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At least 703 records · Page 39Linked to original sources
PubMed2005

The CELO adenovirus Gam1 protein enhances transient and stable recombinant protein expression in Chinese hamster ovary cells.

The Gam1 protein of the avian CELO adenovirus activates transcription through inhibition of histone deacetylase 1 (HDAC1). We investigated the effect of Gam1 on both transient and stable transgene expression in Chinese hamster ovary (CHO) cells, one of the most commonly used mammalian hosts for the large-scale production of recombinant proteins. Transient expression of Gam1 increased reporter protein levels up to 4-fold in suspension cultures of CHO DG44 cells co-transfected with a reporter gene and up to 20-fold in recombinant CHO DG44-derived cell lines. The highest levels of activation were observed when the transgene was under the control of the human cytomegalovirus (HCMV) immediate early promoter/enhancer. Increases in recombinant protein expression in the presence of Gam1 were not accompanied by an enhancement of cell growth or viability. We conclude that Gam1 may serve as a useful genetic tool for increasing recombinant protein expression in CHO DG44 cells.

Animals↗
PubMed2005

Genetic manipulations to study reproduction.

Fertility disorders affect approximately 15% of individuals worldwide. With the imminent completion of the human and mouse genome sequence, it will be more feasible to identify the relevant genes underlying many fertility disorders. Already, the mouse has been utilized extensively as a genetic tool for the dissection of gene function, often providing significant insights into the relationship between gene and disease. In fact, there are over 200 mouse models that display reproductive defects. However, the available mouse mutant resources provide functional information for a mere 10% of the total number of genes in the mouse or human genomes at best. The improvement of available genome annotations together with more powerful techniques to manipulate the mouse genome provide substantial improvements in our ability to identify genes involved in reproduction, and in the future will likely benefit patients with fertility problems.

Animals↗
PubMed2006

Mouse models for gonadotropins: a 15-year saga.

Pituitary gonadotropins FSH and LH are heterodimeric glycoproteins consisting of a common alpha and a hormone-specific beta subunit that are non-covalently linked. Both these hormones bind G-protein coupled receptors and regulate multiple processes in the gonads. Advances in the past two decades in manipulating the mouse genome by random and site-specific mutagenesis have been invaluable to our understanding of the biology of gonadotropins. Using these transgenic and gene targeting approaches, both gain-of-function transgenic as well as knockout mice lacking the hormone-specific gonadotropin subunits, and hence functional dimeric hormones were generated. Furthermore, knockout mice lacking regulators of gonadotropin production have also been characterized. These mice are useful to delineate the distinct in vivo biological roles of FSH and LH, and provide valuable genetic tools to study the signaling mechanisms within the gonads. Here, I will discuss our work on different ways to manipulate gonadotropin ligand function in the mouse.

Animals↗
PubMed2005

Altered dopaminergic innervation and amphetamine response in adult Otx2 conditional mutant mice.

Here, we have investigated the neurological consequences of restricted inactivation of Otx2 in adult En1(cre/+); Otx2(flox/flox) mice. In agreement with the crucial role of Otx2 in midbrain patterning, the mutants had a substantial reduction in tyrosine hydroxylase containing neurons. Although the reduction in the number of DAergic neurons was comparable between the SNc and the VTA, we found an unexpected selectivity in the deinnervation of the terminal fields affecting preferentially the ventral striatum and the olfactory tubercle. Interestingly, the mutants showed no abnormalities in exploratory activity or motor coordination. However, the absence of normal DA tone generated significant alterations in DA D1-receptor signalling as indicated by increased mutant striatal levels of phosphorylated DARPP-32 and by an altered motor response to amphetamine. Therefore, we suggest that the En1(cre/+); Otx2(flox/flox) mutant mouse model represents a genetic tool for investigating molecular and behavioural consequences of developmental neuronal dysfunction in the DAergic system.

Amphetamines↗
PubMed2024

Dissecting host-microbe interactions with modern functional genomics.

Interrogation of host-microbe interactions has long been a source of both basic discoveries and benefits to human health. Here, we review the role that functional genomics approaches have played in such efforts, with an emphasis on recent examples that have harnessed technological advances to provide mechanistic insight at increased scale and resolution. Finally, we discuss how concurrent innovations in model systems and genetic tools have afforded opportunities to interrogate additional types of host-microbe relationships, such as those in the mammalian gut. Bringing these innovations together promises many exciting discoveries ahead.

Genomics↗
PubMed2005

The expression pattern of the Drosophila vesicular glutamate transporter: a marker protein for motoneurons and glutamatergic centers in the brain.

To determine the functions of genes in distinct tissues during the development of Drosophila, it is often desirable to have genetic tools for targeted gene expression in restricted subsets of cells. Here, we report the identification of the enhancer trap line OK371-Gal4, which is expressed in a defined subset of neurons from embryonic stage 15 to adulthood. In the ventral nerve chord, it is expressed almost exclusively in motoneurons and in the brain in a limited number of neuronal clusters. The OK371 enhancer trap element is inserted in the proximity of the annotated gene CG9887, which encodes a Drosophila vesicular glutamate transporter (DVGLUT). In situ hybridization experiments using antisense probes against the mRNAs of DVGLUT and neighboring genes confirm that OK371-Gal4 detects an enhancer of DVGLUT. DVGLUT-specific antibodies detect its expression in identifiable motoneurons, which are known to be glutamatergic in Drosophila. DVGLUT initially appears in small cytoplasmic punctae in the somata of these motoneurons. As development proceeds, DVGLUT-positive particles are transported along motor axons and become concentrated at neuromuscular junctions (NMJs), where they colocalize with the synaptic vesicle marker synaptotagmin. We find that the DVGLUT-specific antibodies are valuable tools for the identification of motoneurons and other glutamatergic neurons. In addition, the OK371-Gal4 line can be used for the targeted expression of any gene in these cells. Given that vesicular glutamate transporters are essential for the uptake of the neurotransmitter glutamate into synaptic vesicles these tools provide a means to test gene function in these functionally important neurons.

Amino Acid Sequence↗
PubMed2006

Molecular genetic transfection of the coccidian parasite Sarcocystis neurona.

Sarcocystis neurona is an apicomplexan parasite that is the major cause of equine protozoal myeloencephalitis (EPM). The biology of this pathogen remains poorly understood in part due to unavailability of molecular genetic tools. Hence, with an objective to develop DNA transfection capabilities for S. neurona, the 5' flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules beta-galactosidase (beta-gal) and yellow fluorescent protein (YFP) could be detected in electroporated S. neurona, thereby confirming the feasibility of transgene expression in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of Toxoplasma gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express beta-gal and YFP. As shown in this study, these transgenic clones can be useful for analyzing growth rate of parasites in vitro and for assessing drug sensitivities. More importantly, the DNA transfection methods described herein should greatly facilitate studies examining intracellular parasitism by this important coccidian pathogen.

5' Flanking Region↗
PubMed2005

Synaptic mitochondria are critical for mobilization of reserve pool vesicles at Drosophila neuromuscular junctions.

In a forward screen for genes affecting neurotransmission in Drosophila, we identified mutations in dynamin-related protein (drp1). DRP1 is required for proper cellular distribution of mitochondria, and in mutant neurons, mitochondria are largely absent from synapses, thus providing a genetic tool to assess the role of mitochondria at synapses. Although resting Ca2+ is elevated at drp1 NMJs, basal synaptic properties are barely affected. However, during intense stimulation, mutants fail to maintain normal neurotransmission. Surprisingly, FM1-43 labeling indicates normal exo- and endocytosis, but a specific inability to mobilize reserve pool vesicles, which is partially rescued by exogenous ATP. Using a variety of drugs, we provide evidence that reserve pool recruitment depends on mitochondrial ATP production downstream of PKA signaling and that mitochondrial ATP limits myosin-propelled mobilization of reserve pool vesicles. Our data suggest a specific role for mitochondria in regulating synaptic strength.

Animals↗
PubMed2006

Thoughts for food: brain mechanisms and peripheral energy balance.

The past decade has witnessed dramatic advancements regarding the neuroendocrine control of food intake and energy homeostasis and the effects of peripheral metabolic signals on the brain. The development of molecular and genetic tools to visualize and selectively manipulate components of homeostatic systems, in combination with well-established neuroanatomical, electrophysiological, behavioral, and pharmacological techniques, are beginning to provide a clearer picture of the intricate circuits and mechanisms of these complex processes. In this review, we attempt to provide some highlights of these advancements and pinpoint some of the shortcomings of the current understanding of the brain's involvement in the regulation of daily energy homeostasis.

Animals↗
PubMed2005

Developmental abnormalities in the Nuc1 rat retina: a spontaneous mutation that affects neuronal and vascular remodeling and retinal function.

The retina serves as an excellent model in which to study vertebrate CNS development. We have discovered a spontaneous mutation in the Sprague-Dawley rat that results in a novel and unusual ocular phenotype, including retinal abnormalities, that we have named Nuc1. We have previously shown that the Nuc1 mutation appears to suppress programmed cell death in the developing retina. Here we report that maturation of both the retinal neurons and the retinal vessels is abnormal in Nuc1 homozygous rats. The developmental changes in the retinal neurons and vasculature are correlated with regard to degree of abnormality. As Nuc1 homozygotes mature, focal retinal detachment begins at approximately 3 months after birth, and near total traction retinal detachment, associated with pre-retinal fibrosis and neovascularization, is evident by 18 months. Electroretinographic studies at 2.5 months of age indicate that functional retinal degeneration precedes retinal detachment. The functional abnormality is most evident in rods and the inner retina, and is present in homozygous but not heterozygous mutants. Immunocytochemical studies of rod and cone photoreceptors indicate abnormalities in rod, but not cone, photoreceptors in Nuc1 homozygotes, consistent with the electroretinographic findings. In Nuc1 animals, the Muller cells are activated. Although such activation may result from inflammation, Muller cells in Nuc1 may be reacting to a neuronal influence. It appears that the Nuc1 mutation plays a regulatory role in both developing and maturing ocular tissues. The Nuc1 mutation may also serve as an important genetic tool to explore the relationships that may exist among gliosis, normal neuronal development, and normal vascular development and how abnormalities in these associations lead to common retinal diseases.

Amacrine Cells↗
PubMed2005

Towards the rational design of cereal starches.

A major challenge in cereal biotechnology is to achieve the rational design of renewable polymers to meet specific requirements for improving human health, nutrition, and food quality, to increase the energy supply, and to provide safer and more profitable industrial inputs. The field of starch synthesis research has advanced at a rapid pace over the past decade, and many core observations about the pathway are well established over a range of species. Owing to the complexity of the starch-synthesis process, in which suites of enzymes act at the interface between soluble and insoluble phases, the rational design of starch granules with specific functionality is still in its infancy. Our fundamental biochemical knowledge of starch biosynthesis has recently advanced, and this new information could be exploited to create novel variability in carbohydrate polymers in cereal grains. We propose two strategies for moving more rapidly towards truly rational design of starch. First, the focusing of fundamental research on processes that are involved in the regulation of starch synthesis and granule assembly. Second, the development of iterative strategies, exploiting new molecular genetics tools, to screen for desired properties in high-throughput systems.

Biotechnology↗
PubMed2005

Identification of linear plasmid pAM1 in the flavonoid degrading strain Actinoplanes missouriensis(T) (DSM 43046).

By pulsed-field gel electrophoresis, a linear DNA element of about 100 kb was identified in Actinoplanes missouriensis(T) DSM 43046, which grows on the flavonoids hesperidin, rutin and quercetin, and which contains a CO forming quercetinase. Among six Actinoplanes species and strains tested, including A. globisporus(T) DSM 43857, A. philippinensis(T) DSM 43019, A. brasiliensis(T) DSM 43805, A. auranticolor(T) DSM 43031, and A. utahensis(T) DSM 43147, only the A. missouriensis strain exhibited such a genetic element. The linear plasmid, named pAM1, has proteins covalently attached to its 5'-ends like other linear replicons of actinomycetes. Attempts to cure pAM1 failed, however a mutant with reduced plasmid content was obtained, which showed reduced ability to degrade the flavonoid rutinosides rutin and hesperidin. Plasmid pAM1 is the first extrachromosomal genetic element identified in an Actinoplanes species and may be useful to develop genetic tools for biotechnologically important Actinoplanes strains.

Biodegradation, Environmental↗
PubMed2005

Thirty years of olfactory learning and memory research in Drosophila melanogaster.

The last 30 years have witnessed tremendous progress in elucidating the basic mechanisms underlying a simple form of olfactory learning and memory in Drosophila. The application of the mutagenic approach to the study of olfactory learning and memory in Drosophila has yielded insights into the participation of a large number of genes in both the development of critical brain regions as well as in the physiology underlying the acquisition, storage, and retrieval of memory. Newer sophisticated molecular-genetic tools have further allowed for the specification and functional dissection of the neuronal circuitry involved in these processes at a systems level. With these advances in our understanding of the genes, neurons, and circuits involved in learning and memory, the field of Drosophila memory research is nearing a state of integration of the bottom up and top down approaches to understanding this form of behavioral plasticity.

Adaptation, Physiological↗
PubMed2006

Construction of transposition insertion libraries and specific gene inactivation in the pathogen Lactococcus garvieae.

This paper reports the development of genetic tools in Lactococcus garvieae, an important Gram-positive bacterial pathogen affecting both fish and mammals. The vector pGKV210, a broad host range vector, was introduced by electroporation into L. garvieae UNIUD074. The maximal frequency obtained was 3.2 x 10(5) transformants/mug of DNA. Moreover, this effect is highly reproducible and appears to be constant, since all L. garvieae strains tested were transformed. Once the optimal transformation procedure was established, it was used to generate isogenic and transposition mutants. Insertional mutagenesis of the L. garvieae SA9H10L gene, similar to a Streptococcus pyogenes gene encoding the M protein (emm64), was carried out using the conditional replication plasmid pORI19. Transposition mutagenesis using the streptococcal temperature-sensitive suicide vector pTV408 to deliver Tn917 into the chromosome of L. garvieae was also achieved at a frequency of ca. 10(-4). Transposon flanking DNA sequences were obtained by plasmid rescue in Escherichia coli and their sequencing analysis demonstrated that the transposon was inserted at different chromosomal loci. Tn917 also made it possible to select a mutant in the operon involved in mannitol fermentation in this microorganism. The results obtained in the present study lay the foundation for future research on the virulence mechanisms of L. garvieae.

Chromosomes, Bacterial↗
PubMed2004

Building a projection map for photoreceptor neurons in the Drosophila optic lobes.

The sensory tasks performed by the eye are diverse and complex. In Drosophila, the eye performs motion detection for navigation as well as detection of the quality of light (color and polarized light). Both types of inputs are processed separately, as different photoreceptors are specialized in these tasks and contact different target cell layers in the optic lobe. However, their respective outputs are likely to be integrated in higher brain centers. Here, we discuss the cell diversity and potential role of the several ganglia that form the fly optic lobe. We also discuss the power of modern genetic tools to provide the potential to trace the visual neural networks.

Animals↗
PubMed2006

Contrasting mechanisms of stem cell maintenance in Drosophila.

Stem cells are self-renewing multipotent cells essential for development or homeostasis of many tissues. Stem cell populations can be found in most multicellular plants and animals. The mechanisms by which these populations are maintained are diverse, utilizing both intrinsic and extrinsic factors to regulate cell division and differentiation. The genetic tools of the fruitfly, Drosophila melanogaster, have permitted detailed characterization of two stem cell populations. In this review, we will examine these contrasting stem cell model systems from Drosophila and their relevance to stem cell populations in other organisms.

Animals↗
PubMed2005

Pigmented-anaerobic bacteria associated with canine periodontitis.

The etiology of human periodontal disease has been the focus of considerable research, yet relatively little is known about the causative agents of companion animal periodontitis. In humans, Porphyromonas gingivalis, a black-pigmented anaerobic bacteria (BPAB), has been implicated as the primary periopathogen. It has been demonstrated that BPAB are also found in companion animal periodontal pockets. While some animal BPAB have been individually identified, a study to identify the most frequently isolated subgingival BPAB has not been completed using genetic tools. The objective of this work was to identify the types and relative frequencies of pigmented anaerobic bacteria found in the periodontal pockets of dogs. Porphyromonas salivosa, Porphyromonas denticanis (a novel species) and Porphyromonas gulae were found to be the most frequently isolated BPAB associated with canine periodontitis.

Animals↗