Development of the central nervous system is controlled by both intrinsic and extrinsic signals that guide neuronal migration to form laminae. Although defects in neuronal mobility have been well documented as a mechanism for abnormal laminar formation, the role of radial glia, which provide the environmental cues, in modulating neuronal migration is less clear. We provide evidence that loss of PTEN in Bergmann glia leads to premature differentiation of this crucial cell population and subsequently to extensive layering defects. Accordingly, severe granule neuron migration defects and abnormal laminar formation are observed. These results uncover an unexpected role for PTEN in regulating Bergmann glia differentiation, as well as the importance of time-dependent Bergmann glia differentiation during cerebellar development.
Epstein-Barr virus (EBVEpstein-Barr virus (EBV) is a gammaherpesvirus that preferentially infects human B lymphocytes (16,17,26). The latent form of EBV is capable of lifelong persistence as a circular episome in human B cells. Stable maintenance of the 165-kb EBV episome is conferred mainly by the abilities of EBV episomes to replicate once per S phase and to segregate efficiently into daughter cells during cell division (44). Both mechanisms require the interaction of EBV nuclear antigen 1 (EBNA-1) protein with the EBV origin of replication (oriP) (23). oriP consists of two groups of EBNA-1 binding sites, the family of repeats (FR) and the dyad symmetry element (DS) (31, 43). The FR is composed of 20 imperfect copies of a 30-bp repeat, each one able to bind an EBNA-1 dimer. The binding of EBNA-1 to the FR forms a bridge between chromosomes and oriP-containing plasmids (25). During mitosis, EBV episomes are tethered to metaphase chromosomes through EBNA-1, allowing the efficient segregation of episomes to daughter cells. The association of EBNA-1 with the FR also increases the nuclear import and retention of EBV plasmids and may target the plasmids to nuclear regions conducive to the efficient expression of EBV episome sequences (20).The DS consists of four copies of the EBNA-1 binding site (43). Upon binding of EBNA-1 through its C-terminal domain, the DS confers the initiation of DNA replication once per S phase (17). In the absence of the DS, EBV episomes fail to undergo replication unless they contain a different source of a replication origin, such as human genomic DNA. Krysan et al. (19) showed that replacement of the DS in EBV plasmids with large human chromosomal fragments resulted in efficient replication and segregation. Further studies demonstrated that the size of the genomic insert affected replication efficiency, favoring inserts of about 20 kb in length (12). EBV plasmids containing human genomic DNA were shown to replicate efficiently in rodent cells, in contrast to plasmids containing EBV-derived oriP (18). The transformation of various cell lines with EBV episomes in vitro has revealed the potential to confer long-term transgene expression and correct genetic defects in deficient cell lines (3,15,18,21,44).The delivery of EBV episomes is commonly performed by transfection. However, for quantitative delivery of EBV episomes, particularly in vivo, different techniques are required. Adenovirus recombinants have been shown to efficiently infect a wide variety of mammalian cells and tissues in vitro and in vivo (1). The expression of transgenes from adenovirus vectors is usually temporary, since the adenovirus DNA remains episomal in the majority of infected cells and is only integrated and therefore stably maintained at a frequency of 0.01 to 1% (11). In vivo, immune responses against viral antigens can cause the elimination of infected cells (8,30,41). Compared to first-generation adenovirus recombinants, helper-dependent adenovirus (HDA) vectors have no viral coding sequences and
To make a safe, long-lasting gene delivery vehicle, we developed a hybrid vector that leverages the relative strengths of adenovirus and Epstein-Barr virus (EBV). A fully gene-deleted helper-dependent adenovirus (HDAd) is used as the delivery vehicle for its scalability and high transduction efficiency. Upon delivery, a portion of the HDAd vector is recombined to form a circular plasmid. This episome includes two elements from EBV: an EBV nuclear antigen 1 (EBNA1) expression cassette and an EBNA1 binding region. Along with a human replication origin, these elements provide considerable genetic stability to the episome in replicating cells while avoiding insertional mutagenesis. Here, we demonstrate that this hybrid approach is highly efficient at delivering EBV episomes to target cells in vivo. We achieved nearly 100% transduction of hepatocytes after a single intravenous injection in mice. This is a substantial improvement over the transduction efficiency of previously available physical and viral methods. Bioluminescent imaging of vector-transduced mice demonstrated that luciferase transgene expression from the hybrid was robust and compared well to a traditional HDAd vector. Quantitative PCR analysis confirmed that the EBV episome was stable at approximately 30 copies per cell for up to 50 weeks and that it remained circular and extrachromosomal. Approaches for adapting the HDAd-EBV hybrid to a variety of disease targets and the potential benefits of this approach are discussed.
Purpose Our goal is to develop a simple, quantitative, robust method to compare the efficacy of imaging reporter genes in culture and in vivo. We describe an adenoviral vector-liver transduction procedure, and compare the luciferase reporter efficacies. Procedures Alternative reporter genes are expressed in a common adenoviral vector. Vector amounts used in vivo are based on cell culture titrations, ensuring the same transduction efficacy is used for each vector. After imaging, in vivo and in vitro values are normalized to hepatic vector transduction using quantitative real-time PCR. Results We assayed standard firefly luciferase (FLuc), enhanced firefly luciferase (EFLuc), luciferase 2 (Luc2), humanized Renilla luciferase (hRLuc), Renilla luciferase 8.6-535 (RLuc8.6), and a membrane-bound Gaussia luciferase variant (extGLuc) in cell culture and in vivo. We observed a greater that 100-fold increase in bioluminescent signal for both EFLuc and Luc2 when compared to FLuc, and a greater than 106-fold increase for RLuc8.6 when compared to hRLuc. ExtGLuc was not detectable in liver. Conclusions Our findings contrast, in some cases, with conclusions drawn in prior comparisons of these reporter genes, and demonstrate the need for a standardized method to evaluate alternative reporter genes in vivo. Our procedure can be adapted for reporter genes that utilize alternative imaging modalities (fluorescence, bioluminescence, MRI, SPECT, PET).
Epstein-Barr virus (EBV) evolved an episomal system for maintaining life-long, latent infection of human B lymphocytes. Circular episomes engineered from EBV components required for this latent form of infection have the capacity to persist in most types of replicating mammalian cells without DNA integration and the pitfalls of insertional mutagenesis. EBV episomes are typically transduced using low-efficiency methods. Here we present a method for efficient delivery of EBV episomes to nuclei of hepatocytes in living mice using a helper-dependent adenoviral vector and Cre-mediated recombination in vivo to generate circular EBV episomes following infection. Cre is transiently expressed from a hepatocyte-specific promoter so that vector generation and transgene expression are tissue specific. We show long-term persistence of the circularized vector DNA and expression of a reporter gene in hepatocytes of immunocompetent mice. Gene Therapy (2010) Of several genes expressed by Epstein-Barr virus (EBV) in latently infected B cells, only EBV nuclear antigen 1 (EBNA-1) is required to confer persistence in mitotic cells to a plasmid bearing the EBV oriP region.1-4 EBNA-1 is a sequence-specific DNA-binding protein that binds to multiple sites in oriP. A dyad symmetry (DS) of inverted EBNA-1 binding sites at one end of oriP functions as an origin of DNA replication in primate cells.4 EBNA-1 bound to the DS recruits cellular DNA pre-replication complex components that result in one firing of the origin per S phase, maintaining EBV episomes at a constant number per cell. 4 Binding to 21-30 bp imperfect direct repeats (the family of repeats, or FR) that make up most of oriP ensures proper episomal segregation of EBV episomes during mitosis. EBNA-1 bound to the FR interacts with metaphase chromosomes, effectively tethering daughter episomes generated during S phase to host daughter chromosomes. This EBNA-1-mediated tethering confers segregation of the episomes to each daughter cell during mitosis, ensuring nuclear retention in replicating cells.4,5 A plasmid containing both oriP and an EBNA-1 expression cassette can persist in replicating cultured human cells with B95% episome retention per cell cycle without selection. 2,4 Although circular plasmids containing the EBV oriP and an EBNA-1 expression cassette are maintained in replicating primate and canine cultured cells, they are not efficiently maintained in cultured rodent cells. 2,4 However, when the region of DS is replaced by a large fragment of human DNA (B20 kb), maintenance in replicating cultured rodent fibroblasts is restored, probably because the region functions as a replication origin.
We describe an inducible genetic model for degeneration of midbrain dopaminergic neurons in adults. In previous studies, knock-in mice expressing hypersensitive M2 domain Leu9'Ser (L9'S) alpha4 nicotinic receptors (nAChR) at near-normal levels displayed dominant neonatal lethality and dopaminergic deficits in embryonic midbrain, because the hypersensitive nAChR is excitotoxic. However, heterozygous L9'S mice that retain the neomycin resistance cassette (neo) in a neighboring intron express low levels of the mutant allele (approximately 25% of normal levels), and these neo-intact mice are therefore viable and fertile. The neo cassette is flanked by loxP sites. In adult animals, we locally injected helper-dependent adenovirus (HDA) expressing cre recombinase. Local excision of the neo cassette, via cre-mediated recombination, was verified by genomic analysis. In L9'S HDA-cre injected animals, locomotion was reduced both under baseline conditions and after amphetamine application. There was no effect in L9'S HDA-control treated animals or in wild-type (WT) littermates injected with either virus. Immunocytochemical analyses revealed marked losses (> 70%) of dopaminergic neurons in L9'S HDA-cre injected mice compared to controls. At 20-33 days postinjection in control animals, the coexpressed marker gene, yellow fluorescent protein (YFP), was expressed in many neurons and few glial cells near the injection, emphasizing the neurotropic utility of the HDA. Thus, HDA-mediated gene transfer into adult midbrain induced sufficient functional expression of cre in dopaminergic neurons to allow for postnatal deletion of neo. This produced increased L9'S mutant nAChR expression, which in turn led to nicotinic cholinergic excitotoxicity in dopaminergic neurons.
Purpose To use a simple, quantitative method to compare the HSV1sr39TK/18F-FHBG PET reporter gene/PET reporter probe (PRG/PRP) system with PRGs derived from human nucleoside kinases. Procedures The same adenovirus vector is used to express alternative PRGs. Equal numbers of vectors are injected intravenously into mice. After PRP imaging, quantitative hepatic PET signals are normalized for transduction by measuring hepatic viral genomes. Results The same adenovirus vector was used to express equivalent amounts of HSV1sr39TK, mutant human thymidine kinase 2 (TK2-DM), and mutant human deoxycytidine kinase (dCK-A100VTM) in mouse liver. HSV1sr39TK expression was measured with 18F-FHBG; TK2-DM and dCK-A100VTM with 18F-L-FMAU. TK2-DM/18F-L-FMAU and HSV1sr39TK/18F-FHBG had equivalent sensitivities; dCK-A100VTM/18F-L-FMAU was twice as sensitive as HSV1sr39TK/18F-FHBG. Conclusions The human PRG/PRP sensitivities are comparable and/or better than HSV1sr39TK/18F-FHBG. However, for clinical use, identification of the best PRP substrate for each enzyme, characterization of probe distribution, and consequences of over-expressing nucleoside kinases must be evaluated.
Prostaglandin-endoperoxide synthase 2 (PTGS2), also known as cyclooxygenase 2 (COX-2), plays a critical role in many normal physiological functions and modulates a variety of pathological conditions. The ability to turn endogenous COX-2 on and off in a reversible fashion, at specific times and in specific cell types, would be a powerful tool in determining its role in many contexts. To achieve this goal, we took advantage of a recently developed RNA interference system in mice. An shRNA targeting the Cox2 mRNA 3′untranslated region was inserted into a microRNA expression cassette, under the control of a tetracycline response element (TRE) promoter. Transgenic mice containing the COX-2-shRNA were crossed with mice encoding a CAG promoter-driven reverse tetracycline transactivator, which activates the TRE promoter in the presence of tetracycline/doxycycline. To facilitate testing the system, we generated a knockin reporter mouse in which the firefly luciferase gene replaces the Cox2 coding region. Cox2 promoter activation in cultured cells from triple transgenic mice containing the luciferase allele, the shRNA and the transactivator transgene resulted in robust luciferase and COX-2 expression that was reversibly down-regulated by doxycycline administration. In vivo, using a skin inflammation-model, both luciferase and COX-2 expression were inhibited over 80% in mice that received doxycycline in their diet, leading to a significant reduction of infiltrating leukocytes. In summary, using inducible RNA interference to target COX-2 expression, we demonstrate potent, reversible Cox2 gene silencing in vivo. This system should provide a valuable tool to analyze cell type-specific roles for COX-2.
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