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.

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.

Increasing hippocampal estrogen receptor alpha levels via viral vectors increases MAP kinase activation and enhances memory in aging rats in the absence of ovarian estrogens.

We previously demonstrated that aged ovariectomized rats that had received prior estradiol treatment in middle-age exhibited increased levels of estrogen receptor alpha (ERα) in the hippocampus as well as enhanced hippocampal dependent memory as compared to aged rats that had not received mid-life estradiol treatment. These effects persisted long after the estradiol treatment had been terminated. The goal of the current experiment was to determine if increased expression of ERα in the hippocampus, in the absence of exogenously administered estrogens, can impact the hippocampus and cognitive function in aging ovariectomized rats. Middle-aged rats were trained for 24 days on an eight-arm radial maze spatial memory task. All rats were then ovariectomized. Forty days later, rats received either lentiviral delivery to the hippocampus of the gene encoding ERα (lenti-ERα) or a control virus. Rats were tested on delay trials in the radial-maze in which delays of varying lengths were imposed between the fourth and fifth arm choices. Following behavior testing, hippocampi were immunostained using western blotting for ERα, the ERα-regulated protein choline acetyltransferase, and phosphorylation of the ERα-regulated kinases, ERK/MAPK and Akt. Results revealed that aging ovariectomized rats that received delivery of lenti-ERα to the hippocampus exhibited enhanced spatial memory as indicated by increased arm-choice accuracy across delays as compared to ovariectomized rats that received control virus. Western blot data revealed that lenti-ERα delivery significantly increased levels of ERα and phosphorylated ERK/MAPK and had no impact on levels of ChAT or phosphorylation of Akt. Results indicate that increasing hippocampal levels of ERα in aging females in the absence of ovarian or exogenously administered estrogens leads to increases in phosphorylation of ERK/MAPK as well as in enhanced memory.

A preclinical assessment of neural stem cells as delivery vehicles for anti-amyloid therapeutics.

Transplantation of neural stems cells (NSCs) could be a useful means to deliver biologic therapeutics for late-stage Alzheimer's disease (AD). In this study, we conducted a small preclinical investigation of whether NSCs could be modified to express metalloproteinase 9 (MMP9), a secreted protease reported to degrade aggregated Aß peptides that are the major constituents of the senile plaques. Our findings illuminated three issues with using NSCs as delivery vehicles for this particular application. First, transplanted NSCs generally failed to migrate to amyloid plaques, instead tending to colonize white matter tracts. Second, the final destination of these cells was highly influenced by how they were delivered. We found that our injection methods led to cells largely distributing to white matter tracts, which are anisotropic conduits for fluids that facilitate rapid distribution within the CNS. Third, with regard to MMP9 as a therapeutic to remove senile plaques, we observed high concentrations of endogenous metalloproteinases around amyloid plaques in the mouse models used for these preclinical tests with no evidence that the NSC-delivered enzymes elevated these activities or had any impact. Interestingly, MMP9-expressing NSCs formed substantially larger grafts. Overall, we observed long-term survival of NSCs in the brains of mice with high amyloid burden. Therefore, we conclude that such cells may have potential in therapeutic applications in AD but improved targeting of these cells to disease-specific lesions may be required to enhance efficacy.

Bicistronic lentiviruses containing a viral 2A cleavage sequence reliably co-express two proteins and restore vision to an animal model of LCA1.

The disease processes underlying inherited retinal disease are complex and are not completely understood. Many of the corrective gene therapies designed to treat diseases linked to mutations in genes specifically expressed in photoreceptor cells restore function to these cells but fail to stop progression of the disease. There is growing consensus that effective treatments for these diseases will require delivery of multiple therapeutic proteins that will be selected to treat specific aspects of the disease process. The purpose of this study was to design a lentiviral transgene that reliably expresses all of the proteins it encodes and does so in a consistent manner among infected cells. We show, using both in vitro and in vivo analyses, that bicistronic lentiviral transgenes encoding two fluorescent proteins fused to a viral 2A-like cleavage peptide meet these expression criteria. To determine if this transgene design is suitable for therapeutic applications, we replaced one of the fluorescent protein genes with the gene encoding guanylate cyclase-1 (GC1) and delivered lentivirus carrying this transgene to the retinas of the GUCY1*B avian model of Leber congenital amaurosis-1 (LCA1). GUCY1*B chickens carry a null mutation in the GC1 gene that disrupts photoreceptor function and causes blindness at hatching, a phenotype that closely matches that observed in humans with LCA1. We found that treatment of these animals with the 2A lentivector encoding GC1 restored vision to these animals as evidenced by the presence of optokinetic reflexes. We conclude that 2A-like peptides, with proper optimization, can be successfully incorporated into therapeutic vectors designed to deliver multiple proteins to neural retinal. These results highlight the potential of this vector design to serve as a platform for the development of combination therapies designed to enhance or prolong the benefits of corrective gene therapies.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The 5' flanking sequence of the human retGC1 gene acquires a photoreceptor cell restricted activity pattern over the course of retinal development.

PURPOSE: Specific mutations of the retinal guanylyl cyclase-1 (retGC1) gene have been linked to Leber congenital amaurosis type 1 (LCA1) and cone-rod dystrophies in humans, diseases that are amenable to treatments using molecular based therapies. As a step towards developing a therapeutic transgene for LCA1, we analyzed the cell specific and developmental activity profiles of fragments of the human retGC1 5' flanking region in vivo. METHODS: We generated self inactivating lentiviral vector constructs carrying three different fragments of the human retGC1 promoter fused to a nuclear localized beta-galactosidase reporter gene (nlacZ). The transgenes were packaged into lentiviral vectors, which were then used to transduce retinal progenitor cells of the developing chick. We monitored the expression of nlacZ in the retina over the course of development and in the retina, brain and pineal gland just prior to hatching. RESULTS: A 1.8 kb fragment of the retGC1 5' flanking region upstream of Exon 2 was capable of targeting nlacZ expression to photoreceptor cells in vivo and its activity was augmented by the presence of intron 1. We also demonstrated that the cell specific activity of this fragment arises, at least in part, by silencing expression in non-photoreceptor cells during the final stages of retinal development. CONCLUSIONS: We have identified a human retGC1 promoter fragment that exhibits photoreceptor cell specific activity in vivo. Our results suggest that an element located in the proximal promoter may play a role in silencing expression of this gene in non-photoreceptor cells, thereby by shaping the restricted expression pattern of GC1 in the retina.

Cone cell survival and downregulation of GCAP1 protein in the retinas of GC1 knockout mice.

PURPOSE: To examine the spatial and temporal characteristics of cone cell survival and the expression of guanylate cyclase-activating proteins (GCAPs) in the guanylate cyclase (GC)-1 knockout (KO) mouse retina. METHODS: Immunohistochemical analyses with peanut agglutinin and an antibody specific for cone transducin were used to examine cone cell survival in the GC1 KO retina at 4, 5, 9, 16, and 24 weeks of age. Immunohistochemical and Northern and Western blot analyses were used to examine the expression of GCAP1 and GCAP2 in 4- to 5-week-old mice. RESULTS: The number of cone cells appeared normal throughout the superior and inferior retinal regions in 4- and 5-week-old GC1 KO mice but gradually decreased by 6 months. Cone cell loss was exacerbated in the inferior retinal region, with only 2% to 8% remaining by 6 months of age; however, 40% to 70% of the cone cells survived in the superior region at this age. GCAP1 and GCAP2 protein levels were downregulated in GC1 KO retinas at 4 weeks of age and GCAP1 immunostaining was absent from the photoreceptor outer segments. CONCLUSIONS: The results of this study show that the rate of cone cell loss in the GC1 KO mouse is comparable to that previously described in the GUCY1*B chicken and in humans with Leber congenital amaurosis (LCA)-1. The GCAP expression data, when combined with those of previous electrophysiological studies of the GC1 KO mouse retina, provide evidence that GC1-GCAP1 interactions are essential for cone cell function in mice and that GC2 and GCAP2 activities contribute to the rod cell response in the absence of GC1.

Circadian oscillator function in embryonic retina and retinal explant cultures.

Retinal circadian oscillators regulate many aspects of retinal function. Investigations of these oscillators and the biochemical cascades that entrain them would be greatly facilitated if experimental paradigms could be identified that permit long-term monitoring of retinal circadian oscillator function in vitro. The purpose of this study was to determine if chicken retinas maintained in explant culture conditions could serve in this capacity. Retinal circadian oscillator function was studied by monitoring iodopsin transcription under cyclic light, constant dark, and following reversal of the light cycle. Rhythms observed in the explant cultures were compared to those observed in retinas of embryos (in ovo) and post-hatch chickens. Robust iodopsin transcript rhythms were observed for up to 9 days in explant cultures maintained under cyclic light. These rhythms persisted for 48 h in constant darkness and the time course for re-entrainment of the rhythm to a reversed light/dark cycle was similar to that observed in post-hatch chicken retinas. These results show that circadian oscillators located within the retina play a key role in the regulation of iodopsin transcription in retinal explant cultures and in retinas of post-hatch chickens. Interestingly, our data show that iodopsin transcription in retinas of intact embryos is primarily, if not entirely, driven by light. These results show that the circadian oscillators driving iodopsin transcription in embryonic retinal explant cultures exhibit functional characteristics similar to those found in post-hatch chicken retina, supporting use of this paradigm in further studies of entrainment of these oscillators in retina.

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.

Analyses of the guanylate cyclase activating protein-1 gene promoter in the developing retina.

PURPOSE: To determine the activity, cell specificity, and developmental expression profiles of fragments of the chicken guanylate cyclase activating protein (GCAP)-1 promoter. METHODS: The intrinsic activities of five GCAP1 promoter-luciferase constructs were measured in transiently transfected primary chicken embryonic retinal cultures. Lentivirus vectors carrying GCAP1 promoter-nlacZ transgenes were used to examine the cell specificities and temporal expression characteristics of selected promoter fragments in developing retina. RESULTS: Three of the five GCAP1 promoter fragments exhibited significant activity in vitro. The expression characteristics of the promoter fragments in vivo varied as a function of promoter length. Expression of nlacZ driven by the 0.6- and 1.7-kb GCAP1 promoter fragments was first observed on embryonic day (E)12 and was restricted to the inner nuclear layer (INL). By E16, nlacZ staining was also detected in the outer nuclear layer (ONL). Expression of nlacZ driven by the 4.2-kb GCAP1 promoter fragment was not observed until E16 and was restricted to the ONL. CONCLUSIONS: The general organization of regulatory cis elements within the GCAP1 promoter is different from other photoreceptor-specific gene promoters. Elements located within 0.3 kb upstream of the transcription start point are capable of producing efficient gene expression; however, additional elements located within 4.0 kb upstream of the transcription start point are necessary to confer on the fragment the cell specificity and developmental expression characteristics of the native GCAP1 promoter. The results of the current study show that the lentiviral vector system is a useful tool for the characterization of the promoters of genes expressed in neural retina.