Levels Of Green Fluorescent Protein Research Articles

Transformation of the rodent malaria parasite Plasmodium chabaudi and generation of a stable fluorescent line PcGFPCON

BackgroundThe rodent malaria parasite Plasmodium chabaudi has proven of great value in the analysis of fundamental aspects of host-parasite-vector interactions implicated in disease pathology and parasite evolutionary ecology. However, the lack of gene modification technologies for this model has precluded more direct functional studies.MethodsThe development of in vitro culture methods to yield P. chabaudi schizonts for transfection and conditions for genetic modification of this rodent malaria model are reported.ResultsIndependent P. chabaudi gene-integrant lines that constitutively express high levels of green fluorescent protein throughout their life cycle have been generated.ConclusionGenetic modification of P. chabaudi is now possible. The production of genetically distinct reference lines offers substantial advances to our understanding of malaria parasite biology, especially interactions with the immune system during chronic infection.

The tissue plasminogen activator gene promoter: a novel tool for radiogenic gene therapy of the prostate?

Radiation therapy is a treatment modality routinely used in cancer management so it is not unexpected that radiation-inducible promoters have emerged as an attractive tool for controlled gene therapy. The human tissue plasminogen activator gene promoter (t-PA) has been proposed as a candidate for radiogenic gene therapy, but has not been exploited to date. The purpose of this study was to evaluate the potential of this promoter to drive the expression of a reporter gene, the green fluorescent protein (GFP), in response to radiation exposure. To investigate whether the promoter could be used for prostate cancer gene therapy, we initially transfected normal and malignant prostate cells. We then transfected HMEC-1 endothelial cells and ex vivo rat tail artery and monitored GFP levels using Western blotting following the delivery of single doses of ionizing radiation (2, 4, 6 Gy) to test whether the promoter could be used for vascular targeted gene therapy. The t-PA promoter induced GFP expression up to 6-fold in all cell types tested in response to radiation doses within the clinical range. These results suggest that the t-PA promoter may be incorporated into gene therapy strategies driving therapeutic transgenes in conjunction with radiation therapy.

High-Level Antigen Expression and Sustained Antigen Presentation in Dendritic Cells Nucleofected with Wild-Type Viral mRNA but Not DNA

Dendritic cells (DC) are potent antigen-presenting cells that hold promise as cell-based therapeutic vaccines for infectious diseases and cancer. Ideally, DC would be engineered to express autologous viral or tumor antigens to ensure the presentation of relevant antigens to host T cells in vivo; however, expression of wild-type viral genes in primary cell lines can be problematic. Nucleofection is an effective means of delivering transgenes to primary cell lines, but its use in transfecting DNA or mRNA into DC has not been widely investigated. We show that nucleofection is a superior means of transfecting human and monkey monocyte-derived DC with DNA and mRNA compared to lipofection and conventional electroporation. However, the delivery of DNA and mRNA had significantly different outcomes in transfected DC. DC nucleofected with DNA encoding green fluorescent protein (GFP) had poor antigen expression and viability and were refractory to maturation with CD40 ligand. In contrast, >90% of DC expressed uniform and high levels of GFP from 3 h to 96 h postnucleofection with mRNA while maintaining a normal maturation response to CD40 ligation. Monkey DC nucleofected with wild-type, non-codon-optimized mRNA encoding simian immunodeficiency virus Gag stimulated robust antigen-specific effector T-cell responses at 24 h and 48 h postnucleofection, reflecting sustained antigen presentation in transfected DC, whereas no detectable T-cell response was noted when DC were nucleofected with DNA encoding the same Gag sequence. These data indicate that mRNA nucleofection may be an optimal means of transfecting DC with autologous tumor or viral antigen for DC-based immunotherapy.

Structural basis for RNA‐silencing suppression by Tomato aspermy virus protein 2b

The 2b proteins encoded by cucumovirus act as post-transcriptional gene silencing suppressors to counter host defence during infection. Here we report the crystal structure of Tomato aspermy virus 2b (TAV2b) protein bound to a 19 bp small interfering RNA (siRNA) duplex. TAV2b adopts an all alpha-helix structure and forms a homodimer to measure siRNA duplex in a length-preference mode. TAV2b has a pair of hook-like structures to recognize simultaneously two alpha-helical turns of A-form RNA duplex by fitting its alpha-helix backbone into two adjacent major grooves of siRNA duplex. The conserved pi-stackings between tryptophan and the 5'-terminal base of siRNA duplex from both ends enhance the recognition. TAV2b further oligomerizes to form a dimer of dimers through the conserved leucine-zipper-like motif at its amino-terminal alpha-helix. Biochemical experiments suggest that TAV2b might interfere with the post-transcriptional gene silencing pathway by directly binding to siRNA duplex.

Surface Display of GFP by Pseudomonas Syringae Truncated Ice Nucleation Protein in Attenuated Vibrio Anguillarum Strain

Microbial cell surface display of foreign proteins has been widely developed for many potential applications in live vaccine construction, whole-cell biocatalysts, and bioadsorption. To investigate the feasibility of displaying heterologous proteins on the surface of attenuated Vibrio anguillarum strain for potential multivalent live vaccine development, different display systems were built upon a truncated ice nucleation protein (INP) from Pseudomonas syringae ICMP3023 whose N- and C-terminal domains were considered to be the putative membrane-anchoring motifs. Green fluorescent protein (GFP), as a reporter, was fused with the display systems in different forms of N-GFP, NC-GFP, and N-GFP-C. Analysis of the total expression level and surface localization of GFP demonstrated that the truncated P. syringae INP could be used to display foreign protein in V. anguillarum, while the system of N-GFP showed the higher levels of total expression and surface display based on unit cell density among the three and might be available for further carrier vaccine development.

Characterization of the promoter activity of a poxvirus conserved element

The conserved sequence element (CSE) is a highly conserved 42-bp poxvirus sequence that can function as a poxvirus promoter element. The CSE is composed of 2 repeats, each containing the highly conserved late poxvirus promoter sequence TAAAT. To define the location of the nucleotides critical for promoter function, polymerase chain reaction was carried out using primers that inserted modified versions of the CSE upstream of the green fluorescent protein (GFP), and the constructs were transiently transfected into cells by using GFP levels as a measure of promoter function. The results of this analysis revealed that the second TAAAT sequence, but not the first TAAAT sequence, is critical for promoter function of the CSE. Furthermore, deletion of half of the intervening sequence, i.e., from 10 to 5 nt, increases the promoter strength of the CSE as compared with the wild-type CSE. These results indicate the potential of this novel poxvirus promoter for driving high levels of gene expression.

UNC-108/Rab2 Regulates Postendocytic Trafficking inCaenorhabditis elegans

After endocytosis, membrane proteins are often sorted between two alternative pathways: a recycling pathway and a degradation pathway. Relatively little is known about how trafficking through these alternative pathways is differentially regulated. Here, we identify UNC-108/Rab2 as a regulator of postendocytic trafficking in both neurons and coelomocytes. Mutations in the Caenorhabditis elegans Rab2 gene unc-108, caused the green fluorescent protein (GFP)-tagged glutamate receptor GLR-1 (GLR-1::GFP) to accumulate in the ventral cord and in neuronal cell bodies. In neuronal cell bodies of unc-108/Rab2 mutants, GLR-1::GFP was found in tubulovesicular structures that colocalized with markers for early and recycling endosomes, including Syntaxin-13 and Rab8. GFP-tagged Syntaxin-13 also accumulated in the ventral cord of unc-108/Rab2 mutants. UNC-108/Rab2 was not required for ubiquitin-mediated sorting of GLR-1::GFP into the multivesicular body (MVB) degradation pathway. Mutations disrupting the MVB pathway and unc-108/Rab2 mutations had additive effects on GLR-1::GFP levels in the ventral cord. In coelomocytes, postendocytic trafficking of the marker Texas Red-bovine serum albumin was delayed. These results demonstrate that UNC-108/Rab2 regulates postendocytic trafficking, most likely at the level of early or recycling endosomes, and that UNC-108/Rab2 and the MVB pathway define alternative postendocytic trafficking mechanisms that operate in parallel. These results define a new function for Rab2 in protein trafficking.

Trans-scleral Delivery of Antiangiogenic Proteins

In this study, we investigated the penetration of various proteins into the mouse eye after a periocular injection of the protein or an adenoviral vector (Ad) expressing the protein. At several time points after the injection, the retina, retinal pigmented epithelium/choroid, and sclera were dissected and enzyme-linked immunosorbent assays were performed. After a periocular injection of AdsFlt-1.10, AdTGFbeta.10, or AdPEDF.11, choroidal levels of pigment epithelium-derived factor (PEDF) and transforming growth factor-beta (TGF-beta) were not significantly different from scleral levels, and choroidal levels of sFlt-1 (soluble Flt-1 or soluble VEGF receptor 1) were only moderately reduced from scleral levels, indicating that each of these proteins penetrate the sclera well. In contrast, retinal levels of each of the three proteins were low compared to choroidal levels, suggesting poor penetration into the retina. Levels of PEDF in the choroid peaked 2 h after a periocular injection of PEDF protein and returned to baseline between 6 and 24 h, and peak levels in the retina were 8.6% of peak choroidal levels. Levels of green fluorescent protein, a protein unlikely to have any binding sites in mouse tissues, peaked in the choroid 2 h after the periocular injection and were undetectable by 4 h, while peak levels in the retina were 64.3% of peak choroidal levels. These data suggest that size and binding characteristics of proteins are likely to influence their ability to penetrate the eye from the periocular space, but in general, proteins as large as 50-75 kDa penetrate well into the choroid, but not into the retina. Periocular injections are feasible for the treatment of choroidal neovascularization with proteins or vectors that express them, but additional investigations are needed before they can be considered for treatment of retinal diseases.

AAV8, 9, Rh10, Rh43 Vector Gene Transfer in the Rat Brain: Effects of Serotype, Promoter and Purification Method

We compared adeno-associated virus (AAV) serotypes for expression levels of green fluorescent protein (GFP) in the adult rat hippocampus by biophotonic imaging. Preparations of AAV serotypes 8, 9, Rh10, and Rh43 incorporating cytomegalovirus (CMV) promoter-driven GFP were purified by a CsCl method. Neither AAV Rh10 nor AAV Rh43 produced greater levels of GFP than AAV8, which was used as a reference. For AAV9, there was an increase relative to AAV8. The CsCl-purified AAV8 displayed an astroglial transduction pattern in contrast to the expected neuronal expression of other AAVs. After preparing the same CMV-GFP plasmid in AAV8 with an iodixanol purification method, the expected neuronal pattern resulted. The astroglial expression with the CsCl AAV8 was probably due to relatively high levels of protein impurities. We compared the CMV promoter with the CMV/chicken beta-actin (CBA) promoter in the context of AAV8, both prepared by iodixanol, and found the CBA promoter to produce stronger GFP expression. At two doses of vectors optimized for serotype, promoter and purification, we did not observe serotype differences among AAV8, AAV9, or AAV Rh10. The purification method can therefore impact the transduction pattern as well as the results when comparing serotype strengths.

Establishment of a Labeling System for Hematopoietic Stem Cells by Nucleostemin Enhancer/Promoter-GFP Transgenic Mice.

A labeling system for tissue stem cells is a powerful tool for stem cell research and for development of stem cell-based regenerative medicine to human disease. Here we generated transgenic mice expressing green fluorescent protein (GFP) under control of enhancer/promoter activity to Nucleostemin (NS) gene. NS, a nucleolar GTP-binding protein, expresses at high levels in embryonic stem cells and neural stem cells, suggesting that NS expresses in various tissue stem cells. In fact, we found that NS highly expressed in mouse immature hematopoietic cells containing hematopoietic stem cells (HSCs) and progenitor cells compared with differentiated hematopoietic cells. To examine whether HSCs could be labeled by using enhancer/promoter activity to NS gene, we generated transgenic mice harboring putative genomic enhancer/promoter region of NS gene followed by GFP cDNA-polyA in the 3′ end (NS-GFP tg mice). Fluorescence activated cell sorting (FACS) analysis indicated that most bone marrow (BM) mononuclear cells (MNCs) showed green fluorescence in NS-GFP tg mice. To validate the relationship between fluorescent intensity of GFP and expression levels of endogenous NS mRNA, BM MNCs from NS-GFP tg mice were separated into four fractions depending on their fluorescent intensity (i.e., GFP−, GFP+, GFP++, and GFP+++) by FACS, and these cells were subjected to quantitative real-time PCR analysis. The fluorescent intensity of GFP certainly reflected the amount of endogenous NS mRNA, indicating that the enhancer/promoter region used in this study is sufficient for monitoring the expression levels of endogenous NS mRNA in vivo. We next characterized expression levels of cell-surface markers on the four fractions of BM MNCs. FACS analysis showed that the GFP+++ cells, but not the other cell populations, lost the expression of mature hematopoietic cell markers (myeloid, B cells, T cells and erythroid cells). The fluorescent intensity of GFP in c-Kit+ Sca-1+ Lineage− (KSL) cells, which represent a primitive hematopoietic cell fraction containing HSCs and progenitor cells, was much higher than that in the other cell populations, indicating that the immature hematopoietic cells highly express GFP. To investigate the biological properties of the GFP-high expressing cells, we further performed colony-forming assay in vitro and BM transplantation assay in vivo. When the sorted BM MNCs were cultured in methylcellulose medium supplemented with cytokines, GFP+++ cells showed the highest number of colony formation. To assess the repopulating capacity as stem cell function, the sorted BM MNCs were transplanted into irradiated recipient mice. Notably, only GFP+++ cells had long-term reconstitution capacity of hematopoiesis in the recipient mice on the competitive reconstitution assay. In addition, the GFP+++ cells that were sorted from the GFP+++ cells-transplanted mouse retained the reconstitution capacity after secondary BM transplantation. The reconstitutive capacity of the GFP+++ cells into multi-lineages was confirmed by detection of donor-derived B, T, and myeloid cells in the recipient mice. These results demonstrate that HSCs with self-renewal capacity and differentiation potential for multi-lineages are enriched in GFP+++ cell population. The NS-GFP tg mouse system may provide a useful tool for effective enrichment of HSCs in combination with other cell-surface markers for HSCs.

The cHS4 chromatin insulator reduces gammaretroviral vector silencing by epigenetic modifications of integrated provirus

The cHS4 chromatin insulator has been shown to improve the expression of integrating gene transfer vectors by reducing the impact of silencing chromosomal position effects. To better understand the underlying mechanisms of this protection, we investigated the influence of this element on the epigenetic modifications of a gammaretroviral reporter vector. In HT1080 cells, we found that a fourfold increase in the level of green fluorescent protein (GFP) reporter expression from the cHS4-insulated vector was correlated with a twofold increase in acetylation at lysines 9 and 14 of histone H3, but not with CpG methylation. In a mouse bone marrow transduction and transplantation model, we found that a 10-fold increase in the likelihood of GFP expression from the cHS4-insulated vector was correlated with an eightfold increase in histone H3 acetylation, as well as a fourfold decrease in CpG methylation. Histone hyperacetylation peaked at the cHS4 core, and in vivo diminished nearly threefold through the central portion of the vector. Taken together, these studies demonstrate that the cHS4 chromatin insulator reduces gammaretroviral vector silencing by modulating epigenetic modifications of integrated provirus, and identify a specific topological distribution of these modifications that may prove informative for future vector designs.

BAC transgenic expression efficiency: bicistronic versus ATG‐fusion strategies

Bacterial artificial chromosome (BAC)-based transgene can be expressed bicistronically with the target gene or fused to its translation start codon. To compare the transgene expression efficiencies of these two methods, mice were created that expressed green fluorescent protein (GFP) from the genomic locus of nucleostemin using the bicistronic (NSiGFP) or the ATG-fusion approach (NSmGFP). Three lines with 1, 2, and 4 copies of the NSiGFP transgene, and two lines with 1 copy of the NSmGFP transgene were generated. Of the three NSiGFP lines, only the 4-copy line can match the NSmGFP lines in their GFP protein expression levels. Analyses of the GFP and nucleostemin RNA transcript levels exclude IRES inefficiency and suggest premature termination of the bicistronic message as the cause for low GFP expression in the NSiGFP mice. This work provides important information for designing BAC transgenics when the transgene expression level is crucial.

Activation of Heat Shock Protein 70 Promoter with meso-Tetrahydroxyphenyl Chlorin Photodynamic Therapy Reported by Green Fluorescent Protein In Vitro and In Vivo¶

Cellular responses to photodynamic therapy (PDT) include induction of heat shock proteins (HSP). We examined meso-tetrahydroxyphenyl chlorin (mTHPC) PDT–mediated HSP activation in EMT6 cells stably transfected with a plasmid containing the gene for green fluorescent protein (GFP) driven by an hsp70 promoter. mTHPC incubation induced concentration-dependent GFP expression. Irradiation of cells exposed to a sensitizer concentration that induced a slight increase in GFP and no loss of cell viability resulted in fluence-dependent GFP accumulation. In response to drug only and to PDT, GFP levels increased to a maximum of four- to five-fold above control levels with increasing drug or fluence and then decreased at higher doses. A trypan blue–exclusion assay confirmed that decreased GFP levels in both cases were due to a loss of cell viability. For initial evaluation in vivo, HSP70/GFP–transfected EMT6 tumors were grown in BALB/c mice and subjected to mTHPC-PDT with a fluence of 1 J/cm2. Six hours after PDT, GFP fluorescence was imaged in these tumors through the intact skin in vivo. These results indicate that sublethal doses of mTHPC-PDT stimulate GFP expression under the control of an hsp70 promoter and illustrate the potential of noninvasively monitoring reporter protein fluorescence as a measure of molecular response to PDT.

형질전환된 벼세포배양에서 green fluorescent protein (GFP) 생산

Green fluorescent protein (GFP) is an attractive reporter for bioprocess monitoring. A fluorescence-based method was developed to quantify GFP levels in transgenic plants and protein extracts. In this study, GFP was produced and secreted from suspension cells derived from transgenic rice. The RAmy3E promoter placed before the GFP gene controlled by sugars such as sucrose. The effects of sucrose concentration on the secretion of GFP and total protein into the medium were investigated in batch suspension culture. It was possible, therefore, to induce the expression of the GFP by removing sucrose from the cultured media or by allowing the rice suspension cells to deplete sucrose catabolically. The dry cell weight (7.06 g/L) and GFP level were detected as highest at 12%, 3% sucrose after 20 day culture, respectively. However secreted GFP fluorescence at the other sucrose concentrations (6%, 12%, 18% and 24%) were a little amount in media.

Multicopy Integration of Heterologous Genes, Using the Lactococcal Group II Intron Targeted to Bacterial Insertion Sequences

Group II introns are mobile genetic elements that can be redirected to invade specific genes. Here we describe the use of the lactococcal group II intron, Ll.ltrB, to achieve multicopy delivery of heterologous genes into the genome of Lactococcus lactis IL1403-UCD without the need for selectable markers. Ll.ltrB was retargeted to invade three transposase genes, the tra gene found in IS904 (tra904), tra981, and tra983, of which 9, 10, and 14 copies, respectively, were present in IL1403-UCD. Intron invasion of tra904, tra981, and tra983 allele groups occurred at high frequencies, and individual segregants possessed anywhere from one to nine copies of intron in the respective tra alleles. To achieve multicopy delivery of a heterologous gene, a green fluorescent protein (GFP) marker was cloned into the tra904-targeted Ll.ltrB, and the resultant intron (Ll.ltrB::GFP) was induced to invade the L. lactis tra904 alleles. Segregants possessing Ll.ltrB::GFP in three, four, five, six, seven, and eight copies in different tra904 alleles were obtained. In general, increasing the chromosomal copy number of Ll.ltrB::GFP resulted in strains expressing successively higher levels of GFP. However, strains possessing the same number of Ll.ltrB::GFP copies within different sets of tra904 alleles exhibited differential GFP expression, and segregants possessing seven or eight copies of Ll.ltrB::GFP grew poorly upon induction, suggesting that GFP expression from certain combinations of alleles was detrimental. The highest level of GFP expression was observed from a specific six-copy variant that produced GFP at a level analogous to that obtained with a multicopy plasmid. In addition, the high level of GFP expression was stable for over 120 generations. This work demonstrates that stable multicopy integration of heterologous genes can be readily achieved in bacterial genomes with group II intron delivery by targeting repeated elements.

Demonstration and Characterization of Mutations Induced by Helicobacter pylori Organisms in Gastric Epithelial Cells

Helicobacter pylori gastritis increases gastric cancer risk. Microsatellite instability-type mutations are secondary to deficient DNA mismatch repair. H. pylori gastritis is more frequent in patients with microsatellite instability-positive gastric cancers, and H. pylori organisms independently of inflammation can reduce DNA mismatch repair protein levels, raising the hypothesis that H. pylori organisms might lead to mutagenesis during infection. Mutations were detected using a green fluorescent protein reporter vector (pEGFP-CA13). Gastric cancer AGS cells transfected with pEGFP-CA13 were cocultured with H. pylori or Escherichia coli. The numbers of green fluorescent protein (GFP)-positive cells were determined, and GFP, hMSH2, and hMLH1 protein levels were measured by Western blot. The effect of H. pylori on CpG methylation status of hMLH1 was determined by methylation-specific polymerase chain reaction. GFP levels and GFP-positive cell numbers in AGS cells cocultured with H. pylori significantly increased, as the levels of hMLH1 and hMSH2 dropped. H. pylori cocultures induced low-level CpG methylation of the hMLH1 promoter. Sequence analysis of cells cocultured with H. pylori showed an increased number of frameshift mutations and point mutations as compared to cells not cocultured with H. pylori (p = .03 and p = .001, respectively). This is the first report showing that H. pylori bacteria may lead to accumulation of genomic mutations, independently of underlying inflammation. This is associated with reduced DNA mismatch repair, and is at least in part associated with CpG methylation of the hMLH1 promoter. These data support the notion that H. pylori-induced mutations and epigenetic alterations in gastric epithelial cells during chronic gastritis may contribute to an increased risk of gastric cancer associated with H. pylori infection.

Gene activation in plastids by the CRE site-specific recombinase

We developed a novel system for gene activation in plastids that uses the CRE/loxP site-specific recombination system to create a translatable reading frame by excision of a blocking sequence. To test the system, we introduced an inactive gfp* gene into the tobacco plastid genome downstream of the selectable spectinomcyin resistance (aadA) marker gene. The aadA gene is the blocking sequence, and is flanked by directly oriented loxP sites for excision by the CRE. In the non-activated state, gfp* is transcribed from the aadA promoter, but the mRNA is not translated due to the lack of an AUG translation initiation codon. Green Fluorescent Protein (GFP) expression is activated by excision of the aadA coding segment to link up the gfp* coding region with the translation initiation codon of aadA. Tobacco plants that carry the inactive gfp* gene do not contain detectable levels of GFP. However, activation of gfp* resulted in GFP accumulation, proving the utility of CRE-induced protein expression in tobacco chloroplasts. The gene activation system described here will be useful to probe plastid gene function and for the production of recombinant proteins in chloroplasts.

Characterization of baculovirus Autographa californica multiple nuclear polyhedrosis virus infection in mammalian cells

The baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) is used as a vector in many gene therapy studies. Wild-type AcMNPV infects many mammalian cell types in vitro, but does not replicate. We investigated the dynamics of AcMNPV genomic DNA in infected mammalian cells and used flow cytometric analysis to demonstrate that recombinant baculovirus containing a cytomegalovirus immediate early promoter/enhancer with green fluorescent protein (GFP) expressed high levels of GFP in Huh-7 cells, but not B16, Raw264.7, or YAC-1 cells. The addition of butyrate, a deacetylase inhibitor, markedly enhanced the percentage of GFP-expressing Huh-7 and B16 cells, but not Raw264.7 and YAC-1 cells. The addition of 5-aza-2′-deoxycytidine, a DNA methylation inhibitor, had no enhancing effect. Polymerase chain reaction analysis using AcMNPV- gp64-specific primers indicated that AcMNPV infected not only Huh-7 and B16 cells, but also Raw264.7 and YAC-1 cells in vitro. The genomic DNA was detected in Huh-7 and B16 cells 96 h after infection. Genomic AcMNPV DNA in YAC-1 cells was not transported to the nucleus. Luciferase assay indicated that AcMNPV p35 gene mRNA and p35 promoter activity were clearly expressed only in Huh-7 and B16 cells. These results suggest that viral genomic DNA expression is restricted by different host cell factors, such as degradation, deacetylation, and inhibition of nuclear transport, depending on the mammalian cell type.

Virally expressed siRNA mediates localized gene silencing in specific cardiovascular (CV) regulatory nuclei of mouse brain

Dissecting the molecular underpinnings of central neuro-cardiovascular control is challenging due to the involvement of many genes in multiple circuits. siRNA is an effective strategy for gene silencing; however, in vivo applications require efficient intracellular transduction in target tissues. Here we tested the hypothesis that adenoviral-mediated delivery of siRNA would induce efficient, stable and localized knockdown in CV regions of mouse brain. Ad-siGFP was designed to express both a U6-driven siRNA hairpin targeted against green fluorescent protein (GFP) and LacZ as a reporter for viral expression. GFP transgenic mice underwent injection of Ad-siGFP or a control virus (Ad-Con) into the paraventricular nucleus (PVN) or ICV to target the subfornical organ (SFO). Mice were sacrificed at 3, 14 or 28 days, and alternating brain sections were processed for GFP visualization or X-gal. GFP levels in PVN were reduced by 3 days in Ad-siGFP mice (38±3%, n=4) compared to Ad-con mice (0±6%, n=4, p<0.05), with further marked knockdown at 2 wks (Ad-siGFP 60±2% vs Ad-con 0±11%, n=5 each, p<0.05). Robust and region-specific LacZ was observed in Ad-siGFP mice at these times. GFP levels in PVN nearly had returned to baseline in Ad-siGFP mice after 4 wks. A similar GFP/LacZ profile was seen in SFO. These results establish proof-of-principle that efficient, stable and localized gene silencing can be achieved in CV regions of mouse brain using viral-delivered siRNA.

Efficient Neuronal Gene Transfer with AAV8 Leads to Neurotoxic Levels of Tau or Green Fluorescent Proteins

Adeno-associated virus (AAV) serotype 8 appears to be the strongest of the natural serotypes reported to date for gene transfer in liver and muscle. In this study, we evaluated AAV8 in the brain by several methods, including biophotonic imaging of green fluorescent protein (GFP). In the adult rat hippocampus, levels of GFP expressed were clearly greater with AAV8 than with AAV2 or AAV5 by Western blot and biophotonic imaging and slightly but significantly greater than AAV1 by Western blot. In the substantia nigra, the GFP expression conferred by AAV8 was toxic to dopamine neurons, although toxicity could be avoided with dose titration. At the low dose at which there was no GFP toxicity from the GFP vector, another AAV8 vector for a disease-related (P301L) form of the microtubule-associated protein tau caused a 78% loss of dopamine neurons and significant amphetamine-stimulated rotational behavior. The AAV8 tau vector-induced cell loss was greater than that from AAV2 or AAV5 tau vectors, demonstrating that the increased gene transfer was functional. While the toxicity observed with GFP expression warrants great caution, the efficient AAV8 is promising for animal models of neurodegenerative diseases and potentially as well for gene therapy of brain diseases.