Adenoviral Vector-mediated Gene Transfer Research Articles

Canine adenoviral vector-mediated gene transfer to the guinea pig brain

Pre-clinical testing of gene transfer tools is often first conducted in small animal models such as laboratory mice or rats, before extension into a larger animal such as non-human primates. The guinea pig brain may provide an intermediary model in which to examine neurodevelopmental processes given that guinea pigs are considered precocial. Here, we characterized the efficacy and biodistribution following injection of a canine adenovirus type 2 (CAV-2) vector in the young adult guinea pig brain. Despite the development of mild fevers post-treatment in the vector-treated animals, widespread transduction was evident throughout the brain parenchyma. Neutralizing antibodies against CAV-2 were not detected. Our data demonstrate that the combination of CAV-2 vectors and guinea pigs provide a clinically relevant model for evaluation of therapeutics.

Adenoviral vector-mediated GM-CSF gene transfer improves anti-mycobacterial immunity in mice – role of regulatory T cells

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a hematopoietic growth factor involved in differentiation, survival and activation of myeloid and non-myeloid cells with important implications for lung antibacterial immunity. Here we examined the effect of pulmonary adenoviral vector-mediated delivery of GM-CSF (AdGM-CSF) on anti-mycobacterial immunity in M. bovis BCG infected mice. Exposure of M. bovis BCG infected mice to AdGM-CSF either applied on 6h, or 6h and 7days post-infection substantially increased alveolar recruitment of iNOS and IL-12 expressing macrophages, and significantly increased accumulation of IFNγpos T cells and particularly regulatory T cells (Tregs). This was accompanied by significantly reduced mycobacterial loads in the lungs of mice. Importantly, diphtheria toxin-induced depletion of Tregs did not influence mycobacterial loads, but accentuated immunopathology in AdGM-CSF-exposed mice infected with M. bovis BCG. Together, the data demonstrate that AdGM-CSF therapy improves lung protective immunity against M. bovis BCG infection in mice independent of co-recruited Tregs, which however critically contribute to limit lung immunopathology in BCG-infected mice. These data may be relevant to the development of immunomodulatory strategies to limit immunopathology-based lung injury in tuberculosis in humans.

Formulation and in vitro and in vivo evaluation of a cationic emulsion as a vehicle for improving adenoviral gene transfer

Advancements in the use of adenoviral vectors in gene therapy have been limited by the need for specific receptors on targeted cell types, immunogenicity and hepatotoxicity following systemic administration. In an effort to overcome the current limitations of adenovirus-mediated gene transfer, cationic emulsions were explored as a vehicle to improve adenoviral vector-mediated gene transfer. Complexation of adenovirus with emulsions containing the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) enhanced the potency of adenoviral gene transfer as compared to DOTAP liposomes. Among the various emulsion formulations examined, those containing the iodized oil, Lipiodol, as an inner core and stabilized by DOTAP/cholesterol/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(poly-ethylene glycol)-5000 most efficiently enhanced adenovirus-mediated gene transfer. Optimized Lipiodol-containing emulsions appear to be more strongly associated with adenoviral particles, exhibiting higher complex stability compared to other formulations. They provide the adenovirus with an additional cellular entry mechanism through caveolae-dependent endocytosis, thereby increasing adenovirus entry into cells. Furthermore, adenovirus–emulsion complexation significantly reduced transgene expression in the liver following systemic administration. These findings indicate that emulsion complexation may be a promising strategy for overcoming many of the challenges associated with the use of adenoviruses in gene therapy. Additionally, the observation of increased transgene expression in lung together with reduced expression in liver demonstrates that the adenovirus–emulsion complex may act as a lung-targeting adenoviral gene delivery system.

507. Adenoviral Vector-Mediated CFTR Gene Transfer and Anion Transport Correction of CF Pig Airways In Vivo

507. Adenoviral Vector-Mediated CFTR Gene Transfer and Anion Transport Correction of CF Pig Airways In Vivo

Switching a Replication-Defective Adenoviral Vector into a Replication-Competent, Oncolytic Adenovirus

The adenovirus immediate early gene E1A initiates the program of viral gene transcription and reprograms multiple aspects of cell function and behavior. For adenoviral (Ad) vector-mediated gene transfer and therapy approaches, where replication-defective (RD) gene transfer is required, E1A has thus been the primary target for deletions. For oncolytic gene therapy for cancer, where replication-competent (RC) Ad viral gene expression is needed, E1A has been either mutated or placed under tumor-specific transcriptional control. A novel Ad vector that initially infected target tumor cells in an RD manner for transgene expression but that could be "switched" into an RC, oncolytic state when needed might represent an advance in vector technology. Here, we report that we designed such an Ad vector (proAdΔ24.GFP), where initial Ad replication is silenced by a green fluorescent protein (GFP) transgene that blocks cytomegalovirus (CMV)-mediated transcription of E1A. This vector functions as a bona fide E1A-deleted RD vector in infected tumor cells. However, because the silencing GFP transgene is flanked by FLP recombination target (FRT) sites, we show that it can be efficiently excised by Flp recombinase site-specific recombination, either when Flp is expressed constitutively in cells or when it is provided in trans by coinfection with a second RD herpes simplex virus (HSV) amplicon vector. This switches the RD Ad, proAdΔ24.GFP, into a fully RC, oncolytic Ad (rAdΔ24) that lyses tumor cells in culture and generates oncolytic progeny virions. In vivo, coinfection of established flank tumors with the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon leads to generation of RC, oncolytic rAdΔ24. In an orthotopic human glioma xenograft tumor model, coinjection of the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon also led to a significant increase in animal survival, compared to controls. Therefore, Flp-FRT site-specific recombination can be applied to switch RD Ad into fully oncolytic RC Ad for tumor therapy and is potentially applicable to a variety of gene therapy approaches.

Effect of human 15-lipoxygenase-1 metabolites on vascular function in mouse mesenteric arteries and hearts

Lipoxygenases regulate vascular function by metabolizing arachidonic acid (AA) to dilator eicosanoids. Previously, we showed that endothelium-targeted adenoviral vector-mediated gene transfer of the human 15-lipoxygenase-1 (h15-LO-1) enhances arterial relaxation through the production of vasodilatory hydroxyepoxyeicosatrienoic acid (HEETA) and trihydroxyeicosatrienoic acid (THETA) metabolites. To further define this function, a transgenic (Tg) mouse line that overexpresses h15-LO-1 was studied. Western blot, immunohistochemistry and RT-PCR results confirmed expression of 15-LO-1 transgene in tissues, especially high quantity in coronary arterial wall, of Tg mice. Reverse-phase HPLC analysis of [14C]-AA metabolites in heart tissues revealed enhanced 15-HETE synthesis in Tg vs. WT mice. Among the 15-LO-1 metabolites, 15-HETE, erythro-13-H-14,15-EETA, and 11(R),12(S),15(S)-THETA relaxed the mouse mesenteric arteries to the greatest extent. The presence of h15-LO-1 increased acetylcholine- and AA-mediated relaxation in mesenteric arteries of Tg mice compared to WT mice. 15-LO-1 was most abundant in the heart; therefore, we used the Langendorff heart model to test the hypothesis that elevated 15-LO-1 levels would increase coronary flow following a short ischemia episode. Both peak flow and excess flow of reperfused hearts were significantly elevated in hearts from Tg compared to WT mice being 2.03 and 3.22 times greater, respectively. These results indicate that h15-LO-1-derived metabolites are highly vasoactive and may play a critical role in regulating coronary blood flow.

Ligand-bound Thyroid Hormone Receptor Contributes to Reprogramming of Pancreatic Acinar Cells into Insulin-producing Cells

One goal of diabetic regenerative medicine is to instructively convert mature pancreatic exocrine cells into insulin-producing cells. We recently reported that ligand-bound thyroid hormone receptor α (TRα) plays a critical role in expansion of the β-cell mass during postnatal development. Here, we used an adenovirus vector that expresses TRα driven by the amylase 2 promoter (AdAmy2TRα) to induce the reprogramming of pancreatic acinar cells into insulin-producing cells. Treatment with l-3,5,3-triiodothyronine increases the association of TRα with the p85α subunit of phosphatidylinositol 3-kinase (PI3K), leading to the phosphorylation and activation of Akt and the expression of Pdx1, Ngn3, and MafA in purified acinar cells. Analyses performed with the lectin-associated cell lineage tracing system and the Cre/loxP-based direct cell lineage tracing system indicate that newly synthesized insulin-producing cells originate from elastase-expressing pancreatic acinar cells. Insulin-containing secretory granules were identified in these cells by electron microscopy. The inhibition of p85α expression by siRNA or the inhibition of PI3K by LY294002 prevents the expression of Pdx1, Ngn3, and MafA and the reprogramming to insulin-producing cells. In immunodeficient mice with streptozotocin-induced hyperglycemia, treatment with AdAmy2TRα leads to the reprogramming of pancreatic acinar cells to insulin-producing cells in vivo. Our findings suggest that ligand-bound TRα plays a critical role in β-cell regeneration during postnatal development via activation of PI3K signaling.

Neo-islet formation in liver of diabetic mice by helper-dependent adenoviral vector-mediated gene transfer.

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes.Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression1. They lack chronic toxicity because they lack viral coding sequences2-5 and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes.In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.

Neo-Islet Formation in Liver of Diabetic Mice by Helper-dependent Adenoviral Vector-Mediated Gene Transfer

Type 1 diabetes is caused by T cell-mediated autoimmune destruction of insulin-producing cells in the pancreas. Until now insulin replacement is still the major therapy, because islet transplantation has been limited by donor availability and by the need for long-term immunosuppression. Induced islet neogenesis by gene transfer of Neuogenin3 (Ngn3), the islet lineage-defining specific transcription factor and Betacellulin (Btc), an islet growth factor has the potential to cure type 1 diabetes. Adenoviral vectors (Ads) are highly efficient gene transfer vector; however, early generation Ads have several disadvantages for in vivo use. Helper-dependent Ads (HDAds) are the most advanced Ads that were developed to improve the safety profile of early generation of Ads and to prolong transgene expression1. They lack chronic toxicity because they lack viral coding sequences2-5 and retain only Ad cis elements necessary for vector replication and packaging. This allows cloning of up to 36 kb genes. In this protocol, we describe the method to generate HDAd-Ngn3 and HDAd-Btc and to deliver these vectors into STZ-induced diabetic mice. Our results show that co-injection of HDAd-Ngn3 and HDAd-Btc induces 'neo islets' in the liver and reverses hyperglycemia in diabetic mice.

Immunological tolerance of human hepatocyte xenograft induced by adenovirus vector-mediated CTLA4Ig gene transfer

Systemic administration of CTLA4Ig has been applied in inducing immunological tolerance of hepatocyte implants, but has potential for systemic immune inhibition. This study was designed to induce hepatocyte immunological tolerance by locally expressing CTLA4Ig in an attempt to improve the effectiveness of cell transplantation. A normal human liver cell line (L02) was transfected with adenovirus vector containing the CTLA4Ig gene (Ad-CTLA4Ig-EGFP) in vitro, and the expression of CTLA4Ig by transfected cells was assessed by fluorescent imaging and immunocytochemical staining. Transfected cells then were injected into the spleen of Sprague-Dawley rats, the survival of cells was determined by immunohistochemistry, and the immune status was examined through CD4+ and CD69+ T cell-counts and ELISA detection of IL-2 in peripheral blood. L02 cells expressed CTLA4Ig in the cytoplasm for >4 weeks. Surviving L02 cells were observed in the experimental group at 3 and 4 weeks post-transplantation, while none was detected in the control group. Furthermore, the percentages of CD4+ and CD4+CD69+ T cells in the CTLA4-transfected group were 24.5% and 45.1%, markedly lower than those in the control group at 4 weeks post-transplantation (P<0.01). Furthermore, the IL-2 level was also lower in the CTLA4-transfected group than in the control group. Adenovirus-mediated CTLA4Ig gene transfer into human hepatocytes has the potential to become an effective method of inducing immunological tolerance in hepatocyte transplantation.

Overexpression of the human ubiquitin E3 ligase CUL4A alleviates hypoxia–reoxygenation injury in pheochromocytoma (PC12) cells

The ubiquitin E3 ligase CUL4A plays important roles in diverse cellular processes including carcinogenesis and proliferation. It has been reported that the expression of CUL4A can be induced by hypoxic-ischemic injury. However, the effect of elevated expression of CUL4A on hypoxia–reoxygenation injury is currently unclear. In this study, human CUL4A (hCUL4A) was expressed in rat pheochromocytoma (PC12) cells using adenoviral vector-mediated gene transfer, and the effects of hCUL4A expression on hypoxia–reoxygenation injury were investigated. In PC12 cells subjected to hypoxia and reoxygenation, we found that hCUL4A suppresses apoptosis and DNA damage by regulating apoptosis-related proteins and cell cycle regulators (Bcl-2, caspase-3, p53 and p27); consequently, hCUL4A promotes cell survival. Taken together, our results reveal the beneficial effects of hCUL4A in PC12 cells upon hypoxia–reoxygenation injury.

Transcription Factor Rational Design Improves Directed Differentiation of Human Mesenchymal Stem Cells Into Skeletal Myocytes

There is great interest in transdifferentiating cells from one lineage into those of another and in dedifferentiating mature cells back into a stem/progenitor cell state by deploying naturally occurring transcription factors (TFs). Often, however, steering cellular differentiation pathways in a predictable and efficient manner remains challenging. Here, we investigated the principle of combining domains from different lineage-specific TFs to improve directed cellular differentiation. As proof-of-concept, we engineered the whole-human TF MyoDCD, which has the NH(2)-terminal transcription activation domain (TAD) and adjacent DNA-binding motif of MyoD COOH-terminally fused to the TAD of myocardin (MyoCD). We found via reporter gene and marker protein assays as well as by a cell fusion readout system that, targeting the TAD of MyoCD to genes normally responsive to the skeletal muscle-specific TF MyoD enforces more robust myogenic reprogramming of nonmuscle cells than that achieved by the parental, prototypic master TF, MyoD. Human mesenchymal stem cells (hMSCs) transduced with a codon-optimized microdystrophin gene linked to a synthetic striated muscle-specific promoter and/or with MyoD or MyoDCD were evaluated for complementing the genetic defect in Duchenne muscular dystrophy (DMD) myocytes through heterotypic cell fusion. Cotransduction of hMSCs with MyoDCD and microdystrophin led to chimeric myotubes containing the highest dystrophin levels.

Combination treatment using adenovirus vector-mediated tumor necrosis factor-alpha gene transfer and a NF-κB inhibitor for pancreatic cancer in mice

Gene therapy using an adenoviral vector expressing tumor necrosis factor-alpha (TNF-α) is a new therapeutic approach for pancreatic cancer. However, the efficacy of TNF-α is limited, because it activates nuclear factor-κB (NF-κB). We investigated the combined use of AxCAhTNF-α, adenoviral vector-expressing human TNF-α, and nafamostat mesilate, a serine-protease inhibitor, a NF-κB inhibitor, using pancreatic cancer in mice. In vitro, nafamostat mesilate inhibited TNF-α-induced NF-κB activation and enhanced TNF-α-induced apoptosis in human cancer cell line (MIAPaCa-2). In vivo, we assessed combination treatment of AxCAhTNF-α and nafamostat mesilate using xenograft models in nude mice by subcutaneous injection of MIAPaCa-2. When the implanted tumor size reached 8.0 mm, TNF-α group ( n = 12), was injected AxCAhTNF-α intra-tumorally once a week, while combination group ( n = 12), was injected AxCAhTNF-α intra-tumorally once a week and nafamostat mesilate intraperitoneally thrice a week. In combination group, tumor growth was significantly slower, and the number of apoptosis cells was significantly greater than those in AxCAhTNF-α group ( p < 0.05). In conclusion, adenovirus vector-mediated TNF-α gene therapy combined with nafamostat mesilate was effective for pancreatic cancer in mice.

Efficient Generation of Hepatoblasts From Human ES Cells and iPS Cells by Transient Overexpression of Homeobox Gene HEX

Human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the potential to differentiate into all cell lineages, including hepatocytes, in vitro. Induced hepatocytes have a wide range of potential application in biomedical research, drug discovery, and the treatment of liver disease. However, the existing protocols for hepatic differentiation of PSCs are not very efficient. In this study, we developed an efficient method to induce hepatoblasts, which are progenitors of hepatocytes, from human ESCs and iPSCs by overexpression of the HEX gene, which is a homeotic gene and also essential for hepatic differentiation, using a HEX-expressing adenovirus (Ad) vector under serum/feeder cell-free chemically defined conditions. Ad-HEX-transduced cells expressed α-fetoprotein (AFP) at day 9 and then expressed albumin (ALB) at day 12. Furthermore, the Ad-HEX-transduced cells derived from human iPSCs also produced several cytochrome P450 (CYP) isozymes, and these P450 isozymes were capable of converting the substrates to metabolites and responding to the chemical stimulation. Our differentiation protocol using Ad vector-mediated transient HEX transduction under chemically defined conditions efficiently generates hepatoblasts from human ESCs and iPSCs. Thus, our methods would be useful for not only drug screening but also therapeutic applications.

Anti-Tumor Effect of Adenoviral Vector-Mediated p53 Gene Transfer on the Growth of Canine Osteosarcoma Xenografts in Nude Mice

We evaluated the anti-tumor effect of adenoviral vector-mediated p53 gene therapy on the growth of canine osteosarcoma xenografts formed in nude mice. Nude mice were subcutaneously transplanted with cells of 2 P53 mutant canine osteosarcoma cell lines, POS and CHOS. The osteosarcoma xenografts were injected with either an adenoviral vector that expresses canine wild-type P53 (AxCA-cp53) or LacZ (AxCA-LacZ). Tumor growth was significantly inhibited in the xenografts injected with AxCA-cp53 in comparison to those injected with AxCA-LacZ or PBS during the observation period of 27 days. An increase of the amount of p21(WAF1/CDKN1A) mRNA, and the number of apoptotic cells was shown in the tumors injected with AxCA-cp53 in comparison to those injected with AxCA-LacZ or PBS. The present study revealed that the adenoviral vector-mediated p53 gene transfer had an anti-tumor effect in canine osteosarcoma xenografts formed in nude mice.

Intra-arterial Targeted Islet-specific Expression of Sirt1 Protects β Cells From Streptozotocin-induced Apoptosis in Mice

Gene therapy provides a promising approach to curing diabetes. However, an effective route for islet-specific targeting has yet to be established. Toward this end, the pancreatic blood circulation system in Balb/c mice was determined by the injection of rhodamine-containing beads. The efficiency of islet targeting was then measured by the injection of adenoviral vectors carrying a green fluorescence gene via the celiac trunk (C.T.). The results showed that >95% of islets and about 60% of β cells within the pancreatic body and tail could be labeled 3 days after surgery. α-Cell labeling was not as efficient, whereas labeling of nonendocrine tissues was barely detectable. For proof of principle, adenoviral vectors carrying a Sirtuin transgene were injected similarly to test the islet protection effect in the streptozotocin (STZ)-induced type 1 diabetic model. The results demonstrated that overexpression of Sirtuin in STZ-treated mice reduced the level of β-cell death and extent of glucose intolerance. This study reports on efficient islet-specific targeting by using adenoviral injection. This procedure could be invaluable to the treatment of diabetes and the study of islet biology.

Improvement of adenoviral vector-mediated gene transfer to airway epithelia by folate-modified anionic liposomes

Despite remarkable progress in the development of both viral and nonviral gene delivery vectors for airway disease treatment, poor gene transfer efficiency to the airway epithelium is a major obstacle in clinical application. To take advantage of the unique features of viral and nonviral vectors, we have developed complexes of adenovirus vector and anionic liposomes (AL-Ad5) by the calcium-induced phase change method. In the current study, based on the fact that there are overexpressed folate receptors on the surface of airway epithelia, we further modified the AL-Ad5 complexes with folate (F-AL-Ad5) to improve the transduction ability of Ad5 in airway epithelia. The transduction efficiencies of the obtained F-AL-Ad5 and AL-Ad5 complexes were assessed in primary-cultured airway epithelia in vitro. Our results indicated that compared with naked adenovirus vector, both AL-Ad5 and F-AL-Ad5 could significantly enhance the gene transduction efficiency of adenovirus vector in primary-cultured airway epithelial cells. Moreover, the enhancement mediated by F-AL-Ad5 was more dramatic than that by AL-Ad5. These results suggested that F-AL-Ad5 may be a useful strategy to deliver therapeutic genes to the airway epithelia and is promising in clinical application.

143. Systemic Delivery of Bioactive Glucagon-Like Peptide 1 Following Adenoviral Vector-Mediated Gene Transfer to Murine Submandibular Glands

143. Systemic Delivery of Bioactive Glucagon-Like Peptide 1 Following Adenoviral Vector-Mediated Gene Transfer to Murine Submandibular Glands

Prevention of hepatic ischemia–reperfusion injury by pre-administration of catalase-expressing adenovirus vectors

Liver ischemia/reperfusion (I/R) injury, which is mainly caused by the generation of reactive oxygen species (ROS) during the reperfusion, remains an important clinical problem associated with liver transplantation and major liver surgery. Therefore, ROS should be detoxified to prevent hepatic I/R-induced injury. Delivery of antioxidant genes into liver is considered to be promising for prevention of hepatic I/R injury; however, therapeutic effects of antioxidant gene transfer to the liver have not been fully examined. The aim of this study was to examine whether adenovirus (Ad) vector-mediated catalase gene transfer in the liver is an effective approach for scavenging ROS and preventing hepatic I/R injury. Intravenous administration of Ad vectors expressing catalase, which is an antioxidant enzyme scavenging H 2O 2, resulted in a significant increase in catalase activity in the liver. Pre-injection of catalase-expressing Ad vectors dramatically prevented I/R-induced elevation in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and hepatic necrosis. The livers were also protected in another liver injury model, CCl 4-induced liver injury, by catalase-expressing Ad vectors. Furthermore, the survival rates of mice subjected to both partial hepatectomy and I/R treatment were improved by pre-injection of catalase-expressing Ad vectors. On the other hand, control Ad vectors expressing β-galactosidase did not show any significant preventive effects in the liver on the models of I/R-induced or CCl 4-induced hepatic injury described above. These results indicate that hepatic delivery of the catalase gene by Ad vectors is a promising approach for the prevention of oxidative stress-induced liver injury.

Feline Immunodeficiency Virus as a Gene Transfer Vector in the Rat Nucleus Tractus Solitarii

Gene transfer has been used to examine the role of putative neurotransmitters in the nucleus tractus solitarii (NTS). Most such studies used adenovirus vector-mediated gene transfer although adenovirus vector transfects both neuronal and non-neuronal cells. Successful transfection in the NTS has also been reported with lentivirus as the vector. Feline immunodeficiency virus (FIV), a lentivirus, may preferentially transfect neurons and could be a powerful tool to delineate physiological effects produced by altered synthesis of transmitters in neurons. However, it has not been studied in NTS. Therefore, we sought to determine whether FIV transfects rat NTS cells and to define the type of cell transfected. We found that injection of FIV encoding LacZ gene (FIVLacZ) into the NTS led to transfection of numerous NTS cells. Injection of FIVLacZ did not alter immunoreactivity (IR) for neuronal nitric oxide synthase, which we have shown resides in NTS neurons. A majority (91.7 +/- 3.9%) of transfected cells contained IR for neuronal nuclear antigen, a neuronal marker; 2.1 +/- 3.8% of transfected cells contained IR for glial fibrillary acidic protein, a glial marker. No transfected neurons or fibers were observed in the nodose ganglion, which sends afferents to the NTS. We conclude that FIV almost exclusively transfects neurons in the rat NTS from which it is not retrogradely transported. The cell-type specificity of FIV in the NTS may provide a molecular method to study local physiological functions mediated by potential neurotransmitters in the NTS.