Pattern Of Green Fluorescent Protein Expression Research Articles

The Long Noncoding RNA Cardiac Mesoderm Enhancer-Associated Noncoding RNA (Carmn) Is a Critical Regulator of Gastrointestinal Smooth Muscle Contractile Function and Motility

Visceral smooth muscle cells (SMCs) are an integral component of the gastrointestinal (GI) tract that regulate GI motility. SMC contraction is regulated by posttranslational signaling and the state of differentiation. Impaired SMC contraction is associated with significant morbidity and mortality, but the mechanisms regulating SMC-specific contractile gene expression, including the role of long noncoding RNAs (lncRNAs), remain largely unexplored. Herein, we reveal a critical role of Carmn (cardiac mesoderm enhancer-associated noncoding RNA), an SMC-specific lncRNA, in regulating visceral SMC phenotype and contractility of the GI tract. Genotype-Tissue Expression and publicly available single-cell RNA sequencing (scRNA-seq) data sets from embryonic, adult human, and mouse GI tissues were interrogated to identify SMC-specific lncRNAs. The functional role of Carmn was investigated using novel green fluorescent protein (GFP) knock-in (KI) reporter/knock-out (KO) mice. Bulk RNA-seq and single nucleus RNA sequencing (snRNA-seq) of colonic muscularis were used to investigate underlying mechanisms. Unbiased in silico analyses and GFP expression patterns in Carmn GFP KI mice revealed that Carmn is highly expressed in GI SMCs in humans and mice. Premature lethality was observed in global Carmn KO and inducible SMC-specific KO mice due to GI pseudo-obstruction and severe distension of the GI tract, with dysmotility in cecum and colon segments. Histology, GI transit, and muscle myography analysis revealed severe dilation, significantly delayed GI transit, and impaired GI contractility in Carmn KO vs control mice. Bulk RNA-seq of GI muscularis revealed that loss of Carmn promotes SMC phenotypic switching, as evidenced by up-regulation of extracellular matrix genes and down-regulation of SMC contractile genes, including Mylk, a key regulator of SMC contraction. snRNA-seq further revealed SMC Carmn KO not only compromised myogenic motility by reducing contractile gene expression but also impaired neurogenic motility by disrupting cell-cell connectivity in the colonic muscularis. These findings may have translational significance, because silencing CARMN in human colonic SMCs significantly attenuated contractile gene expression, including MYLK, and decreased SMC contractility. Luciferase reporter assays showed that CARMN enhances the transactivation activity of the master regulator of SMC contractile phenotype, myocardin, thereby maintaining the GI SMC myogenic program. Our data suggest that Carmn is indispensable for maintaining GI SMC contractile function in mice and that loss of function of CARMN may contribute to human visceral myopathy. To our knowledge this is the first study showing an essential role of lncRNA in the regulation of visceral SMC phenotype.

Development of a screening system for agents that modulate taste receptor expression with the CRISPR-Cas9 system in medaka

Taste recognition mediated by taste receptors is critical for the survival of animals in nature and is an important determinant of nutritional status and quality of life in humans. However, many factors including aging, diabetes, zinc deficiency, infection with influenza or cold viruses, and chemotherapy can trigger dysgeusia, for which a standard treatment has not been established. We here established an engineered strain of medaka (Oryzias latipes) that expresses green fluorescent protein (GFP) from the endogenous taste 1 receptor 3 (T1R3) gene locus with the use of the CRISPR-Cas9 system. This T1R3-GFP knock-in (KI) strain allows direct visualization of expression from this locus by monitoring of GFP fluorescence. The pattern of GFP expression in the T1R3-GFP KI fish thus mimicked that of endogenous T1R3 gene expression. Furthermore, exposure of T1R3-GFP KI medaka to water containing monosodium glutamate or the anticancer agent 5-fluorouracil resulted in an increase or decrease, respectively, in GFP fluorescence intensity, effects that also recapitulated those on T1R3 mRNA abundance. Finally, screening for agents that affect GFP fluorescence intensity in T1R3-GFP KI medaka identified tryptophan as an amino acid that increases T1R3 gene expression. The establishment of this screening system for taste receptor expression in medaka provides a new tool for the development of potential therapeutic agents for dysgeusia.

DNA demethylation facilitates the specific transcription of the mouse X-linked Tsga8 gene in round spermatids†.

Some X-linked genes necessary for spermiogenesis are specifically activated in the postmeiotic germ cells. However, the regulatory mechanism about this activation is not clearly understood. Here, we examined the potential mechanism controlling the transcriptional activation of the mouse testis specific gene A8 (Tsga8) gene in round spermatids. We observed that the Tsga8 expression was negatively correlated with the methylation level of the CpG sites in its core promoter. During spermatogenesis, the Tsga8 promoter was methylated in spermatogonia, and then demethylated in spermatocytes. The demethylation status of Tsga8 promoter was maintained through the postmeiotic germ cells, providing a potentially active chromatin for Tsga8 transcription. In vitro investigation showed that the E12 and Spz1 transcription factors can enhance the Tsga8 promoter activity by binding to the unmethylated E-box motif within the Tsga8 promoter. Additionally, the core Tsga8 promoter drove green fluorescent protein (GFP) expression in the germ cells of Tsga8-GFP transgenic mice, and the GFP expression pattern was similar to that of endogenous Tsga8. Moreover, the DNA methylation profile of the Tsga8-promoter-driven transgene was consistent with that of the endogenous Tsga8 promoter, indicating the existence of a similar epigenetic modification for the Tsga8 promoter to ensure its spatiotemporal expression in vivo. Taken together, this study reports the details of a regulatory mechanism that includes DNA methylation and transcription factors to mediate the postmeiotic expression of an X-linked gene.

Quantification of green fluorescent protein expression in mouse retinal ganglion cells following intravitreal injection of recombinant adeno‐associated virus

PurposeTo determine the transduction efficiency and spatial pattern of green fluorescent protein (GFP) expression in mouse retinal ganglion cells (RGCs) following intravitreal injection of recombinant adeno‐associated virus (rAAV2).Methods16 adult C57bL/6 mice received intravitreal injections of rAAV2‐CAG‐GFP at 3 different titres: 1 × 10E11, 1 × 10E12 or 1 × 10E13 genomic particles/ml. 2 μl of the virus was injected at each concentration. To determine the effect of volume on transduction efficiency, a subset of animals received 1 μl of the highest viral titre.Animals were sacrificed 21 days after injection. Retinal wholemounts were immunostained with Brn3a to identify RGCs and quantification carried out using imageJ software. The percentage of GFP positive RGCs was measured at each titre using Volocity software at 20× magnification. A further 20–25 images at 10× magnification were captured for each titre and merged using to reconstruct the entire wholemount to assess the spatial pattern of expression.ResultsRGC transduction rate increased with viral titre; 10% at 1 × 10E11, 53% and 1 × 10E12 and 64% at 1 × 10E13 genomic particles/ml. The volume injected did not appear to affect the transduction efficiency, with 64% of RGCs transduced at the highest titre using either 1 or 2 ul injection. GFP intensity also increased with viral titre with the spatial pattern of GFP expression more extensive at the highest titre. GFP expression at lower titres tended to localise around the injection site.ConclusionsGFP transduction efficiency of RGCs can be quantified efficiently using Volocity software. We have demonstrated an increase in GFP expression and spread at higher viral titres with similar transduction efficiency at a lower volume.

Generation of BAC transgenic tadpoles enabling live imaging of motoneurons by using the urotensin II-related peptide (ust2b) gene as a driver.

Xenopus is an excellent tetrapod model for studying normal and pathological motoneuron ontogeny due to its developmental morpho-physiological advantages. In mammals, the urotensin II-related peptide (UTS2B) gene is primarily expressed in motoneurons of the brainstem and the spinal cord. Here, we show that this expression pattern was conserved in Xenopus and established during the early embryonic development, starting at the early tailbud stage. In late tadpole stage, uts2b mRNA was detected both in the hindbrain and in the spinal cord. Spinal uts2b+ cells were identified as axial motoneurons. In adult, however, the uts2b expression was only detected in the hindbrain. We assessed the ability of the uts2b promoter to drive the expression of a fluorescent reporter in motoneurons by recombineering a green fluorescent protein (GFP) into a bacterial artificial chromosome (BAC) clone containing the entire X. tropicalis uts2b locus. After injection of this construction in one-cell stage embryos, a transient GFP expression was observed in the spinal cord of about a quarter of the resulting animals from the early tailbud stage and up to juveniles. The GFP expression pattern was globally consistent with that of the endogenous uts2b in the spinal cord but no fluorescence was observed in the brainstem. A combination of histological and electrophysiological approaches was employed to further characterize the GFP+ cells in the larvae. More than 98% of the GFP+ cells expressed choline acetyltransferase, while their projections were co-localized with α-bungarotoxin labeling. When tail myotomes were injected with rhodamine dextran amine crystals, numerous double-stained GFP+ cells were observed. In addition, intracellular electrophysiological recordings of GFP+ neurons revealed locomotion-related rhythmic discharge patterns during fictive swimming. Taken together our results provide evidence that uts2b is an appropriate driver to express reporter genes in larval motoneurons of the Xenopus spinal cord.

Green fluorescent protein (GFP) expression patterns in the olfactory epithelium of GFP transgenic cloned Jinhua pigs

Domestic pigs possess a well-developed sense of smell. However, the morphology of the porcine olfactory epithelium (OE) is poorly understood. Recently, several strains of transgenic cloned pigs that are presumed to ubiquitously express green fluorescent protein (GFP) have been created. Thus, the purpose of this study was to elucidate the features of porcine OE using the tissues of GFP transgenic cloned pigs. Based on observations of Hematoxylin and Eosin staining and measurements of thickness, porcine OE tissue portions were classified into three categories (thick, standard, and thin). Cryosections revealed that the prominent GFP signals were expressed in olfactory sensory neurons (OSN), Bowman's glands, and olfactory nerve. A few GFP-expressing sustentacular cells were seen; however, the intensity of GFP fluorescence was slight. In the thick portion, numerous GFP-expressing polygonal OSN that did not possess dendrites were found. In the standard portions, GFP-expressing cells had longitudinal dendrites. A few GFP-expressing cells were found in the thin portion. In the thick and standard portions, most of the prominent GFP-expressing cells were positive for olfactory marker protein. Moreover, double immunofluorescence staining with boiled GFP and Sox2 antibody revealed that GFP expression patterns in OSN are synchronized with Sox2 immunoreactive patterns.

Early postnatal virus inoculation into the scala media achieved extensive expression of exogenous green fluorescent protein in the inner ear and preserved auditory brainstem response thresholds

Gene transfer into the inner ear is a promising approach for treating sensorineural hearing loss. The special electrochemical environment of the scala media raises a formidable challenge for effective gene delivery at the same time as keeping normal cochlear function intact. The present study aimed to define a suitable strategy for preserving hearing after viral inoculation directly into the scala media performed at various postnatal developmental stages. We assessed transgene expression of green fluorescent protein (GFP) mediated by various types of adeno-associated virus (AAV) and lentivirus (LV) in the mouse cochlea. Auditory brainstem responses were measured 30 days after inoculation to assess effects on hearing. Patterns of GFP expression confirmed extensive exogenous gene expression in various types of cells lining the endolymphatic space. The use of different viral vectors and promoters resulted in specific cellular GFP expression patterns. AAV2/1 with cytomegalovirus promoter apparently gave the best results for GFP expression in the supporting cells. Histological examination showed normal cochlear morphology and no hair cell loss after either AAV or LV injections. We found that hearing thresholds were not significantly changed when the injections were performed in mice younger than postnatal day 5, regardless of the type of virus tested. Viral inoculation and expression in the inner ear for the restoration of hearing must not damage cochlear function. Using normal hearing mice as a model, we have achieved this necessary step, which is required for the treatment of many types of congenital deafness that require early intervention.

A Robust and Sensitive Synthetic Sensor to Monitor the Transcriptional Output of the Cytokinin Signaling Network in Planta

Cytokinins are classic plant hormones that orchestrate plant growth, development, and physiology. They affect gene expression in target cells by activating a multistep phosphorelay network. Type-B response regulators, acting as transcriptional activators, mediate the final step in the signaling cascade. Previously, we have introduced a synthetic reporter, Two Component signaling Sensor (TCS)::green fluorescent protein (GFP), which reflects the transcriptional activity of type-B response regulators. TCS::GFP was instrumental in uncovering roles of cytokinin and deepening our understanding of existing functions. However, TCS-mediated expression of reporters is weak in some developmental contexts where cytokinin signaling has a documented role, such as in the shoot apical meristem or in the vasculature of Arabidopsis (Arabidopsis thaliana). We also observed that GFP expression becomes rapidly silenced in TCS::GFP transgenic plants. Here, we present an improved version of the reporter, TCS new (TCSn), which, compared with TCS, is more sensitive to phosphorelay signaling in Arabidopsis and maize (Zea mays) cellular assays while retaining its specificity. Transgenic Arabidopsis TCSn::GFP plants exhibit strong and dynamic GFP expression patterns consistent with known cytokinin functions. In addition, GFP expression has been stable over generations, allowing for crosses with different genetic backgrounds. Thus, TCSn represents a significant improvement to report the transcriptional output profile of phosphorelay signaling networks in Arabidopsis, maize, and likely other plants that display common response regulator DNA-binding specificities.

Trap identification of the constitutive promoter-like sequences from the bacterial fish pathogen, as exemplified by Edwardsiella tarda

A trap identification system for isolating functional sequences to allow the constitutive expression of foreign protein from Edwardsiella tarda was developed. Using the green fluorescent protein (GFP) reporter-based trap system, various functional sequences to drive heterologous expression of the GFP were selectable in Escherichia coli host. However from the bioinformatic sequence analysis, all the segments predicted as regulatory regions were not native promoters actually existing upstream of endogenous E. tarda genes. Instead, a number of non-authentic sequences, possibly resulted from the random shuffling and/or intermolecular ligation were also proven to be able to display a potent GFP expression in the recombinant E. coli. Further analysis with selected clones showed that both authentic and non-authentic sequences could function in as a constitutive promoter, leading quite a consistent and stable GFP expression after repetitive subcultures. Microscopic examination also confirmed the uniform pattern of GFP expression in every host bacterium. Semi-quantitative assay of GFP showed that there was no clear relationship between expression levels and organizational features of the promoters trapped. Functional promoter-like elements achieved in the present study could be a good starting material for multivalent genetic engineering of E. tarda in order to produce recombinant vaccines in a cost-effective fashion.

Molecular properties of Kiss1 neurons in the arcuate nucleus of the mouse.

Neurons that produce kisspeptin play a critical role in reproduction. However, understanding the molecular physiology of kisspeptin neurons has been limited by the lack of an in vivo marker for those cells. Here, we report the development of a Kiss1-CreGFP knockin mouse, wherein the endogenous Kiss1 promoter directs the expression of a Cre recombinase-enhanced green fluorescent protein (GFP) fusion protein. The pattern of GFP expression in the brain of the knockin recapitulates what has been described earlier for Kiss1 in the male and female mouse, with prominent expression in the arcuate nucleus (ARC) (in both sexes) and the anteroventral periventricular nucleus (in females). Single-cell RT-PCR showed that the Kiss1 transcript is expressed in 100% of GFP-labeled cells, and the CreGFP transcript was regulated by estradiol in the same manner as the Kiss1 gene (i.e. inhibited in the ARC and induced in the anteroventral periventricular nucleus). We used this mouse to evaluate the biophysical properties of kisspeptin (Kiss1) neurons in the ARC of the female mouse. GFP-expressing Kiss1 neurons were identified in hypothalamic slice preparations of the ARC and patch clamped. Whole-cell (and loose attached) recordings revealed that Kiss1 neurons exhibit spontaneous activity and expressed both h- (pacemaker) and T-type calcium currents, and hyperpolarization-activated cyclic nucleotide-regulated 1-4 and CaV3.1 channel subtypes (measured by single cell RT-PCR), respectively. N-methyl-D-aspartate induced bursting activity, characterized by depolarizing/hyperpolarizing oscillations. Therefore, Kiss1 neurons in the ARC share molecular and electrophysiological properties of other CNS pacemaker neurons.

Robust In Vivo Transduction of Nervous System and Neural Stem Cells by Early Gestational Intra Amniotic Gene Transfer Using Lentiviral Vector

Presently, in vivo methods to efficiently and broadly transduce all major cell types throughout both the central (CNS) and peripheral adult nervous system (PNS) are lacking. In this study, we hypothesized that during early fetal development neural cell populations, including neural stem cells (NSCs), may be accessible for gene transfer via the open neural groove. To test this hypothesis, we injected lentiviral vectors encoding a green fluorescent protein (GFP) marker gene into the murine amniotic cavity at embryonic day 8. This method (i) efficiently and stably transduced the entire nervous system for at least 80% of the lifespan of the mice, (ii) transduced all major neural cell types, and (iii) transduced adult NSCs of the subventricular zone (SVZ) and subgranular zones (SGZs). This simple approach has broad applications for the study of gene function in nervous system development and adult NSCs and may have future clinical applications for treatment of genetic disorders of the nervous system.

Transgenic rabbit production with simian immunodeficiency virus-derived lentiviral vector

Transgenic rabbit is the preferred disease model of atherosclerosis, lipoprotein metabolism and cardiovascular diseases since upon introducing genetic mutations of human genes, rabbit models reflect human physiological and pathological states more accurately than mouse models. Beyond that, transgenic rabbits are also used as bioreactors to produce pharmaceutical proteins in their milk. Since in the laboratory rabbit the conventional transgenesis has worked with the same low efficiency in the last twenty five years and truly pluripotent embryonic stem cells are not available to perform targeted mutagenesis, our aim was to adapt lentiviral transgenesis to this species. A simian immunodeficiency virus based replication defective lentiviral vector was used to create transgenic rabbit through perivitelline space injection of fertilized oocytes. The enhanced green fluorescent protein (GFP) gene was placed under the ubiquitous CAG promoter. Transgenic founder rabbits showed mosaic pattern of GFP expression. Transgene integration and expression was revealed in tissues derived from all three primary germ layers. Transgene expression was detected in the developing sperm cells and could get through the germ line without epigenetic silencing, albeit with very low frequency. Our data show for the first time, that lentiviral transgenesis could be a feasible and viable alternative method to create genetically modified laboratory rabbit.

TGF-beta: A retrograde signaling molecule involved in the formation of neuromuscular junctions in C. elegans

Event Abstract Back to Event TGF-beta: A retrograde signaling molecule involved in the formation of neuromuscular junctions in C. elegans Abir Rahman1* and Bill Walthall1 1 Georgia State University, Biology Dep, United States The aim of this research is to investigate the role of Transforming Growth Factor Beta (TGF-B) in the formation of neuromuscular junctions in the nematode. Laser ablation of selected embryonic myoblasts removes guidance markers used by post-embryonic muscles (1). The absence of these guideposts results in migrating muscle losing their way and differentiating in ectopic locations. These ectopic muscle strands become innervated by processes from the DD motor neurons (mns). We postulate that a retrograde signaling molecule released by the muscles induces neurite sprouting from the DD mns, resulting in the formation of ectopic neuromuscular junctions (nmjs). An earlier report, combined with our own observations focusing on post-embryonic muscle, indicate that the transient expression pattern of TGF- B corresponds to the nmj formation in the DD mns (2). Current experiments combining laser ablations in various mutant backgrounds, with green fluorescent protein expression patterns should shed light onto th1e communication between presynaptic and the post-synaptic elements at the nmj.

In Vivo Analysis of Cone Survival in Mice

To identify individual cone photoreceptors in a transgenic mouse line in vivo based on selective expression of green fluorescent protein (GFP) using cSLO (confocal scanning laser ophthalmoscopy) and to use this approach to monitor cone cell fate in mouse models of retinal degeneration. Transgenic mice expressing GFP under the control of a red-green opsin promoter (RG-GFP mice) were analyzed in vivo with respect to GFP expression in cone cells using cSLO and functional integrity using electroretinography (ERG). Histology was performed to correlate the pattern of GFP expression with light microscopic data. Longitudinal monitoring of cone survival was evaluated in crossbreds of RG-GFP mice with cpfl1 and Rpe65(-/-) mutant mice, respectively. The authors found that RG-GFP transgenic mice had a stable GFP expression that did not interfere with retinal function up to at least 3 months of age. Thus, a longitudinal analysis of cone degeneration in individual RG cpfl1 and RG Rpe65(-/-) cross-bred mice in vivo was successfully performed and demonstrated distinct time frames of cone survival in the particular mouse model. Monitoring GFP expression in cone photoreceptor cells, such as in the RG-GFP mouse, is a promising in vivo approach for the analysis of cone survival in mice.

Vsx1 3′ untranlated region-mediated translation difference at differentdevelopmental stages of goldfish embryos

Visual system homeobox-1 (vsx1) is important in retinal progenitor proliferation, differentiation, and function maintenance of bipolar cells in vertebrates. Recent study in Xenopus laevis has shown that vsx1 3' untranslated region (3' UTR) can mediate cell-specific translation of vsx1 mRNA in the bipolar cells of the retinal inner nuclear layer (INL). vsx1 is also transcribed at the early developmental stages prior to eye formation and its spatiotemporal expression patterns are conserved in all the examined vertebrates. In order to determine whether the vsx1 3' UTR has a role in regulating the spatiotemporal expression of vsx1 during early embryogenesis, we constructed a vsx1 UTR-controlled green fluorescent protein (GFP) reporter gene system and examined the GFP expression pattern in goldfish, Carassius auratus, at different developmental stages. Our results indicated that both the vsx1 5' UTR and the vsx1 3' UTR remarkably repressed GFP expression at transcription level but did not regulate tissue-specific translation at early developmental stages. GFP protein was ubiquitously expressed in the embryos injected with GFP-sensors containing vsx1 UTRs before 60 h post-fertilization (hpf). From hatching stage (72 hpf) onwards, however, GFP protein was specifically expressed in the bipolar cells of the retinal INL in the vsx1 3'UTR-GFP-sensor embryos, but was still ubiquitously expressed in the embryos injected with GFP-sensor lacking vsx1 3' UTR. These observations showed a significant difference of vsx1 3' UTR-mediated translation between early and late developmental stages and suggested that vsx1 3' UTR might not be involved in regulating the spatiotemporal expression of vsx1 until hatching stage during embryogenesis.

Optimization of the transient transformation of Catharanthus roseus cells by particle bombardment and its application to the subcellular localization of hydroxymethylbutenyl 4-diphosphate synthase and geraniol 10-hydroxylase

The monoterpene indole alkaloids (MIA) synthesized in Catharanthus roseus are highly valuable metabolites due to their pharmacological properties. In planta, the MIA biosynthetic pathway exhibits a complex compartmentation at the cellular level, whereas subcellular data are sparse. To gain insight into this level of organization, we have developed a high efficiency green fluorescent protein (GFP) imaging approach to systematically localize MIA biosynthetic enzymes within C. roseus cells following a biolistic-mediated transient transformation. The biolistic transformation protocol has been first optimized to obtain a high number of transiently transformed cells with a ~12-fold increase compared to previous protocols and thus to clearly and easily identify the fusion GFP expression patterns in numerous cells. On the basis of this protocol, the subcellular localization of hydroxymethylbutenyl 4-diphosphate synthase (HDS), a methyl erythritol phosphate pathway enzyme and geraniol 10-hydroxylase (G10H), a monoterpene-secoiridoid pathway enzyme has been next characterized. Besides showing the accumulation of HDS within plastids of C. roseus cells, we also provide evidences of the presence of HDS in long stroma-filled thylakoid-free extensions budding from plastids, i.e. stromules that are in close association with other organelles such as endoplasmic reticulum (ER) or mitochondria in agreement with their proposed function in enhancing interorganelle metabolite exchanges. Furthermore, we also demonstrated that G10H is an ER-anchored protein, consistent with the presence of a transmembrane helix at the G10H N-terminal end, which is both necessary and sufficient to drive the ER anchoring.

Large-scale gene expression pattern analysis, in situ, in Caenorhabditis elegans

Observation of gene expression in situ provides a direct connection between the genetic information in the genome sequence and the fully determined developmental cell lineage of Caenorhabditis elegans. Green Fluorescent Protein (GFP) reporters have been fused with many C. elegans genes, in large-scale projects, by conventional DNA ligation, PCR stitching, Gateway recombination and recombineering. These reporter gene fusions have then been used in C. elegans transformation either by microinjection or microprojectile bombardment. So far, the developmental distributions of GFP, as driven by the C. elegans DNA to which the reporter gene has been attached, have been determined simply from direct examination of the transgenic strains by epifluorescence microscopy. Automation of GFP expression pattern determination promises improvements in both quality and quantity of this data type, facilitating the handling of such expression pattern data within computer databases. As with the descriptions of the developmental cell lineage and the genome sequence, a complete description of gene expression patterns will provide a vital knowledge framework through which a full understanding of the development of this animal can emerge.

Visualization of monoaminergic neurons and neurotoxicity of MPTP in live transgenic zebrafish

We describe an enhancer trap transgenic zebrafish line, ETvmat2:GFP, in which most monoaminergic neurons are labeled by green fluorescent protein (GFP) during embryonic development. The reporter gene of ETvmat2:GFP was inserted into the second intron of vesicular monoamine transporter 2 ( vmat2) gene, and the GFP expression pattern recapitulates that of the vmat2 gene. The GFP positive neurons include the large and pear-shaped tyrosine hydroxylase positive neurons (TH populations 2 and 4) in the posterior tuberculum of ventral diencephalon (PT neurons), which are thought to be equivalent to the midbrain dopamine neurons in mammals. We found that these PT neurons and two other GFP labeled non-TH type neuronal groups, one in the paraventricular organ of the posterior tuberculum and the other in the hypothalamus, were significantly reduced after exposure to MPTP, while the rest of GFP-positive neuronal clusters, including those in telencephalon, pretectum, raphe nuclei and locus coeruleus, remain largely unchanged. Furthermore, we showed that the effects of hedgehog signaling pathway inhibition on the development of monoaminergic neurons can be easily visualized in individual living ETvmat2:GFP embryos. This enhancer trap line should be useful for genetic and pharmacological analyses of monoaminergic neuron development and processes underlying Parkinson's disease.

Human Blue-Opsin Promoter Preferentially Targets Reporter Gene Expression to Rat S-Cone Photoreceptors

To develop a gene therapy system that specifically targets transgene expression to S-cones of the mammalian retina, the authors coupled recombinant AAV-mediated delivery with the use of a human blue-opsin (HB) promoter to drive expression. Two regions of the HB promoter sequence, HB569 and HB996, were amplified from human DNA, cloned into an AAV vector cassette upstream of the green fluorescent protein (GFP) gene, and packaged into AAV2 and AAV5 capsids. Eyes of postnatal day (P) 40 to P48 Sprague-Dawley rats were subretinally injected with 2 muL vector. Animals were humanely killed 2 to 3 weeks or 20 months after injection, and the pattern and persistence of GFP expression were analyzed in the treated retinas by immunohistochemistry, Western blotting, and RT-PCR. AAV5.HB.GFP vectors targeted photoreceptor transduction with an efficiency 20-fold higher than analogous serotype 2 vector. Both AAV5.HB.GFP vectors exhibited similar transduction efficiencies with patterns of GFP expression that did not vary depending on the size of the HB promoter used. Transgene expression was exclusively localized to photoreceptors of retinas treated with either vector. Furthermore, GFP expression was observed for at least 20 months. Dual GFP immunostaining with S- or M-opsin antibodies and GFP/PNA labeling revealed that cones coexpressing S-opsin/GFP or M-opsin/GFP constituted 37.5% +/- 8% and 13.5% +/- 3% of the GFP-positive photoreceptors, respectively, whereas rods constituted 49% +/- 5% of the GFP-positive photoreceptors. Because cones constitute approximately 1% of adult rat retinal photoreceptors, it was estimated that the relative transduction efficiency of AAV5.HB.GFP vectors was approximately 100:1 for cones versus rods. AAV5.HB.GFP vector injected into the subretinal space of Sprague-Dawley rats targeted gene expression to photoreceptor cells with an efficiency approximately 20-fold higher than that for AAV2.HB.GFP. Transgene expression regulated by the human blue cone-promoter persisted at least for 20 months. Cones coexpressing S-opsin and the GFP transgene appeared to prevail, confirming that in addition to having properties of the AAV serotype, the promoter choice is key to fine-tuning transgene delivery and expression in specific retinal cells. The system described here may be effective in a therapeutic setting in which strong S-cone transgene expression is required.

DEVELOPMENT OF NOVEL APPROACH FOR GENE TRANSFER OF RODENT FETAL LUNG

Introduction In utero gene therapy might have potential interest for treatment of congenital lung diseases. Aim To develop a new method for accurate injections into specific compartments of the rodent fetuses during early stages of lung development and to assess transduction efficiency of adenoviral and lentiviral vectors after in utero injection. Methods Guided by a 40 mHz ultrasound scanhead we performed intra-amniotic, intra-parenchymal and intra-pleural injections in rodent fetuses (n=131) at E15.5–E16.5. For injection, we used PBS, lentivirus and adenovirus with green fluorescent protein (GFP) as a reporter gene. We evaluated survival rate, morphometric parameters and pattern of viral transfection at E16.5; E18.5; E21.5 and P7. Results Dramatically different patterns of GFP expression were observed ranging from visceral and parietal pleural expression, to focal intraparenchymal expression, to diffuse airway and alveolar expression. Adenoviral vectors appeared more efficient than lentiviral vectors for lung transduction. Conclusions We describe a new model of image guided microinjection of viral vectors into rodent fetuses targeting the fetal lung as early as pseudoglandular stage. These results should facilitate studies of fetal lung gene therapy as well as the effect of growth factors on lung development.