mRFP Expression Research Articles

Abstract PR-010: Asc1lb progenitor-specific RB conditional inactivation in zebrafish models rare CNS primitive neuroectodermal tumors

Abstract PR-010: Asc1lb progenitor-specific RB conditional inactivation in zebrafish models rare CNS primitive neuroectodermal tumors

Cre/lox regulated conditional rescue and inactivation with zebrafish UFlip alleles generated by CRISPR-Cas9 targeted integration.

The ability to regulate gene activity spatially and temporally is essential to investigate cell-type-specific gene function during development and in postembryonic processes and disease models. The Cre/lox system has been widely used for performing cell and tissue-specific conditional analysis of gene function in zebrafish. However, simple and efficient methods for isolation of stable, Cre/lox regulated zebrafish alleles are lacking. Here, we applied our GeneWeld CRISPR-Cas9 targeted integration strategy to generate floxed alleles that provide robust conditional inactivation and rescue. A universal targeting vector, UFlip, with sites for cloning short homology arms flanking a floxed 2A-mRFP gene trap, was integrated into an intron in rbbp4 and rb1. rbbp4off and rb1off integration alleles resulted in strong mRFP expression,>99% reduction of endogenous gene expression, and recapitulated known indel loss-of-function phenotypes. Introduction of Cre led to stable inversion of the floxed cassette, loss of mRFP expression, and phenotypic rescue. rbbp4on and rb1on integration alleles did not cause phenotypes in combination with a loss-of-function mutation. Addition of Cre led to conditional inactivation by stable inversion of the cassette, gene trapping and mRFP expression, and the expected mutant phenotype. Neural progenitor Cre drivers were used for conditional inactivation and phenotypic rescue to showcase how this approach can be used in specific cell populations. Together these results validate a simplified approach for efficient isolation of Cre/lox-responsive conditional alleles in zebrafish. Our strategy provides a new toolkit for generating genetic mosaics and represents a significant advance in zebrafish genetics.

A Synthetic Biology Approach Using Engineered Bacteria to Detect Perfluoroalkyl Substance (PFAS) Contamination in Water.

Per- and polyfluoroalkyl substances (PFAS) are a class of synthetic compounds used industrially for a wide variety of applications. These PFAS compounds are very stable and persist in the environment. The PFAS contamination is a growing health issue as these compounds have been reported to impact human health and have been detected in both domestic and global water sources. Contaminated water found on military bases poses a potentially serious health concern for active duty military, their families, and the surrounding communities. Previous detection methods for PFAS in contaminated water samples require expensive and time-consuming testing protocols that limit the ability to detect this important global pollutant. The main objective of this work was to develop a novel detection system that utilizes a biological reporter and engineered bacteria as a way to rapidly and efficiently detect PFAS contamination. The United States Air Force Academy International Genetically Engineered Machine team is genetically engineering Rhodococcus jostii strain RHA1 to contain novel DNA sequences composed of a propane 2-monooxygenase alpha (prmA) promoter and monomeric red fluorescent protein (mRFP). The prmA promoter is activated in the presence of PFAS and transcribes the mRFP reporter. The recombinant R. jostii containing the prmA promoter and mRFP reporter respond to exposure of PFAS by activating gene expression of the mRFP. At 100 µM of perfluorooctanoic acid, the mRFP expression was increased 3-fold (qRT-PCR). Rhodococcus jostii without exposure to PFAS compounds had no mRFP expression. This novel detection system represents a synthetic biology approach to more efficiently detect PFAS in contaminated samples. With further refinement and modifications, a similar system could be readily deployed in the field around the world to detect this critical pollutant.

Building the vertebrate codex using the gene breaking protein trap library.

One key bottleneck in understanding the human genome is the relative under-characterization of 90% of protein coding regions. We report a collection of 1200 transgenic zebrafish strains made with the gene-break transposon (GBT) protein trap to simultaneously report and reversibly knockdown the tagged genes. Protein trap-associated mRFP expression shows previously undocumented expression of 35% and 90% of cloned genes at 2 and 4 days post-fertilization, respectively. Further, investigated alleles regularly show 99% gene-specific mRNA knockdown. Homozygous GBT animals in ryr1b, fras1, tnnt2a, edar and hmcn1 phenocopied established mutants. 204 cloned lines trapped diverse proteins, including 64 orthologs of human disease-associated genes with 40 as potential new disease models. Severely reduced skeletal muscle Ca2+ transients in GBT ryr1b homozygous animals validated the ability to explore molecular mechanisms of genetic diseases. This GBT system facilitates novel functional genome annotation towards understanding cellular and molecular underpinnings of vertebrate biology and human disease.

NOX2 Derived Superoxide Induces Pro-Tumoral p16INK4a Driven Senescence in the AML Bone Marrow Microenvironment

BackgroundAcute myeloid leukaemia (AML) is an age-related disease that is highly dependent on the bone marrow (BM) microenvironment. Senescence is the irreversible arrest of cell proliferation and is associated with the senescence associated secretary phenotype (SASP). The senescent phenotype has been associated with pro-tumoral changes that support proliferation and survival of malignant cells. Because AML is an age-related disease and highly dependent on the BM microenvironment, which naturally becomes senescent with age, we hypothesized, and subsequently tested the idea, that AML not only favors a senescent microenvironment but might actively shape a senescent microenvironment to promote tumor proliferation and survival.MethodsPrimary AML and bone marrow stroma was isolated from bone marrow samples obtained from patients following informed consent and non-malignant CD34+ cells were isolated from cord blood following informed consent. All primary tissue was used under approval from the UK Health Research Authority. NSG, p16-3MR(Demaria et al. 2014 Cell Development) and wildtype C57Bl/6 animals were used and experiments were conducted with approval from the UK Home Office and University of East Anglia Animal Welfare and Ethical Review Board. Primary AML and non-malignant human CD34+ cells were engrafted into NSG mice (AML-NSG and hu-NSG). Bone marrow stromal cells (BMSCs) were isolated by cell sorting for mouse CD45-, Ter119-, CD31-, CD105+, CD140a+. Syngeneic AML cells were generated from lineage negative cells transduced with the oncogene MN1. Senescence was determined by p16, p21 mRNA expression, SASP expression (IL-6 and MIP3a), senescence associated β-Galactosidase activity and using themonomeric red fluorescent protein (mRFP) component of the p16-3MR mouse model. Oxidative stress and superoxide levels were measured using 2‘,7‘-dichlorofluorescein (DCF) fluorescence and the Amplex Red assay. NOX-2 in AML was inhibited by RNAi.ResultsFirst, we isolated and analysed BMSC from AML-NSG and hu-NSG. Using real-time PCR we detected high expression of p16 and p21 in the BMSC isolated from AML-NSG but not hu-NSG. Moreover, we detected high mouse IL-6 and MIP3a cytokine levels from serum isolated from AML-NSG but not hu-NSG. MN1 engrafted into the p16-3MR model induced mRFP expression in the BMSC population. This shows that in both a xenograft and syngeneic model of AML the BMSC population becomes senescent. Using the p16-3MR model we selectively depleted senescent BMSC via the viral thymidine kinase death switch. Treatment of p16-3MR mice engrafted with MN1 with ganciclovir reduced tumour volume and prolonged animal survival. Since hydrogen peroxide has been shown to induce senescence we hypothesised that NOX2 derived superoxide from AML drives the BMSC senescence. BMSCs isolated from the MN1 engrafted p16-3MR model isolated BMSC show increased DCF fluorescence and isolated BM shows elevated superoxide compared to BMSC and BM isolated from control mice. NOX2 knock down resulted in significant reduction in BM derived superoxide and BMSC DCF fluorescence and this resulted in a reduced BMSC senescent phenotype.ConclusionHere we show that in AML, NOX2 derived superoxide induces a p16INK4a drivensenescent phenotype in the BMSC. Moreover, we find that this process forms a fundamental part of the pro-tumoral pathophysiology of the disease. The senescent bone marrow microenvironment may therefore present new treatment targets in AML. DisclosuresNo relevant conflicts of interest to declare.

Novel Spatiotemporal Analysis for Exploring a-Cell Differentiation

Aims: In vivo studies have demonstrated that pancreatic α-cells have potency to transdifferentiate into insulin-producing β cells, which could lead to potential therapy to cure diabetes. In addition, inappropriate glucagon secretion from α cells has been shown to be involved in the pathophysiology of diabetes. Although such findings highlight the need for better understanding of α-cell biology, it remains to be elucidated where α cells originate and what regulates α cell fate: α cell neogenesis, maturation and dedifferentiation. To address this issue, we generated a novel reporter mouse “Gcg-Timer.” Methods: We have previously generated Neurog3-Timer mouse (Miyatsuka et al. Diabetes 2009), in which newborn endocrine cells can be sorted by FACS separately from differentiated cells. However, we failed to obtain spatiotemporal information of newborn cells due to the low fluorescent intensity of DsRed-E5. To overcome this difficulty, we used a new reporter construct eGFP-IRES-mRFP and inserted it into the proglucagon gene. In this Gcg-Timer mouse, green fluorescence can be detected first, because mRFP expression is controlled by IRES and therefore lower than eGFP expression. As a result, newly-generated α cells exhibit green fluorescence and can be distinguished from more differentiated α cells exhibiting both green and red fluorescence. Results: Both microscopic analysis and flow cytometry revealed that green-dominant α cells were observed in Gcg-Timer mice at embryonic and neonatal stages until postnatal day 3 (P3), but not at P14 and adult stages. Intriguingly, red dominant cells, which were negative for glucagon staining, were detected at P14, indicating that α-to-non-α transition occurs at this stage. Conclusions: Gcg-Timer mice enabled us to label not only newly-generated α cells but also dedifferentiated α cells in a time-dependent manner. The α-cell neogenesis occurs specifically during embryonic and early neonatal stages under physiological condition. Disclosure M. Himuro: None. T. Miyatsuka: Research Support; Self; Eli Lilly and Company, Japan Society for the Promotion of Science, Daiichi Sankyo Company, Limited, Novartis Pharma K.K., Astellas Pharma Inc., MSD K.K., Takeda Pharmaceutical Company Limited.. L. Suzuki: None. M. Miura: Research Support; Self; Novo Nordisk Inc.. Speaker's Bureau; Self; Sanofi K.K.. Research Support; Self; Kowa Pharmaceuticals America, Inc.. T. Katahira: None. M. Takahashi: None. Y. Nishida: Research Support; Self; Novo Nordisk Inc.. Speaker's Bureau; Self; Daiichi Sankyo Company, Limited, AstraZeneca. S. Oshima: None. R. Okamoto: None. H. Watada: Advisory Panel; Self; AstraZeneca. Consultant; Self; Astellas Pharma US, Inc., AstraZeneca, Boehringer Ingelheim GmbH, Daiichi Sankyo Company, Limited, Sumitomo Dainippon Pharma Co., Ltd., Eli Lilly and Company, Kissei Pharmaceutical Co., Ltd., Kowa Pharmaceuticals America, Inc., Kyowa Hakko Kirin Co., Ltd., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novo Nordisk A/S, Novartis AG, Ono Pharmaceutical Co., Ltd., Sanofi, Sanwa Kagaku Kenkyusho Co., Ltd., Takeda Development Center Asia, Pte. Ltd.. Research Support; Self; Abbott, Astellas Pharma US, Inc., AstraZeneca, Bayer AG, Benefit One Health Care Co., Ltd., Boehringer Ingelheim GmbH, Bristol-Myers Squibb Company, Daiichi Sankyo Company, Limited, Sumitomo Dainippon Pharma Co., Ltd., Eli Lilly and Company, Kissei Pharmaceutical Co., Ltd., Kowa Pharmaceuticals America, Inc., Kyowa Hakko Kirin Co., Ltd., Johnson & Johnson Diabetes Institute, LLC., Merck Sharp & Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Mochida Pharmaceutical Co., Ltd., Nitto Boseki Co., Ltd., Novartis AG, Novo Nordisk A/S, Ono Pharmaceutical Co., Ltd., Pfizer Inc., Sanofi, Sanwa Kagaku Kenkyusho Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Development Center Asia, Pte. Ltd., Terumo Medical Corporation.

R/L, a double reporter mouse line that expresses luciferase gene upon Cre-mediated excision, followed by inactivation of mRFP expression.

The Cre/loxP system has become an important tool for the conditional gene knockout and conditional gene expression in genetically engineered mice. The applications of this system depend on transgenic reporter mouse lines that provide Cre recombinase activity with a defined cell type-, tissue-, or developmental stage-specificity. To develop a sensitive assay for monitoring Cre-mediated DNA excisions in mice, we generated Cre-mediated excision reporter mice, designated R/L mice (R/L: mRFP(monomeric red fluorescent protein)/luciferase), express mRFP throughout embryonic development and adult stages, while Cre-mediated excision deletes a loxP-flanked mRFP reporter gene and STOP sequence, thereby activating the expression of the second reporter gene luciferase, as assayed by in vivo and ex vivo bioluminescence imaging. After germ line deletion of the floxed mRFP and STOP sequence in R/L mice by EIIa-Cre mice, the resulting luciferase transgenic mice in which the loxP-mRFP-STOP-loxP cassette is excised from all cells express luciferase in all tissues and organs examined. The expression of luciferase transgene was activated in liver of RL/Alb-Cre double transgenic mice and in brain of RL/Nestin-Cre double transgenic mice when R/L reporter mice were mated with Alb-Cre mice and Nestin-Cre mice, respectively. Our findings reveal that the double reporter R/L mouse line is able to indicate the occurrence of Cre-mediated excision from early embryonic to adult lineages. Taken together, these findings demonstrate that the R/L mice serve as a sensitive reporter for Cre-mediated DNA excision both in living animals and in organs, tissues, and cells following necropsy.

A native promoter and inclusion of an intron is necessary for efficient expression of GFP or mRFP in Armillaria mellea.

Armillaria mellea is a significant pathogen that causes Armillaria root disease on numerous hosts in forests, gardens and agricultural environments worldwide. Using a yeast-adapted pCAMBIA0380 Agrobacterium vector, we have constructed a series of vectors for transformation of A. mellea, assembled using yeast-based recombination methods. These have been designed to allow easy exchange of promoters and inclusion of introns. The vectors were first tested by transformation into basidiomycete Clitopilus passeckerianus to ascertain vector functionality then used to transform A. mellea. We show that heterologous promoters from the basidiomycetes Agaricus bisporus and Phanerochaete chrysosporium that were used successfully to control the hygromycin resistance cassette were not able to support expression of mRFP or GFP in A. mellea. The endogenous A. mellea gpd promoter delivered efficient expression, and we show that inclusion of an intron was also required for transgene expression. GFP and mRFP expression was stable in mycelia and fluorescence was visible in transgenic fruiting bodies and GFP was detectable in planta. Use of these vectors has been successful in giving expression of the fluorescent proteins GFP and mRFP in A. mellea, providing an additional molecular tool for this pathogen.

Expression of the short chain fatty acid receptor GPR41/FFAR3 in autonomic and somatic sensory ganglia

G-protein-coupled receptor 41 (GPR41) also called free fatty acid receptor 3 (FFAR3) is a Gαi-coupled receptor activated by short-chain fatty acids (SCFAs) mainly produced from dietary complex carbohydrate fibers in the large intestine as products of fermentation by microbiota. FFAR3 is expressed in enteroendocrine cells, but has recently also been shown to be present in sympathetic neurons of the superior cervical ganglion.The aim of this study was to investigate whether the FFAR3 is present in other autonomic and sensory ganglia possibly influencing gut physiology. Cryostat sections were cut of autonomic and sensory ganglia of a transgenic reporter mouse expressing the monomeric red fluorescent protein (mRFP) gene under the control of the FFAR3 promoter. Control for specific expression was also done by immunohistochemistry with an antibody against the reporter protein. mRFP expression was as expected found not only in neurons of the superior cervical ganglion, but also in sympathetic ganglia of the thoracic and lumbar sympathetic trunk. Further, neurons in prevertebral ganglia expressed the mRFP reporter. FFAR3-mRFP-expressing neurons were also present in both autonomic and sensory ganglia such as the vagal ganglion, the spinal dorsal root ganglion and the trigeminal ganglion. No expression was observed in the brain or spinal cord. By use of radioactive-labeled antisense DNA probes, mRNA encoding the FFAR3 was found to be present in cells of the same ganglia. Further, the expression of the FFAR3 in the ganglia of the transgenic mice was confirmed by immunohistochemistry using an antibody directed against the receptor protein, and double labeling colocalized mRFP and the FFAR3-protein in the same neurons. Finally, quantitative real-time polymerase chain reaction (qRT-PCR) on extracts from the ganglia supported the presence mRNA encoding the FFAR3 in most of the investigated tissues.These data indicate that FFAR3 is expressed on postganglionic sympathetic and sensory neurons in both the autonomic and somatic peripheral nervous system and that SCFAs act not only through the enteroendocrine system but also directly by modifying physiological reflexes integrating the peripheral nervous system and the gastro-intestinal tract.

Rabies Virus Vector Transgene Expression Level and Cytotoxicity Improvement Induced by Deletion of Glycoprotein Gene

The glycoprotein (G) of rabies virus (RV) is required for binding to neuronal receptors and for viral entry. G-deleted RV vector is a powerful tool for investigating the organization and function of the neural circuits. It gives the investigator the ability to genetically target initial infection to particular neurons and to control trans-synaptic propagation. In this study we have quantitatively evaluated the effect of G gene deletion on the cytotoxicity and transgene expression level of the RV vector. We compared the characteristics of the propagation-competent RV vector (rHEP5.0-CVSG-mRFP) and the G-deleted RV vector (rHEP5.0-ΔG-mRFP), both of which are based on the attenuated HEP-Flury strain and express monomeric red fluorescent protein (mRFP) as a transgene. rHEP5.0-ΔG-mRFP showed lower cytotoxicity than rHEP5.0-CVSG-mRFP, and within 16 days of infection we found no change in the basic electrophysiological properties of neurons infected with the rHEP5.0-ΔG-mRFP. The mRFP expression level of rHEP5.0-ΔG-mRFP was much higher than that of rHEP5.0-CVSG-mRFP, and 3 days after infection the retrogradely infected neurons were clearly visualized by the expressed fluorescent protein without any staining. This may be due to the low cytotoxicity and/or the presumed change in the polymerase gene (L) expression level of the G-deleted RV vector. Although the mechanisms remains to be clarified, the results of this study indicate that deletion of the G gene greatly improves the usability of the RV vector for studying the organization and function of the neural circuits by decreasing the cytotoxicity and increasing the transgene expression level.

Chicken β-globin insulators fail to shield the nkx2.5 promoter from integration site effects in zebrafish

Genetic lineage tracing and conditional mutagenesis are developmental genetics techniques reliant on precise tissue-specific expression of transgenes. In the mouse, high specificity is usually achieved by inserting the transgene into the locus of interest through homologous recombination in embryonic stem cells. In the zebrafish, DNA containing the transgenic construct is randomly integrated into the genome, usually through transposon-mediated transgenesis. Expression of such transgenes is affected by regulatory features surrounding the integration site from general accessibility of chromatin to tissue-specific enhancers. We tested if the 1.2kb cHS4 insulators derived from the chicken β-globin locus can shield a transgene from chromosomal position effects in the zebrafish genome. As our test promoters, we used two different-length versions of the zebrafish nkx2.5. We found that flanking a transgenic construct by cHS4 insulation sequences leads to overall increase in the expression of nkx2.5:mRFP. However, we also observed a very high degree of variability of mRFP expression, indicating that cHS4 insulators fail to protect nkx2.5:mRFP from falling under the control of enhancers in the vicinity of integration site.

The ventral to dorsal BMP activity gradient in the early zebrafish embryo is determined by graded expression of BMP ligands

In the early zebrafish embryo, a ventral to dorsal gradient of bone morphogenetic protein (BMP) activity is established, which is essential for the specification of cell fates along this axis. To visualise and mechanistically determine how this BMP activity gradient forms, we have used a transgenic zebrafish line that expresses monomeric red fluorescent protein (mRFP) under the control of well-characterised BMP responsive elements. We demonstrate that mRFP expression in this line faithfully reports BMP and GDF signalling at both early and late stages of development. Taking advantage of the unstable nature of mRFP transcripts, we use in situ hybridisation to reveal the dynamic spatio-temporal pattern of BMP activity and establish the timing and sequence of events that lead to the formation of the BMP activity gradient. We show that the BMP transcriptional activity gradient is established between 30% and 40% epiboly stages and that it is preceded by graded mRNA expression of the BMP ligands. Both Dharma and FGF signalling contribute to graded bmp transcription during these early stages and it is subsequently maintained through autocrine BMP signalling. We show that BMP2B protein is also expressed in a gradient as early as blastula stages, but do not find any evidence of diffusion of this BMP to generate the BMP transcriptional activity gradient. Thus, in contrast to diffusion/transport-based models of BMP gradient formation in Drosophila, our results indicate that the establishment of the BMP activity gradient in early zebrafish embryos is determined by graded expression of the BMP ligands.

Directed Differentiation of Human Embryonic Stem Cells to Interrogate the Cardiac Gene Regulatory Network

The limited ability of the heart to regenerate has prompted development of new systems to produce cardiomyocytes for therapeutics. While differentiation of human embryonic stem cells (hESCs) into cardiomyocytes has been well documented, the process remains inefficient and/or expensive, and progress would be facilitated by better understanding the early genetic events that cause cardiac specification. By maintaining a transgenic cardiac-specific MYH6-monomeric red fluorescent protein (mRFP) reporter hESC line in conditions that promote pluripotency, we tested the ability of combinations of 15 genes to induce cardiac specification. Screening identified GATA4 plus TBX5 as the minimum requirement to activate the cardiac gene regulatory network and produce mRFP(+) cells, while a combination of GATA4, TBX5, NKX2.5, and BAF60c (GTNB) was necessary to generate beating cardiomyocytes positive for cTnI and α-actinin. Including the chemotherapeutic agent, Ara-C, from day 10 of induced differentiation enriched for cTnI/α-actinin double positive cells to 45%. Transient expression of GTNB for 5-7 days was necessary to activate the cardiogenesis through progenitor intermediates in a manner consistent with normal heart development. This system provides a route to test the effect of different factors on human cardiac differentiation and will be useful in understanding the network failures that underlie disease phenotypes.

Tracing phenotypic reversibility of pancreatic β-cells in vitro.

Aims/Introduction: Studies have suggested that pancreatic β-cells undergo dedifferentiation during proliferation in vitro. However, due to limitations of the methodologies used, the question remains whether such dedifferentiated cells can redifferentiate into β-cells. We have established a method for cell tracing in combination with fluorescence-activated cell sorter (FACS). Using this method, mouse pancreatic β-cells labeled with green fluorescent protein (GFP) under the control of the insulin promoter are collected by FACS. These β-cells can be traced and characterized throughout the culture process, even when insulin becomes undetectable, because the cells are also marked with monomeric red fluorescent protein (mRFP) driven by the CAG promoter. When cultured with fetal mouse pancreatic cells, FACS sorted β-cells lost GFP expression, but retained mRFP expression. The cells also lost expressions of genes characteristic of the β-cell phenotype, such as Pdx1 and glucokinase, indicating dedifferentiation. More than 30% of such dedifferentiated pancreatic β-cells were detected in S or G2/M phase. Furthermore, these dedifferentiated cells redifferentiated into insulin-expressing cells on cultivation with a MEK1/2 inhibitor. Our data provide direct evidence that pre-existing β-cells can undergo dedifferentiation and redifferentiation in vitro, their phenotype is reversible and that dedifferentiation in β-cells is associated with progression of the cell cycle. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2010.00051.x, 2010).

Differences in transductional tropism of adenoviral and lentiviral vectors in the rat brainstem

Adenoviral vectors (AVVs) and lentiviral vectors (LVVs) are highly useful research tools which can be used to investigate the function of specific cell phenotypes in the brain. The transductional tropism of viral vectors has a critical impact upon the transgene expression in different brain areas. This largely depends on the properties of the viral particles, which for AVVs are most commonly analogous to the serotype 5 adenovirus and, in the case of LVVs, are determined by the envelope used for pseudotyping, for example the vesicular stomatitis virus coat (VSVG). We have created a matching set of shuttle plasmids that allow a one-step transfer of an entire expression cassette between the backbones of AVVs and LVVs. This has permitted a fair assessment of the impact of the vector type on tropism for both AVVs and LVVs. Thus, the aims of this study were twofold: (i) to develop and demonstrate the validity of a transgene 'swap' system between AVVs and LVVs; and (ii) using this system, to assess the tropism of AVVs and LVVs for neuronal versus glial cell types. We have constructed AVVs and VSVG-coated LVVs to express monomeric red fluorescent protein (mRFP) driven by the human cytomegalovirus promoter (hCMV). Transgene expression in neurones and glia in the hypoglossal and dorsal vagal motor nuclei of the rat brainstem was compared by determining the colocalization with immunostaining for the neuronal marker NeuN (neuronal nuclear antigen) and the glial marker GFAP (glial fibrillatory acidic protein). We found that 55% of mRFP-expressing cells transduced with AVVs were immunopositive for GFAP, while only 38% were NeuN-immunoreactive. In contrast, when the same expression cassette was delivered by VSVG-coated LVVs, the neurone/glia ratio of mRFP expression was reversed with 56% of mRFP-positive cells identified as neurones and 26% as glia. Thus, the present study provides compelling evidence that VSVG-coated LVVs significantly shift transgene expression towards neurones while transduction with AVVs favours glia.