Use of lentiviral vectors to deliver and express bicistronic transgenes in developing chicken embryos.

The abilities of lentiviral vectors to carry large transgenes (∼8kb) and to efficiently infect and integrate these genes into the genomes of both dividing and non-dividing cells make them ideal candidates for transport of genetic material into cells and tissues. Given the properties of these vectors, it is somewhat surprising that they have seen only limited use in studies of developing tissues and in particular of the developing nervous system. Over the past several years, we have taken advantage of the large capacity of these vectors to explore the expression characteristics of several dual promoter and 2A peptide bicistronic transgenes in developing chick neural retina, with the goal of identifying transgene designs that reliably express multiple proteins in infected cells. Here we summarize the activities of several of these transgenes in neural retina and provide detailed methodologies for packaging lentivirus and delivering the virus into the developing neural tubes of chicken embryos in ovo, procedures that have been optimized over the course of several years of use in our laboratory. Conditions to hatch injected embryos are also discussed. The chicken-specific techniques will be of highest interest to investigators using avian embryos, development and packaging of lentiviral vectors that reliably express multiple proteins in infected cells should be of interest to all investigators whose experiments demand manipulation and expression of multiple proteins in developing cells and tissues.

Role of estrogen receptor α and ß in preserving hippocampal function during aging.

The expression of the ERα and ERß estrogen receptors in the hippocampus may be important in the etiology of age-related cognitive decline. To examine the role of ERα and ERß in regulating transcription and learning, ovariectomized wild-type (WT) and ERα and ERß knockout (KO) mice were used. Hippocampal gene transcription in young ERαKO mice was similar to WT mice 6 h after a single estradiol treatment. In middle-age ERαKO mice, hormone deprivation was associated with a decrease in the expression of select genes associated with the blood-brain barrier; cyclic estradiol treatment increased transcription of these select genes and improved learning in these mice. In contrast to ERαKO mice, ERßKO mice exhibited a basal hippocampal gene profile similar to WT mice treated with estradiol and, in the absence of estradiol treatment, young and middle-age ERßKO mice exhibited preserved learning on the water maze. The preserved memory performance of middle-age ERßKO mice could be reversed by lentiviral delivery of ERß to the hippocampus. These results suggest that one function of ERß is to regulate ERα-mediated transcription in the hippocampus. This model is supported by our observations that knockout of ERß under conditions of low estradiol allowed ERα-mediated transcription. As estradiol levels increased in the absence of ERα, we observed that other mechanisms, likely including ERß, regulated transcription and maintained hippocampal-dependent memory. Thus, our results indicate that ERα and ERß interact with hormone levels to regulate transcription involved in maintaining hippocampal function during aging.

Role of the PAS sensor domains in the Bacillus subtilis sporulation kinase KinA.

Histidine kinases are sophisticated molecular sensors that are used by bacteria to detect and respond to a multitude of environmental signals. KinA is the major histidine kinase required for initiation of sporulation upon nutrient deprivation in Bacillus subtilis. KinA has a large N-terminal region (residues 1 to 382) that is uniquely composed of three tandem Per-ARNT-Sim (PAS) domains that have been proposed to constitute a sensor module. To further enhance our understanding of this "sensor" region, we defined the boundaries that give rise to the minimal autonomously folded PAS domains and analyzed their homo- and heteroassociation properties using analytical ultracentrifugation, nuclear magnetic resonance (NMR) spectroscopy, and multiangle laser light scattering. We show that PAS(A) self-associates very weakly, while PAS(C) is primarily a monomer. In contrast, PAS(B) forms a stable dimer (K(d) [dissociation constant] of <10 nM), and it appears to be the main N-terminal determinant of KinA dimerization. Analysis of KinA mutants deficient for one or more PAS domains revealed a critical role for PAS(B), but not PAS(A), in autophosphorylation of KinA. Our findings suggest that dimerization of PAS(B) is important for keeping the catalytic domain of KinA in a functional conformation. We use this information to propose a model for the structure of the N-terminal sensor module of KinA.

Failed cytokinesis of neural progenitors in citron kinase-deficient rats leads to multiciliated neurons.

Most, if not all, cortical neurons possess a single primary cilium; however, little is known about the mechanisms that control neuronal ciliogenesis. The Citron kinase-deficient (Citron-K(fh/fh)) rat, a model in which failed cytokinesis during development produces cortical neurons containing multiple cellular organelles, provides a unique system in which to examine the relationship between centriole inheritance and neuronal ciliogenesis. In this study, we analyzed the cerebral cortex of these animals using immunohistochemistry, serial confocal, and electron microscopy to determine if the multinucleated neurons present in the cortex of these animals also possess multiple centrioles and cilia. We found that neurons containing multiple nuclei possessed multiple centrioles and cilia whose lengths varied across cortical regions. Despite the presence of multiple cilia, we found that perinatal expression of adenylyl cyclase III, a cilia-specific marker, and somatostatin receptor 3, a receptor enriched in cilia, were preserved in developing Citron-K(fh/fh) brain. Together, these results show that multinucleated neurons arising from defective cytokinesis can extend multiple cilia.

Evidence from artificial septal targeting and site-directed mutagenesis that residues in the extracytoplasmic ß domain of DivIB mediate its interaction with the divisomal transpeptidase PBP 2B.

Bacterial cytokinesis is achieved through the coordinated action of a multiprotein complex known as the divisome. The Escherichia coli divisome is comprised of at least 10 essential proteins whose individual functions are mostly unknown. Most divisomal proteins have multiple binding partners, making it difficult to pinpoint epitopes that mediate pairwise interactions between these proteins. We recently introduced an artificial septal targeting approach that allows the interaction between pairs of proteins to be studied in vivo without the complications introduced by other interacting proteins (C. Robichon, G. F. King, N. W. Goehring, and J. Beckwith, J. Bacteriol. 190:6048-6059, 2008). We have used this approach to perform a molecular dissection of the interaction between Bacillus subtilis DivIB and the divisomal transpeptidase PBP 2B, and we demonstrate that this interaction is mediated exclusively through the extracytoplasmic domains of these proteins. Artificial septal targeting in combination with mutagenesis experiments revealed that the C-terminal region of the ß domain of DivIB is critical for its interaction with PBP 2B. These findings are consistent with previously defined loss-of-function point mutations in DivIB as well as the recent demonstration that the ß domain of DivIB mediates its interaction with the FtsL-DivIC heterodimer. These new results have allowed us to construct a model of the DivIB/PBP 2B/FtsL/DivIC quaternary complex that strongly implicates DivIB, FtsL, and DivIC in modulating the transpeptidase activity of PBP 2B.

Expression characteristics of dual-promoter lentiviral vectors targeting retinal photoreceptors and Müller cells.

PURPOSE: Growing evidence suggests that successful treatment of many inherited photoreceptor diseases will require multi-protein therapies that not only correct the genetic defects linked to these diseases but also slow or halt the related degenerative phenotypes. To be effective, it is likely that therapeutic protein expression will need to be targeted to specific cell types. The purpose of this study was to develop dual-promoter lentiviral vectors that target expression of two proteins to retinal cones and rods, rods only, or Müller cells. METHODS: Dual-promoter lentivectors were constructed using the following promoters: Xenopus opsin promoter (XOPS)1.3, murine opsin promoter (MOPS), interphotoreceptor retinoid binding protein promoter (IRBP156), rhodopsin kinase (RK), neural retina leucine zipper (NRLL), vimentin (VIM), cluster differentiation (CD44), and glial fibrillary acidic protein (GFAP). Vectors were packaged and injected into the neural tubes of chicken embryos. The activities of the promoters alone, in duplicate, or when paired with a different promoter were analyzed in transduced, fully-developed retinas, using direct fluorescent and immunofluorescent microscopy. RESULTS: IRBP156, NRLL, and RK were active in cones and rods while XOPS1.3 was active only in rods. Of the glial promoters, only GFAP activity was restricted to Müller cells; both VIM and CD44 were active in Müller and neural cells. Dual-promoter vectors carrying IRBP156 and RK or XOPS1.3 and MOPS, in the order listed, exhibited robust expression of both reporter transgenes in cones and rods or rods only, respectively. Expression of the upstream transgene was much lower than the downstream transgene in dual-promoter vectors constructed using two copies of either RK or IRBP156. Analyses of the expression of a dual-promoter vector carrying CD44 and VIM in the order listed showed that the activity of the VIM promoter was more restricted to glial cells when paired with the CD44 promoter, while the activity of the CD44 promoter was inhibited to the extent that no CD44-driven reporter protein was detected in transduced cells. CONCLUSIONS: We have identified two dual-promoter vectors, one that targets cones and rods and one that targets rods alone. Both vectors reliably express the two proteins encoded by the transgenes they carry. When two well matched promoters are not available, we found that it is possible to target expression of two proteins to single cells using dual-promoter vectors carrying two copies of the same promoter. These vectors should be useful in studies of retina when co-delivery of a reporter protein with an experimental protein is desired or when expression of two exogenous proteins in targeted cells is required.

Structure of the sporulation histidine kinase inhibitor Sda from Bacillus subtilis and insights into its solution state.

The crystal structure of the DNA-damage checkpoint inhibitor of sporulation, Sda, from Bacillus subtilis, has been solved by the MAD technique using selenomethionine-substituted protein. The structure closely resembles that previously solved by NMR, as well as the structure of a homologue from Geobacillus stearothermophilus solved in complex with the histidine kinase KinB. The structure contains three molecules in the asymmetric unit. The unusual trimeric arrangement, which lacks simple internal symmetry, appears to be preserved in solution based on an essentially ideal fit to previously acquired scattering data for Sda in solution. This interpretation contradicts previous findings that Sda was monomeric or dimeric in solution. This study demonstrates the difficulties that can be associated with the characterization of small proteins and the value of combining multiple biophysical techniques. It also emphasizes the importance of understanding the physical principles behind these techniques and therefore their limitations.

The divisomal protein DivIB contains multiple epitopes that mediate its recruitment to incipient division sites.

Bacterial cytokinesis is orchestrated by an assembly of essential cell division proteins that form a supramolecular structure known as the divisome. DivIB and its orthologue FtsQ are essential members of the divisome in Gram-positive and Gram-negative bacteria respectively. DivIB is a bitopic membrane protein composed of an N-terminal cytoplasmic domain, a single-pass transmembrane domain, and a C-terminal extracytoplasmic region comprised of three separate protein domains. A molecular dissection approach was used to determine which of these domains are essential for recruitment of DivIB to incipient division sites and for its cell division functions. We show that DivIB has three molecular epitopes that mediate its localization to division septa; two epitopes are encoded within the extracytoplasmic region while the third is located in the transmembrane domain. It is proposed that these epitopes represent sites of interaction with other divisomal proteins, and we have used this information to develop a model of the way in which DivIB and FtsQ are integrated into the divisome. Remarkably, two of the three DivIB localization epitopes are dispensable for vegetative cell division; this suggests that the divisome is assembled using a complex network of protein-protein interactions, many of which are redundant and likely to be individually non-essential.

Viral Vector-mediated Delivery of Estrogen Receptor-α to the Hippocampus Improves Spatial Learning in Estrogen Receptor-α Knockout Mice.

Estrogen, which influences both classical genomic and rapid membrane-associated signaling cascades, has been implicated in the regulation of hippocampal function, including spatial learning. Gene mutation studies suggest that estrogen effects are mediated by estrogen receptor-α (ER-α); however, because gonadal steroids influence the organization of the hippocampus during development, it has been difficult to distinguish developmental effects from those specific to adults. In this study we show that lentiviral delivery of the gene encoding ER-α to the hippocampus of adult ER-α-knockout (ER-αKO) mice restores hippocampal responsiveness to estrogen and rescues spatial learning. We propose that constitutive estrogen receptor activity is important for maintaining hippocampus-dependent memory function in adults.

Viral vector-mediated delivery of estrogen receptor-alpha to the hippocampus improves spatial learning in estrogen receptor-alpha knockout mice.

Estrogen, which influences both classical genomic and rapid membrane-associated signaling cascades, has been implicated in the regulation of hippocampal function, including spatial learning. Gene mutation studies suggest that estrogen effects are mediated by estrogen receptor-alpha (ER-alpha); however, because gonadal steroids influence the organization of the hippocampus during development, it has been difficult to distinguish developmental effects from those specific to adults. In this study we show that lentiviral delivery of the gene encoding ER-alpha to the hippocampus of adult ER-alpha-knockout (ER-alphaKO) mice restores hippocampal responsiveness to estrogen and rescues spatial learning. We propose that constitutive estrogen receptor activity is important for maintaining hippocampus-dependent memory function in adults.

Emerging Roles of Primary Cilia in Glioma.

Primary cilia are microtubule-based organelles that are typically present on cells during the G0 or G1-S/G2 phases of the cell cycle. Recent studies of glioblastoma (GBM) biopsies, a brain tumor that is notorious for its aggressive growth and resistance to treatment, show that many cells in the tumor lack cilia. At this point, it remains unclear whether primary cilia promote or suppress glioma tumorigenesis. In this review, we will discuss the different roles that have been proposed for primary cilia in glioma and how cilia may contribute to the resistance of these tumors to current therapies.

The beer and biofuels laboratory: A report on implementing and supporting a large, interdisciplinary, yeast-focused course-based undergraduate research experience.

Course-integrated Undergraduate Research Experiences (CUREs) involve large numbers of students in real research. We describe a late-year microbiology CURE in which students use yeast to address a research question around beer brewing or synthesizing biofuel; the interdisciplinary student-designed project incorporates genetics, bioinformatics, biochemistry, analytical chemistry, and microbiology. Students perceived significant learning gains around multiple technical and "becoming a scientist" aspects of the project. The project is demanding for both the students and the academic implementers. We examine the rich landscape of support and interaction that this CURE both encourages and requires while also considering how we can support the exercise better and more sustainably. The findings from this study provide a picture of a CURE implementation that has begun to reach the limits of both the students' and the academics' capacities to complete it. © 2018 by The International Union of Biochemistry and Molecular Biology, 46(3):213-222, 2018.

Targeted expression of two proteins in neural retina using self-inactivating, insulated lentiviral vectors carrying two internal independent promoters.

PURPOSE: There is increasing interest in developing viral vectors capable of reliably delivering multiple therapeutic genes to targeted cell populations. Currently, bicistronic vectors carrying two transgenes linked by an internal ribosomal entry site (IRES) are the most commonly employed vectors to accomplish this goal. We and others have found that the protein encoded downstream of the IRES in these vectors is not reliably expressed. The purpose of this study was to determine if replacement of the IRES in our self-inactivating, insulated, lentiviral vectors with a second, independent, cell-specific promoter would produce a vector that reliably expressed two proteins in targeted retinal cells in vivo. METHODS: Five dual promoter lentiviral vectors were constructed using our self-inactivating (SIN), insulated, lentiviral backbone. Each vector carried two independent transgenes encoding a fluorescent protein (GFP or tdTomato) whose expression was driven by three photoreceptor promoters (interphotoreceptor retinoid binding protein-IRPB1783; guanylate cyclase activating protein 1-GCAP292; rhodopsin-mOP500) and one ubiquitously expressed promoter (elongation factor 1alpha-EF1alpha). Constructs were packaged and injected into the optic vesicles of developing chicken embryos. The day before hatching, the retinas were removed and examined as whole mount tissues and as frozen sections using fluorescent microscopy. RESULTS: In our first experiment, we characterized the expression of the three photoreceptor promoters in chicken retina. The activities of GCAP292 and IRBP1783 were restricted to cone cells. GCAP292 was also active in a small sub-group of inner nuclear cells. The activity of mOP500 was restricted to rod cells. In our second experiment, we characterized the activity of three dual promoter vectors: GCAP292-GFP-IRBP1783-tdTomato, IRBP-tdTomato-GCAP292-GFP, and IRBP1783-tdTomato-mOP500-GFP. All three vectors produced easily detectable levels of GFP and tdTomato in transduced retinas, a result that prompted further analyses of the expression characteristics of these vectors. In retinas treated with either of the GCAP292/IRBP1783 dual promoter vectors, GFP and tdTomato were only detected in cone cells. No GFP was detected in the inner retina. In retinas treated with IRBP1783-tdTomato-mOP500-GFP, tdTomato was detected only in cone cells and GFP was detected only in rod cells, a result indicating that these promoters retained their intrinsic expression specificities in this dual promoter vector. In our final experiment, the ubiquitously expressed EF1alpha promoter was paired with either GCAP292 or mOP500 creating EF1alpha-tdTomato-GCAP292-GFP and EF1alpha-tdTomato-mOP-GFP. In retinas treated with EF1alpha-tdTomato-GCAP292-GFP, GFP was only detected in cone cells. In retinas treated with EF1alpha-tdTomato-mOP500-GFP, GFP was detected in rod cells and in several cells within the inner retina. CONCLUSIONS: The results of this study show that it is possible to construct dual promoter lentiviral vectors that reliably express two proteins in a cell-specific manner. Among the dual promoter vectors created for this study, we have identified two vectors that specifically target expression of both transgenes to cone cells and one vector that specifically targets expression of one transgene to cone cells and the other transgene to rod cells. The ability to create one lentiviral vector that is capable of targeting expression of multiple genes to single or multiple cells in vivo should prove very useful in the development and delivery of complex, combination therapies to diseased tissues.

Effective visual design and communication practices for research posters: Exemplars based on the theory and practice of multimedia learning and rhetoric.

Evidence shows that science graduates often do not have the communication skills they need to meet workplace standards and expectations. One common mode of science communication is the poster. In a review of the literature we show that poster design is historically problematic, and that the guidance provided to students as they create posters for assessment is frequently inconsistent. To address this inconsistency we provide some guiding design principles for posters that are grounded in communication theory and the fundamentals of rhetoric. We also present three nondiscipline-specific example posters with accompanying notes that explain why the posters are examples of poor, average, and excellent poster design. The subject matter for the posters is a fabricated set of experiments on a topic that could not actually be the subject of research. Instructors may use these resources with their students, secure in the knowledge that they do not and will never represent an answer set to an extant assessment item. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(3):249-261, 2017.

A unique large-scale undergraduate research experience in molecular systems biology for non-mathematics majors.

Systems biology is frequently taught with an emphasis on mathematical modeling approaches. This focus effectively excludes most biology, biochemistry, and molecular biology students, who are not mathematics majors. The mathematical focus can also present a misleading picture of systems biology, which is a multi-disciplinary pursuit requiring collaboration between biochemists, bioinformaticians, and mathematicians. This article describes an authentic large-scale undergraduate research experience (ALURE) in systems biology that incorporates proteomics, bacterial genomics, and bioinformatics in the one exercise. This project is designed to engage students who have a basic grounding in protein chemistry and metabolism and no mathematical modeling skills. The pedagogy around the research experience is designed to help students attack complex datasets and use their emergent metabolic knowledge to make meaning from large amounts of raw data. On completing the ALURE, participants reported a significant increase in their confidence around analyzing large datasets, while the majority of the cohort reported good or great gains in a variety of skills including "analysing data for patterns" and "conducting database or internet searches." An environmental scan shows that this ALURE is the only undergraduate-level system-biology research project offered on a large-scale in Australia; this speaks to the perceived difficulty of implementing such an opportunity for students. We argue however, that based on the student feedback, allowing undergraduate students to complete a systems-biology project is both feasible and desirable, even if the students are not maths and computing majors. © 2016 by The International Union of Biochemistry and Molecular Biology, 45(3):235-248, 2017.

Lentiviral expression of retinal guanylate cyclase-1 (RetGC1) restores vision in an avian model of childhood blindness.

BACKGROUND: Leber congenital amaurosis (LCA) is a genetically heterogeneous group of retinal diseases that cause congenital blindness in infants and children. Mutations in the GUCY2D gene that encodes retinal guanylate cyclase-1 (retGC1) were the first to be linked to this disease group (LCA type 1 [LCA1]) and account for 10%-20% of LCA cases. These mutations disrupt synthesis of cGMP in photoreceptor cells, a key second messenger required for function of these cells. The GUCY1*B chicken, which carries a null mutation in the retGC1 gene, is blind at hatching and serves as an animal model for the study of LCA1 pathology and potential treatments in humans. METHODS AND FINDINGS: A lentivirus-based gene transfer vector carrying the GUCY2D gene was developed and injected into early-stage GUCY1*B embryos to determine if photoreceptor function and sight could be restored to these animals. Like human LCA1, the avian disease shows early-onset blindness, but there is a window of opportunity for intervention. In both diseases there is a period of photoreceptor cell dysfunction that precedes retinal degeneration. Of seven treated animals, six exhibited sight as evidenced by robust optokinetic and volitional visual behaviors. Electroretinographic responses, absent in untreated animals, were partially restored in treated animals. Morphological analyses indicated there was slowing of the retinal degeneration. CONCLUSIONS: Blindness associated with loss of function of retGC1 in the GUCY1*B avian model of LCA1 can be reversed using viral vector-mediated gene transfer. Furthermore, this reversal can be achieved by restoring function to a relatively low percentage of retinal photoreceptors. These results represent a first step toward development of gene therapies for one of the more common forms of childhood blindness.

Light-driven cone arrestin translocation in cones of postnatal guanylate cyclase-1 knockout mouse retina treated with AAV-GC1.

PURPOSE: Cone function and survival are compromised in the guanylate cyclase-1 (GC1) knockout mouse. Disruption of the light-driven translocation of cone arrestin is one of the phenotypes of cone cells in this retina: the cone arrestin in these cells is localized to the outer segments and synaptic terminals, regardless of the state of light adaptation. The purpose of this study was to determine whether the expression of GC1 restores cone arrestin translocation in the cone cells of postnatal GC1 knockout mouse retina. METHODS: Subretinal injections of AAV-GC1 were performed on 3-week-old GC1 KO mice. Electroretinographic and immunohistochemical analyses of treated retinas were carried out 5 weeks after injection. GC1 and cone arrestin antibodies were used to identify photoreceptors transduced by the AAV vector and to localize cone arrestin within cone cells, respectively. RESULTS: Treatment of GC1 knockout retinas with AAV-GC1 restored the light-driven translocation of cone arrestin in transduced cone cells. Staining patterns for cone arrestin in transduced and wild-type cone cells were indistinguishable after dark and light adaptation. In dark-adapted retinas, cone arrestin was distributed throughout the subcellular compartments of the cone cells. In light-adapted retinas, cone arrestin was concentrated in the cone outer segments. Successful restoration of cone arrestin translocation did not translate to a restoration of cone ERG responses, which remained undetectable in the treated retinas. CONCLUSIONS: AAV-mediated expression of GC1 in a subpopulation of cone cells in postnatal GC1 knockout retina restores light-driven translocation of cone arrestin in these cells. These findings, which show that fully developed cone cells that have developed in the absence of GC1 can respond to viral-mediated expression of this enzyme, support further analysis of this animal model of Leber congenital amaurosis type 1 (LCA1), a disease that results from null mutations in the gene encoding this enzyme.

GC1 deletion prevents light-dependent arrestin translocation in mouse cone photoreceptor cells.

PURPOSE: Light-driven translocation of phototransduction regulatory proteins between the inner and outer segments of photoreceptor cells plays a role in the adaptation of these cells to light. The purpose of this study was to examine the effects of the absence of guanylate cyclase 1 (GC1) on light-driven protein translocation in rod and cone cells. Both cell types express GC1, but differ in sensitivity, saturation, and response times to light. METHODS: Immunohistochemical techniques employing antibodies specific for cone and rod transducin alpha (Talpha) subunits and arrestins were used to examine light-driven translocation of these proteins in the retinas of wild-type and GC1 knockout (KO) mice. RESULTS: Translocation of cone arrestin from cone outer segments to the inner cell regions was disrupted in the absence of GC1, whereas translocation of arrestin and Talpha in rods was not affected. Cone Talpha did not translocate in wild-type and GC1 KO mice, but differed in its subcellular distribution in GC1 KO retina, remaining in the cone outer segment in light and in dark. CONCLUSIONS: These results suggest that multiple, independent pathways regulate the translocation of phototransduction proteins and that GC1, and presumably cGMP, are of key importance in signaling the translocation of cone arrestin.

Rhythmic expression of clock-controlled genes in retinal photoreceptors is sensitive to 18-beta-glycyrrhetnic acid and 18-alpha-glycyrrhetnic acid-3-hemisuccinate.

Chicken retina contains circadian oscillators that drive rhythmic transcription of several genes expressed in photoreceptor cells. To determine if gap junctions assist in coordinating these transcript rhythms, we examined the effects of two compounds, 18alpha-glycyrrhetnic acid-3-hemisuccinate (ACO) and 18beta-glycyrrhetnic acid (18beta-GA), on photoreceptor iodopsin and arylalkylamine N-acetyltransferase (AANAT) transcript rhythms in embryonic chicken retinal explant cultures that were maintained under different lighting conditions. Both compounds, whose actions include reversibly block gap junction permeability, produced rapid and sustained reductions in iodopsin and AANAT mRNA levels, but did not alter the levels of guanylate cyclase activating protein-1 (GCAP1) mRNA, a noncircadian-regulated, photoreceptor-specific gene. The iodopsin and AANAT mRNA rhythms re-emerged in the cultured retinas within 24 h of removal of the compounds. These results show that the effects of ACO and 18beta-GA on iodopsin and AANAT mRNA levels were not due to generalized suppression of gene transcription. The dramatic reduction in the levels of iodopsin and AANAT mRNA induced by these compounds suggests a mechanism of action that directly affects the synthesis and/or degradation of these transcripts rather than the synchronization or function of the retinal oscillators that drive transcription of these genes.

Efficient large-scale production and concentration of HIV-1-based lentiviral vectors for use in vivo.

The aim of this study was to develop an efficient method for packaging and concentrating lentiviral vectors that consistently yields high-titer virus on a scale suitable for in vivo applications. Transient cotransfection of 293T packaging cells with DNA plasmids encoding lentiviral vector components was optimized using SuperFect, an activated dendrimer-based transfection reagent. The use of SuperFect allowed reproducible and efficient production of high-titer lentiviral vector at concentrations greater than 1 x 10(7) transducing units per ml (TU/ml) and required less than one-third of the total amount of DNA used in traditional calcium phosphate transfection methods. Viral titers were further increased using a novel concentration protocol that yielded an average final titer of 1.4 x 10(10) TU/ml. Lentiviruses produced using these methods exhibited efficient transduction of central nervous system and peripheral tissues in vivo. The method is reproducible and can be scaled up to facilitate the use of these vectors in animal studies.