Labeling Neural Cells Using Adenoviral Gene Transfer of Membrane-Targeted GFP (original) (raw)
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Experimental Neurology, 1998
The present studies used defined cells of the subfornical organ (SFO) and supraoptic nuclei (SON) as model systems to demonstrate the efficacy of replication-deficient adenovirus (Ad) encoding green fluorescent protein (GFP) for gene transfer. The studies investigated the effects of both direct transfection of the SON and indirect transfection (i.e., via retrograde transport) of SFO neurons. The SON of rats were injected with Ad (2 ؋ 10 6 pfu) and sacrificed 1-7 days later for cell culture of the SON and of the SFO. In the SON, GFP fluorescence was visualized in both neuronal and nonneuronal cells while only neurons in the SFO expressed GFP. Successful in vitro transfection of cultured cells from the SON and SFO was also achieved with Ad (2 ؋ 10 6 to 2 ؋ 10 8 pfu).
Gene Therapy, 1997
In this study, we have evaluated the capacity of recombi-morphological profiles of transduced cells were consistnant adeno-associated virus (rAAV) vectors, containing cell ently neuronal, and there was no evidence of transgene type-specific promoters, to transduce neurons in vivo in the expression in glial elements. Transduction efficiencies for normal adult rat spinal cord. The neuron-specific enolase the NSE and PDGF rAAVs were estimated at 15 and 45 (NSE) promoter and the platelet-derived growth factor infectious particles per GFP-positive neuron, respectively, (PDGF) B-chain promoter were used to direct expression in the absence of detectable adenovirus. This study of a 'humanized' form of the gene for green fluorescent strongly supports a role for rAAV vectors in CNS gene therprotein (GFP). Neuron-specific rAAVs were injected into apy and lays the groundwork for delivery of more functional the mid-cervical regions of adult rat spinal cords. At genes to spinal cord neurons as a possible way to enhance 10-14 days, expression was detected in all animals and spinal cord repair following injury. persisted for up to 15 weeks. Immunocytochemical and
Experimental neurology, 2002
Previous studies demonstrated that the rat neuron-specific enolase (NSE) promoter is effective for transgene expression in the brain in a variety of adeno-associated virus-2 vectors. This study evaluated the dose response and longer time course of this promoter and compared it to two cytomegalovirus/chicken beta-actin hybrid (CBA) promoter-based systems. NSE promoter-driven green fluorescent protein (GFP)-expressing neurons were found at doses as low as 10(7) particles, with expression increasing in a dose-dependent manner over a 3.3-log range. Bicistronic expression of GFP via an internal ribosome entry site coupled to the NSE promoter was also dose dependent, although the potency was decreased by 3.4-fold. The number of GFP-expressing neurons was stable for at least 25 months. The CBA promoter increased the numbers of GFP-expressing cells versus the NSE promoter, although the expression pattern remained neuronal and persisted for at least 18 months. The CBA promoter permitted dete...
Targeting adenoviral transgene expression to neurons
Molecular and Cellular Neuroscience, 2008
Adenovirus (Ad) is an efficient and safe vector for CNS gene delivery since it infects non-replicating neurons and does not cause insertional mutagenesis of host cell genomes. However, the promiscuous Ad CAR receptor targets cells non-specifically and activates a host immune response. Using Ad5 containing an expression cassette encoding the gene for green fluorescent protein, gfp, regulated by the neuron specific promoter synapsin-1 and the woodchuck post-transcriptional regulatory element (WPRE), we demonstrate efficient, prolonged and promoter-restricted gfp expression in neurons of mixed primary adult rat dorsal root ganglion (DRG) and retinal cell cultures. We also demonstrate restricted gfp expression in DRG neurons after direct injections of Ad5 containing the synapsin-1 gfp /WPRE construct into L4 DRG in vivo, while Ad5 CMV gfp transfected both DRG glia and neurons. Moreover, since the effective titres of delivered Ad5 are reduced with this neuron specific promoter/WPRE expression cassette, the viral immune challenge should be attenuated when used in vivo.
2017
Investigation of the neuronalconnectionshave been conducted over a long span of time. Debate between researchers about contiguity or continuity of the nerve elements resulted in a new era in the research of neuronal pathways. The issue has been resolved when synaptic connections were discovered by the electron microscope. This made it possible to use two types of tract tracing methods. First non-transsynaptic and later trans-synaptic tracers were applied. The formerone is suitable to demonstrate direct neuronal connections; the latterare able to describe multisynaptic neuronal circuits.Development of trans-synaptic neurotropic viruses expressing reporter molecules was a great step in this research. GFP, a natural fluorescent protein was discovered in jellyfishmore than 50 years ago by Shimomura. Later it was found that GFP fluorescence wasstable, species-independent and could be monitored noninvasively using the techniques of fluorescence microscopy and flow cytometry. Later it was ...
Imaging Neuronal Subsets in Transgenic Mice Expressing Multiple Spectral Variants of GFP
Neuron, 2000
The first report on GFP expression in heterologous cells illustrated its use as a vital stain for neurons (Chalfie Washington University School of Medicine St. Louis, Missouri 63110 et al., 1994). Since that time, neuroscience has been one of the greatest beneficiaries of GFP technology, and GFP has been used to facilitate the study of neuronal development and plasticity in transgenic worms, flies, fish, and mice (for example, Dynes and Ngai, 1998; Mur-Summary ray et al., 1998; van den Pol and Ghosh, 1998; Knobel et al., 1999; Zito et al., 1999; Rodriguez et al., 1999; We generated transgenic mice in which red, green, yellow, or cyan fluorescent proteins (together termed Higashijima et al., 2000)
A Viral Toolbox of Genetically Encoded Fluorescent Synaptic Tags
iScience, 2020
Fibronectin intrabodies generated with mRNA display (FingRs) are a recently developed tool for labeling excitatory or inhibitory synapses, with the benefit of not altering endogenous synaptic protein expression levels or synaptic transmission. Here, we generated a viral vector FingR toolbox that allows for multi-color, neuron-type-specific labeling of excitatory or inhibitory synapses in multiple brain regions. We screened various fluorophores, FingR fusion configurations, and transcriptional control regulations in adeno-associated virus (AAV) and retrovirus vector designs. We report the development of a red FingR variant and demonstrated dual labeling of excitatory and inhibitory synapses in the same cells. Furthermore, we developed cre-inducible FingR AAV variants and demonstrated their utility, finding that the density of inhibitory synapses in aspiny striatal cholinergic interneurons remained unchanged in response to dopamine depletion. Finally, we generated FingR retroviral vectors, which enabled us to track the development of excitatory and inhibitory synapses in hippocampal adult-born granule cells.
European Journal of Neuroscience, 2001
The cerebellar Purkinje cell has been the focus of numerous studies involving the analysis of development and information processing in the nervous system. Purkinje cells represent less than 0.1% of the total cell content of the cerebellum. To facilitate studies of molecules that are expressed in such a small proportion of neurons, we have established procedures for the puri®cation of these cells. Transgenic mice were developed in which the expression of green¯uorescent protein (GFP) was controlled by the L7 promoter. In adult cerebellum, GFP¯uorescence was only detected in Purkinje cells, where it ®lled dendrites, soma and axons. GFP¯uorescence was detected in Purkinje cells as early as embryonic day 17 and increased during development in vivo and in dissociated cerebellar culture. Mirroring endogenous L7 expression, high levels of GFP were observed in retinal rod bipolar cells. Lower levels of GFP were seen in olfactory periglomerular cells, neurons in the interpeduncular nucleus, and superior colliculus neurons. Cerebella from transgenic mice were dissociated by mild enzymatic treatment and Purkinje cells were isolated by¯uorescence-activated cell sorting (FACS). By selecting optimal parameters, a fraction of viable Purkinje cells that was 94% pure was obtained. These results indicate that FACS is a powerful tool for isolating Purkinje cells from postnatal L7-GFP transgenic mice. GFP-positive neurons will also be useful in the real-time observation of dendritic morphogenesis and axonal outgrowth during development, or after neuronal activity in vitro.
Gene Therapy, 1999
Adenoviruses are highly efficient vectors for gene transfer cerebellum and striatum. Anatomical and immunohistochinto brain cells. Restricting transgene expression to specific emical analyses of the Ad-NSE-stained cells showed that cell types and maintaining long-term expression are major neurons were preferentially transduced. More neurons goals for gene therapy in the central nervous system. We were stained in the hippocampus following infection with targeted gene expression to neurons by constructing an Ad-NSE than with Ad-RSV. Cytotoxicity from Ad-NSE was adenoviral vector that expressed the E. coli LacZ reporter lower than from Ad-RSV. β-Galactosidase gene gene under the control of the rat neuron-specific enolase expression after Ad-NSE infection remained stable for 3. promoter (Ad-NSE). Expression from Ad-NSE was com-months, and was detectable for 6 months. Thus, the NSEpared with that from an adenoviral vector encoding the adenoviral vector can be used to transfer potentially therasame reporter gene under the control of the Rous sarcoma peutic genes into neuronal cells. The use of a cell-specific virus LTR promoter (Ad-RSV). Both recombinant adeno-promoter also resulted in high in vivo efficiency and longviruses were injected stereotactically into rat hippocampus, term transgene expression.