mice model with GFP expression in essentially all tissues could be obtained by crossing nontransgenic athymic nude mice with transgenic GFP mice. This model should greatly expand our knowledge of glioma-host interactions. The data indicated that hGSCs might play a decisive role in tissue remodeling of gliomas as well.