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Revealing the molecular events associated with reprogramming different somatic cell types to pluripotency is critical for understanding the characteristics of induced pluripotent stem cell (iPSC) therapeutic derivatives. Inducible reprogramming factor transgenic cells or animals--designated as secondary (2°) reprogramming systems--not only provide excellent experimental tools for such studies but also offer a strategy to study the variances in cellular reprogramming outcomes due to different in vitro and in vivo environments. To make such studies less cumbersome, it is desirable to have a variety of efficient reprogrammable mouse systems to induce successful mass reprogramming in somatic cell types. Here, we report the development of two transgenic mouse lines from which 2° cells reprogram with unprecedented efficiency. These systems were derived by exposing primary reprogramming cells containing doxycycline-inducible Yamanaka factor expression to a transient interruption in transgene expression, resulting in selection for a subset of clones with robust transgene response. These systems also include reporter genes enabling easy readout of endogenous Oct4 activation (GFP), indicative of pluripotency, and reprogramming transgene expression (mCherry). Notably, somatic cells derived from various fetal and adult tissues from these 2° mouse lines gave rise to highly efficient and rapid reprogramming, with transgene-independent iPSC colonies emerging as early as 1 wk after induction. These mouse lines serve as a powerful tool to explore sources of variability in reprogramming and the mechanistic underpinnings of efficient reprogramming systems. [ABSTRACT FROM AUTHOR]

Genetically modified animals continue to provide important insights into the molecular basis of health and disease. Research has focused mostly on genetically modified mice, although other species like pigs resemble the human physiology more closely. In addition, crossspecies comparisons with phylogenetically distant species such as chickens provide powerful insights into fundamental biological and biomedical processes. One of the most versatile genetic methods applicable across species is CRISPR-Cas9. Here, we report the generation of transgenic chickens and pigs that constitutively express Cas9 in all organs. These animals are healthy and fertile. Functionality of Cas9 was confirmed in both species for a number of different target genes, for a variety of cell types and in vivo by targeted gene disruption in lymphocytes and the developing brain, and by precise excision of a 12.7-kb DNA fragment in the heart. The Cas9 transgenic animals will provide a powerful resource for in vivo genome editing for both agricultural and translational biomedical research, and will facilitate reverse genetics as well as cross-species comparisons. [ABSTRACT FROM AUTHOR]

Polymorphism in the alcohol dehydrogenase (ADH) protein of Drosophila melanogaster, like genetic variation in many other enzymes, has long been hypothesized to bemaintained by a selective trade-off between thermostability and enzyme activity. Two major Adh variants, named Fast and Slow, are distributed along latitudinal clines on several continents. The balancing selection trade-off hypothesis posits that Fast is favored at high latitudes because it metabolizes alcohol faster, whereas Slow is favored at low latitudes because it is more stable at high temperatures. Here we use biochemical and physiological assays of precisely engineered genetic variants to directly test this hypothesis. As predicted, the Fast protein has higher catalytic activity than Slow, and both the Fast protein and regulatory variants linked to it confer greater ethanol tolerance on transgenic animals. But we found no evidence of a temperature-mediated tradeoff: The Fast protein is not less stable or active at high temperatures, and Fast alleles increase ethanol tolerance and survivorship at all temperatures tested. Further, analysis of a population genomic dataset reveals no signature of balancing selection in the Adh gene. These results provide strong evidence against balancing selection driven by a stability/activity trade-off in Adh, and they justify caution about this hypothesis for other enzymes except those for which it has been directly tested. Our findings tentatively suggest that environmentspecific selection for the Fast allele, coupled with demographic history, may have produced the observed pattern of Adh variation. [ABSTRACT FROM AUTHOR]

The neurodegenerative disorder Huntington's disease (HD) is typically characterized by extensive loss of striatal neurons and the midlife onset of debilitating and progressive chorea, dementia, and psychological disturbance. HD is caused by a CAG repeat expansion in the Huntingtin (HTT) gene, translating to an elongated glutamine tract in the huntingtin protein. The pathogenic mechanism resulting in cell dysfunction and death beyond the causative mutation is not well defined. To further delineate the early molecular events in HD, we performed RNA-sequencing (RNA-seq) on striatal tissue from a cohort of 5-y-old OVT73-line sheep expressing a human CAG-expansion HTT cDNA transgene. Our HD OVT73 sheep are a prodromal model and exhibit minimal pathology and no detectable neuronal loss. We identified significantly increased levels of the urea transporter SLC14A1 in the OVT73 striatum, along with other important osmotic regulators. Further investigation revealed elevated levels of the metabolite urea in the OVT73 striatum and cerebellum, consistent with our recently published observation of increased urea in postmortem human brain from HD cases. Extending that finding, we demonstrate that postmortem human brain urea levels are elevated in a larger cohort of HD cases, including those with low-level neuropathology (Vonsattel grade 0/1). This elevation indicates increased protein catabolism, possibly as an alternate energy source given the generalized metabolic defect in HD. Increased urea and ammonia levels due to dysregulation of the urea cycle are known to cause neurologic impairment. Taken together, our findings indicate that aberrant urea metabolism could be the primary biochemical disruption initiating neuropathogenesis in HD. [ABSTRACT FROM AUTHOR]

Growing insight into the pathogenesis of autoimmune diseases and numerous studies in preclinical models highlights the potential of regulatory T cells to restore tolerance. By using non-obese diabetic (NOD) BDC2.5 TCR-transgenic (Tg), and IL-10 and Foxp3 doublereporter mice, we demonstrate that alteration of gut microbiota during cohousing experiments or treatment with anti-CD3 mAb significantly increase intestinal IL-10-producing type 1 regulatory T (Tr1) cells and decrease diabetes incidence. These intestinal antigenspecific Tr1 cells have the ability to migrate to the periphery via a variety of chemokine receptors such as CCR4, CCR5, and CCR7 and to suppress proliferation of Th1 cells in the pancreas. The ability of Tr1 cells to cure diabetes in NOD mice required IL-10 signaling, as Tr1 cells could not suppress CD4+ T cells with a dominant-negative IL-10R. Taken together, our data show a key role of intestinal Tr1 cells in the control of effector T cells and development of diabetes. Therefore, modulating gut-associated lymphoid tissue to boost Tr1 cells may be important in type 1 diabetes management. [ABSTRACT FROM AUTHOR]

Recent studies have indicated that mammalian cells contain a cytosolic protein disaggregation machinery comprised of Hsc70, DnaJ homologs, and Hsp110 proteins, the last of which acts to accelerate a rate-limiting step of nucleotide exchange of Hsc70. We tested the ability of transgenic overexpression of a Thy1 promoterdriven human Hsp110 protein, HspA4L (Apg1), in neuronal cells of a transgenic G85R SOD1YFP ALS mouse strain to improve survival. Notably, G85R is a mutant version of Cu/Zn superoxide dismutase 1 (SOD1) that is unable to reach native form and that is prone to aggregation, with prominent YFP-fluorescent aggregates observed in the motor neurons of the transgenic mice as early as 1 mo of age. The several-fold overexpression of Hsp110 in motor neurons of these mice was associated with an increased median survival from ~5.5 to 7.5 mo and increased maximum survival from 6.5 to 12 mo. Improvement of survival was also observed for a G93A mutant SOD1 ALS strain. We conclude that neurodegeneration associated with cytosolic misfolding and aggregation can be ameliorated by overexpression of Hsp110, likely enhancing the function of a cytosolic disaggregation machinery. [ABSTRACT FROM AUTHOR]

The authors present insights on the regulatory tracts on genetically engineered (GE) animals that are used as food. Topics discussed include the policy of the U.S. on GE animals, the approval given by the U.S. Food and Drug Administration (FDA) on the GE AquAdvantage salmon for use as food, and the U.S. government agencies that regulate GE plants. Also mentioned is the application of the concepts generally recognized as safe (GRAS) and substantial equivalence on the regulation of GE animals.

Avian integumentary organs include feathers, scales, claws, and beaks. They cover the body surface and play various functions to help adapt birds to diverse environments. These keratinized structures are mainly composed of corneous materials made of α-keratins, which exist in all vertebrates, and β-keratins, which only exist in birds and reptiles. Here, members of the keratin gene families were used to study how gene family evolution contributes to novelty and adaptation, focusing on tissue morphogenesis. Using chicken as a model, we applied RNA-seq and in situ hybridization to map α- and β-keratin genes in various skin appendages at embryonic developmental stages. The data demonstrate that temporal and spatial α- and β-keratin expression is involved in establishing the diversity of skin appendage phenotypes. Embryonic feathers express a higher proportion of β-keratin genes than other skin regions. In feather filament morphogenesis, β-keratins show intricate complexity in diverse substructures of feather branches. To explore functional interactions, we used a retrovirus transgenic system to ectopically express mutant α- or antisense β-keratin forms. α- and β-keratins show mutual dependence and mutations in either keratin type results in disrupted keratin networks and failure to form proper feather branches. Our data suggest that combinations of α- and β-keratin genes contribute to the morphological and structural diversity of different avian skin appendages, with feather-β-keratins conferring more possible composites in building intrafeather architecture complexity, setting up a platform of morphological evolution of functional forms in feathers. [ABSTRACT FROM AUTHOR]

Myocardial mitochondrial Ca2+ entry enables physiological stress responses but in excess promotes injury and death. However, tissue-specific in vivo systems for testing the role of mitochondrial Ca2+ are lacking. We developed a mouse model with myocardial delimited transgenic expression of a dominant negative (DN) form of the mitochondrial Ca2+ uniporter (MCU). DN-MCU mice lack MCU-mediated mitochondrial Ca2+ entry in myocardium, but, surprisingly, isolated perfused hearts exhibited higher O2 consumption rates (OCR) and impaired pacing induced mechanical performance compared with wild-type (WT) littermate controls. In contrast, OCR in DN-MCU-permeabilized myocardial fibers or isolated mitochondria in low Ca2+ were not increased compared with WT, suggesting that DN-MCU expression increased OCR by en hanced energetic demands related to extramitochondrial Ca2+ homeostasis. Consistent with this, we found that DN-MCU ventricular cardiomyocytes exhibited elevated cytoplasmic [Ca2+] that was partially reversed by ATP dialysis, suggesting that metabolic defects arising from loss of MCU function impaired physiological intracellular Ca2+ homeostasis. Mitochondrial Ca2+ overload is thought to dissipate the inner mitochondrial membrane potential (Δψm) and enhance formation of reactive oxygen species (ROS) as a consequence of ischemia-reperfusion injury. Our data show that DN-MCU hearts had preserved Δψm and reduced ROS during ischemia reperfusion but were not protected from myocardial death compared withWT. Taken together, our findings show that chronicmyocardial MCU inhibition leads to previously unanticipated compensatory changes that affect cytoplasmic Ca2+ homeostasis, reprogram transcription, increase OCR, reduce performance, and prevent antici- pated therapeutic responses to ischemia-reperfusion injury. [ABSTRACT FROM AUTHOR]

As with other retinal cell types, retinal ganglion cells (RGCs) arise from multipotent retinal progenitor cells (RPCs), and their formation is regulated by a hierarchical gene-regulatory network (GRN). Within this GRN, three transcription factors--atonal homolog 7 (Atoh7), POU domain, class 4, transcription factor 2 (Pou4f2), and insulin gene enhancer protein 1 (Isl1)--occupy key node positions at two different stages of RGC development. Atoh7 is upstream and is required for RPCs to gain competence for an RGC fate, whereas Pou4f2 and Isl1 are downstream and regulate RGC differentiation. However, the genetic and molecular basis for the specification of the RGC fate, a key step in RGC development, remains unclear. Here we report that ectopic expression of Pou4f2 and Isl1 in the Atoh7-null retina using a binary knockin-transgenic system is sufficient for the specification of the RGC fate. The RGCs thus formed are largely normal in gene expression, survive to postnatal stages, and are physiologically functional. Our results indicate that Pou4f2 and Isl1 compose a minimally sufficient regulatory core for the RGC fate. We further conclude that during development a core group of limited transcription factors, including Pou4f2 and Isl1, function downstream of Atoh7 to determine the RGC fate and initiate RGC differentiation. [ABSTRACT FROM AUTHOR]

Huntington disease is a progressive and fatal genetic disorder with debilitating motor and cognitive defects. Chorea, rigidity, dystonia, and muscle weakness are characteristic motor defects of the disease that are commonly attributed to central neurodegeneration. However, no previous study has examined the membrane properties that control contraction in Huntington disease muscle. We show primary defects in ex vivo adult skeletal muscle from the R6/2 transgenic mouse model of Huntington disease. Action potentials in diseased fibers are more easily triggered and prolonged than in fibers from WT littermates. Furthermore, some action potentials in the diseased fibers self-trigger. These defects occur because of decreases in the resting chloride and potassium conductances. Consistent with this, the expression of the muscle chloride channel, ClC-1, in Huntington disease muscle was compromised by improper splicing and a corresponding reduction in total Clcn1 (gene for ClC-1) mRNA. Additionally, the total Kcnj2 (gene for the Kir2.1 potassium channel) mRNA was reduced in disease muscle. The resulting muscle hyperexcitability causes involuntary and prolonged contractions that may contribute to the chorea, rigidity, and dystonia that characterize Huntington disease. [ABSTRACT FROM AUTHOR]

We have developed a unique method for mouse transgenesis. The transposase-enhanced pronuclear microinjection (PNI) technique described herein uses the hyperactive piggyBac transposase to insert a large transgene into the mouse genome. This procedure increased transgene integration efficiency by fivefold compared with conventional PNI or intracytoplasmic sperm injection-mediated transgenesis. Our data indicate that the transposase-enhanced PNI technique additionally requires fewer embryos to be microinjected than traditional methods to obtain transgenic animals. This transposase-mediated approach is also very efficient for single-cell embryo cytoplasmic injections, offering an easy-to-implement transgenesis method to the scientific community. [ABSTRACT FROM AUTHOR]

Transgenic birds embody one of the most potent and exciting research tools in biotechnology for agriculture, medicine, and model animals. To date, retrovirus- or lentivirus-mediated transgenesis has been established in chickens and quail. However, despite having a valid technique for viral transduction to achieve transgenic birds, many obstacles exist for practical applications because of relatively low and variable rates of germ-line transmission and transgenic offspring showing transgene silencing, as well as safety issues related to viral vector use. Thus, the generation of transgenic poultry by nonviral integration is a prerequisite for the introduction of biotechnology to practical applications. Herein, we show that a germ-line-competent chicken primordial germ-cell (PGC) line was established with high efficiency of transmission to offspring and that piggyBac transposition into PGCs improved the efficiency of transgenic chicken production and led to high-level transgene expression. GFP transgene-expressing donor PGC-transferred recipient chickens produced donor-derived progenies, and the germ-line transmission efficiency of donor PGCs was 95.2% on average. Subsequently, half of the donor-derived offspring (52.2%) were transgenic chicks because GFP-expressing donor PGCs, in which a transgene was inserted into one chromosome 20, were heterozygous. In all of the transgenic chickens, GFP expression was constant and strong, regardless of age. Our results demonstrate that piggyBac transposition into the chicken PGC line could be the surest way to generate transgenic chickens safely for practical applications. [ABSTRACT FROM AUTHOR]

The development of a spider silk-manufacturing process is of great interest. However, there are serious problems with natural manufacturing through spider farming, and standard recombinant protein production platforms have provided limited progress due to their inability to assemble spider silk proteins into fibers. Thus, we used piggyBac vectors to create transgenic silkworms encoding chimeric silkworm/spider silk proteins. The silk fibers produced by these animals were composite materials that included chimeric silkworm/spider silk proteins integrated in an extremely stable manner. Furthermore, these composite fibers were, on average, tougher than the parental silkworm silk fibers and as tough as native dragline spider silk fibers. These results demonstrate that silkworms can be engineered to manufacture composite silk fibers containing stably integrated spider silk protein sequences, which significantly improve the overall mechanical properties of the parental silkworm silk fibers. [ABSTRACT FROM AUTHOR]

Genetically modified Metarhizium spp represent a major new arsenal for combating insect pests and insect-borne diseases. However, for these tools to be used safely and effectively, we need a much better understanding of their evolutionary potential and invasion ecology. In order to model natural as well as anthropogenic dispersal scenarios, we investigated evolutionary processes in a green fluorescent protein tagged strain of Metarhizium robertsii following transfer from a semitropical to a temperate soil community. Adaptive changes occurred over four years despite recurrent genetic bottlenecks and lack of recombination with locally well adapted strains. By coupling microarray-based functional analysis with DNA hybridizations we determined that expression of cell wall and stress response genes evolved at an accelerated rate in multiple replicates, whereas virulence determinants, transposons, and chromosome structure were unaltered. The mutable genes were enriched for TATA boxes possibly because they are larger mutational targets. In further field trials, we showed that the new mutations increased the fitness of M. robertsii in the new range by enhancing saprophytic associations, and these benefits were maintained in subsequent years. Consistent with selection being habitat rather than host specific, populations of an avirulent mutant cycled with seasons similarly to the wild type, whereas a mutant unable to adhere to plant roots showed a linear decrease in population. Our results provide a mechanistic basis for understanding postrelease adaptations, show that agents can be selected that lack gene flow and virulence evolution, and describe a means of genetically containing transgenic strains by disrupting the Mad2 gene. [ABSTRACT FROM AUTHOR]

Cells sense and respond to changes in oxygen concentration through gene regulatory processes that are fundamental to survival. Surprisingly, little is known about how anemia affects hypoxia signaling. Because nitric oxide synthases (NOSs) figure prominently in the cellular responses to acute hypoxia, we defined the effects of NOS deficiency in acute anemia. In contrast to endothelial NOS or inducible NOS deficiency, neuronal NOS (nNOS-/- mice demonstrated increased mortality during anemia. Unlike wildtype (WT) animals, anemia did not increase cardiac output (CO) or reduce systemic vascular resistance (SVR) in nNOS-/- mice. At the cellular level, anemia increased expression of HIF-1α protein and HIF-responsive mRNA levels (EPO, VEGF, GLUT1, PDK1) in the brain of WT, but not nNOS-/- mice, despite comparable reductions in tissue PO2. Paradoxically, nNOS-/- mice survived longer during hypoxia, retained the ability to regulate CO and SVR, and increased brain HIF-α protein levels and HIF-responsive mRNA transcripts. Real-time imaging of transgenic animals expressing a reporter HIF-α(ODD)-luciferase chimeric protein confirmed that nNOS was essential for anemia-mediated increases in HIF-α protein stability in vivo. S-nitrosylation effects the functional interaction between HIF and pVHL. We found that anemia led to nNOS-dependent S-nitrosylation of pVHL in vivo and, of interest, led to decreased expression of GSNO reductase. These findings identify nNOS effects on the HIF/ pVHL signaling pathway as critically important in the physiological responses to anemia in vivo and provide essential mechanistic insight into the differences between anemia and hypoxia. [ABSTRACT FROM AUTHOR]

Plants perceive environmental signals such as day length and temperature to determine optimal timing for the transition from vegetative to floral stages. Arabidopsis flowers under long-day conditions through the CONSTANS (CO)-FLOWERING LOCUS T (FT) regulatory module. It is thought that the environmental cues for photoperiodic control of flowering are initially perceived in the leaves. We have previously shown that GIGANTEA (GI) regulates the timing of CO expression, together with FLAVIN-BINDING, KELCH REPEAT, F BOX protein 1. Normally, CO and FT are expressed exclusively in vascular bundles, whereas GI is expressed in various tissues. To better elucidate the role of tissue-specific expression of GI in the flowering pathway, we established transgenic lines in which GI is expressed exclusively in mesophyll, vascular bundles, epidermis, shoot apical meristem, or root. We found that GI expressed in either mesophyll or vascular bundles rescues the late-flowering phenotype of the gi-2 loss-of-function mutant under both short-day and long-day conditions. Interestingly, GI expressed in mesophyll or vascular tissues increases FT expression without up-regulating CO expression under short-day conditions. Furthermore, we examined the interaction between GI and FT repressors in mesophyll. We found that GI can bind to three FT repressors: SHORT VEGETATIVE PHASE (SVP), TEMPRANILLO (TEM)1, and TEM2. Finally, our chromatin immunoprecipitation experiments showed that GI binds to FT promoter regions that are near the SVP binding sites. Taken together, our data further elucidate the multiple roles of GI in the regulation of flowering time. [ABSTRACT FROM AUTHOR]

Antivascular agents have become a standard of treatment for many malignancies. However, most of them target the VEGF pathway and lead to refractoriness. To improve the diversity of options for antivascular therapy, we applied a high-throughput screen for small molecules targeting cell adhesion. We then assayed the resulting antiadhesion hits in a transgenic zebrafish line with endothelial expression of EGFP (Tg(fli1:EGFP)y1) to identify nontoxic molecules with antivascular activity selective to neovasculature. This screen identified dehydro-α-lapachone (DAL), a natural plant product. We found that DAL inhibits vessel regeneration, interferes with vessel anastomosis, and limits plexus formation in zebrafish. Furthermore, DAL induces vascular pruning and growth delay in orthotopic mammary tumors in mice. We show that DAL targets cell adhesion by promoting ubiquitination of the Rho-GTPase Rac1, which is frequently up-regulated in many different cancers. [ABSTRACT FROM AUTHOR]

Extrinsic factors and the interactions of neurons with surrounding tissues are essential for almost every aspect of neuronal development. Here we describe a strategy of gene expression with an independent enhancer-driven cellular marker (GEEM) for studying roles of cell-cell interactions and extrinsic factors in the development of the Drosophila nervous system. Key to this strategy is robust expression of enhancer-driven transgenic markers in specific neurons. To this end, we have created vectors to achieve bright and even labeling of neuronal processes, easy cloning of enhancer elements, and efficient and flexible generation of transgenic animals. We provide examples of enhancer-driven membrane markers for specific neurons in both the peripheral and central nervous systems and their applications in the study of neuronal projections and connections in the Drosophila brain. We further applied GEEM to examine the wrapping of sensory neuron somas by glia during embryonic and larval stages, and neuron-glia interaction during dendrite pruning in live animals, leading to the discovery that glia play critical roles in the severing and degradation of proximal dendrites. The GEEM paradigm should be applicable to the studies of both cell-autonomous and nonautonomous regulations of any cell type. [ABSTRACT FROM AUTHOR]

Mutations in voltage-gated ion channels are responsible for several types of epilepsy. Genetic epilepsies often exhibit variable severity in individuals with the same mutation, which may be due to variation in genetic modifiers. The Scn2aQ54 transgenic mouse model has a sodium channel mutation and exhibits epilepsy with strain-dependent severity. We previously mapped modifier loci that influence Scn2aQ54 phenotype severity and identified Kcnv2, encoding the voltage-gated potassium channel subunit Kv8.2, as a candidate modifier. In this study, we demonstrate a threefold increase in hippocampal Kcnv2 expression associated with more severe epilepsy. In vivo exacerbation of the phenotype by Kcnv2 transgenes supports its identification as an epilepsy modifier. The contribution of KCNV2 to human epilepsy susceptibility is supported by identification of two nonsynonymous variants in epilepsy patients that alter function of Kv2.1/Kv8.2 heterotetrameric potassium channels. Our results demonstrate that altered potassium subunit function influences epilepsy susceptibility and implicate Kcnv2 as an epilepsy gene. [ABSTRACT FROM AUTHOR]

TAR DNA-binding protein-43 (TDP-43), a DNA/RNA-binding protein involved in RNA transcription and splicing, has been associated with the pathophysiology of neurodegenerative diseases, including ALS. However, the function of TDP-43 in motor neurons remains undefined. Here we use both gain- and loss-of-function approaches to determine roles of TDP-43 in motor neurons. Mice expressing human TDP-43 in neurons exhibited growth retardation and premature death that are characterized by abnormal intranuclear inclusions composed of TDP-43 and fused in sarcoma/translocated in liposarcoma (FUS/TLS), and massive accumulation of mitochondria in TDP-43-negative cytoplasmic inclusions in motor neurons, lack of mitochondria in motor axon terminals, and immature neuromuscular junctions. Whereas an elevated level of TDP-43 disrupts the normal nuclear distribution of survival motor neuron (SMN)-associated Gemini of coiled bodies (GEMs) in motor neurons, its absence prevents the formation of GEMs in the nuclei of these cells. Moreover, transcriptome-wide deep sequencing analysis revealed that a decrease in abundance of neurofilament transcripts contributed to the reduction of caliber of motor axons in TDP-43 mice. In concert, our findings indicate that TDP-43 participates in pathways critical for motor neuron physiology, including those that regulate the normal distributions of SMN-associated GEMs in the nucleus and mitochondria in the cytoplasm. [ABSTRACT FROM AUTHOR]

Synaptic vesicle fusion at many synapses has been kinetically sep- arated into two distinct Ca2-dependent temporal components consisting of a rapid synchronous phase followed by a slower asyn- chronous component. Mutations in the synaptic vesicle Ca2 sensor Synaptotagmin 1 (Syt 1) reduce synchronous neurotransmission while enhancing the slower asynchronous phase of release. Syt 1 regulation of vesicle fusion requires interactions mediated by its tandem cytoplasmic C2 domains (C2A and C2B). Although Ca2* bind- ing by Syt 1 is predicted to drive synchronous release, it is unknown if Ca2 interactions with either C2 domain is required for suppres- sion of asynchronous release. To determine if Ca2 binding by Syt 1 regulates these two phases of release independently, we performed electrophysiological analysis of transgenically expressed Syt 1 mu- tated at Ca2 binding sites in C2A or C2B in the background of Dro- sophila Syt 1-null mutants. Transgenic animals expressing mutations that disrupt Ca2 binding to C2A fully restored the synchronous phase of neurotransmitter release, whereas the asynchronous component was not suppressed. In contrast, rescue with Ca2-binding mutants in C2B displayed little rescue of the synchronous release component, but reduced asynchronous release. These results suggest that the tandem (2 domains of Syt 1 play independent roles in neurotrans- mission, as Ca2 binding to C2A suppresses asynchronous release, whereas Ca2 binding to C2B mediates synchronous fusion. [ABSTRACT FROM AUTHOR]

At present, genetically modified rats have not been generated from ES cells because stable ES cells and a suitable injection method are not available. To monitor the pluripotency of rat ES cells, we generated Oct4-Venus transgenic (Tg) rats via a conventional method, in which Venus is expressed by the Oct4 promoter/en- hancer. This monitoring system enabled us to define a significant condition of culture to establish authentic rat ES cells based on a combination of 20% FBS and cell signaling inhibitors for Rho- associated kinase. mitogen-activated protein kinase, TGF-β, and glycogen synthase kinase-3. The rat ES cells expressed ES cell markers such as Oct4, Nanog, Sox2, and Rex1 and retained a normal karyotype. Embryoid bodies and teratomas were also produced from the rat ES cells. All six ES cell lines derived from three different rat strains successfully achieved germline transmission, which strongly depended on the presence of the inhibitors during the injection process. Most importantly, high-quality Tg rats possessing a correct transgene expression pattern were successfully generated via the selection of gene-manipulated ES cell clones through germ- line transmission. Our rat ES cells should be sufficiently able to receive gene targeting as well as Tg manipulation, thus providing valuable animal models for the study of human diseases. [ABSTRACT FROM AUTHOR]

It is commonly assumed but not proven that microRNAs (miRNAs) and their targets coevolve. Under this assumption. miRNAs and targets from different species may interact adversely, resulting in reduced fitness. However, the strength of the adverse interactions may not be detectable because even outright deletions of miRNAs often manifest only subtle fitness effects. We tested and measured the strength of heterospecific interactions by carrying out trans- genic experiments across Drosophila species by overexpressing the miR310s cluster of Drosophila melanogaster (Dm310s) and Dro- sophila pseudoobscura (Dp310s) in D. melanogaster. Flies overex- pressing the heterospecific Dp310s are only one-third as viable as those overexpressing the conspecific Dm310s. The viability effect is easily detectable in comparison to the effect of the deletion of miR3lOs. The number of genes significantly misexpressed under the influence of Dp310s is 3-10 times greater than under Dm310s. Importantly, the numbers of predicted targets are similar between them. Expression analysis of the predicted target genes suggests that miRNAs may sometimes function to buffer fluctuations in the transcriptome output. After the buffering function has evolved, heterospecific combinations may cause adverse effects. [ABSTRACT FROM AUTHOR]

Male moths are endowed with odorant receptors (ORs) to detect species-specific sex pheromones with remarkable sensitivity and selectivity. We serendipitously discovered that an endogenous OR in the fruit fly, Drosophila melanogaster, is highly sensitive to the sex pheromone of the silkworm moth, bombykol. Intriguingly, the fruit fly detectors are more sensitive than the receptors of the silkworm moth, although its ecological significance is unknown. By expression in the "empty neuron" system, we identified the fruit fly bombykol-sensitive OR as DmelOR7a (= DmOR7a). The profiles of this receptor in response to bombykol in the native sensilla (ab4) or expressed in the empty neuron system (ab3 sensilla) are indistinguishable. Both WT and transgenic flies responded with high sensitivity, in a dose-dependent manner, and with rapid signal termination. In contrast, the same empty neuron expressing the moth bombykol receptor, BmorOR1, demonstrated low sensitivity and slow signal inactivation. When expressed in the trichoid sensilla T1 of the fruit fly, the neuron housing BmorOR1 responded with sensitivity comparable to that of the native trichoid sensilla in the silkworm moth. By challenging the native bombykol receptor in the fruit fly with high doses of another odorant to which the receptor responds with the highest sensitivity, we demonstrate that slow signal termination is induced by overdose of a stimulus. As opposed to the empty neuron system in the basiconic sensilla, the structural, biochemical, and/or biophysical features of the sensilla make the T1 trichoid system of the fly a better surrogate for the moth receptor. [ABSTRACT FROM AUTHOR]

To overcome burden of mosquito-borne diseases, multiple control strategies are needed. Population replacement with genetically modified mosquitoes carrying antipathogen effector genes is one of the possible approaches for controlling disease transmission. However, transgenic mosquitoes with antipathogen phenotypes based on overexpression of a single type effector molecule are not efficient in interrupting pathogen transmission. Here, we show that co-overexpression of two antimicrobial peptides (AMP), Cecropin A, and Defensin A, in transgenic Aedes aegypti mosquitoes results in the cooperative antibacterial and antiPlasmodium action of these AMPs. The transgenic hybrid mosquitoes that overexpressed both Cecropin A and Defensin A under the control of the vitellogenin promoter exhibited an elevated resistance to Pseudomonas aeruginosa infection, indicating that these AMPs acted cooperatively against this pathogenic bacterium. In these mosquitoes infected with P. gallinaceum, the number of oocysts was dramatically reduced in midguts, and no sporozoites were found in their salivary glands when the mosquitoes were fed twice to reactivate transgenic AMP production. Infection experiments using the transgenic hybrid mosquitoes, followed by sequential feeding on naive chicken, and then naive wild-type mosquitoes showed that the Plasmodium transmission was completely blocked. This study suggests an approach in generating transgenic mosquitoes with antiPlasmodium refractory phenotype, which is coexpression of two or more effector molecules with cooperative action on the parasite. [ABSTRACT FROM AUTHOR]

Ducks and wild waterfowl perpetuate all strains of influenza viruses in nature. In their natural host, influenza viruses typically cause asymptomatic infection and little pathology. Ducks are often resistant to influenza viruses capable of killing chickens. Here, we show that the influenza virus sensor, RIG-I, is present in ducks and plays a role in clearing an influenza infection. We show evidence suggesting that RIG-I may be absent in chickens, providing a plausible explanation for their increased susceptibility to influenza viruses compared with ducks. RIG-I detects RNA ligands derived from uncapped viral transcripts and initiates the IFN response. In this study, we show that the chicken embryonic.fibroblast cell line, DF-1, cannot respond to a RIG-I ligand. However, transfection of duck RIG-I into DF-1 cells rescues the detection of ligand and induces IFN-β promoter activity. Additionally, DF-1 cells expressing duck RIG-I have an augmented IFN response resulting in decreased influenza replication after challenge with either low or highly pathogenic avian influenza virus. Implicating RIG-I in the antiviral response to an infection in vivo, we found that RIG-I expression is induced 200 fold, early in an innate immune response in ducks challenged with the H5N1 virus A/Vietnam/1203/04. Finding this natural disease resistance gene in ducks opens the possibility of increasing influenza resistance through creation of a transgenic chicken. [ABSTRACT FROM AUTHOR]

G protein-coupled receptors (GPCRs) are ubiquitous mediators of signaling of hormones, neurotransmitters, and sensing. The old dogma is that a one ligand/one receptor complex constitutes the functional unit of GPCR signaling. However, there is mounting evidence that some GPCRs form dimers or oligomers during their biosynthesis, activation, inactivation, and/or internalization. This evidence has been obtained exclusively from cell culture experiments, and proof for the physiological significance of GPCR di/ oligomerization in vivo is still missing. Using the mouse luteinizing hormone receptor (LHR) as a model GPCR, we demonstrate that transgenic mice coexpressing binding-deficient and signaling-deficient forms of LHR can reestablish normal LH actions through intermolecular functional complementation of the mutant receptors in the absence of functional wild-type receptors. These results provide compelling in vivo evidence for the physiological relevance of intermolecular cooperation in GPCR signaling. [ABSTRACT FROM AUTHOR]

Zebra finches are widely used for studying the basic biology of vocal learning. The inability to introduce genetic modifications in these animals has substantially limited studies on the molecular biology of this behavior, however. We used an HIV-based lentivirus to produce germline transgenic zebra finches. The lentivirus encoded the GFP regulated by the human ubiquitin-C promoter [Lois C. Hong EJ, Pease 5, Brown EJ, Baltimore D (2002) Science 295:868872]. which is active in a wide variety of cells. The virus was injected into the very early embryo (blastodisc stage) to target the primordial germllne cells that later give rise to sperm and eggs. A total of 265 fertile eggs were injected with virus, and 35 hatched (13%); 23 of these potential founders (FO) were bred, and three (13%) produced germline transgenic hatchlings that expressed the GFP protein (Fl). Two of these three founders (FO) have produced transgenic young at a rate of 12% and the third at a rate of 6%. Furthermore, two of the Fl generation transgenics have since reproduced, one having five offspring (all GFP positive) and the other four offsping (one GFP positive). [ABSTRACT FROM AUTHOR]

The pseudostratified epithelium of the mouse trachea and human airways contains a population of basal cells expressing Trp-63 (p63) and cytokeratins 5 (Krt5) and Krt14. Using a KRT5-CreERT2 transgenic mouse line for lineage tracing, we show that basal cells generate differentiated cells during postnatal growth and in the adult during both steady state and epithelial repair. We have fractionated mouse basal cells by FACS and identified 627 genes preferentially expressed in a basal subpopulation vs. non-BCs. Analysis reveals potential mechanisms regulating basal cells and allows comparison with other epithelial stem cells. To study basal cell behaviors, we describe a simple in vitro clonal sphere-forming assay in which mouse basal cells self-renew and generate luminal cells, including differentiated ciliated cells, in the absence of stroma. The transcriptional profile identified 2 cell-surface markers, ITGA6 and NGFR, which can be used in combination to purify human lung basal cells by FACS. Like those from the mouse trachea, human airway basal cells both self-renew and generate luminal daughters in the sphere-forming assay. [ABSTRACT FROM AUTHOR]

\Alterations in cellular cholesterol synthesis or content in cultured neurons affect the cleavage of amyloid precursor protein to amyloidogenic Aβ40 and Aβ42 peptides characteristic of Alzheimer's disease. To determine whether a decrease in cholesterol synthesis affects amyloid precursor protein processing in vivo, we crossed cholesterol 24-hydroxylase knockout mice, which exhibit a 50% reduction in brain sterol synthesis, with transgenic mice [B6.Cg-Tg(APPswe, PSEN1E9)85Dbo/J] that develop Alzheimer's disease- like pathology. Amyloid precursor protein expression and amyloid plaque deposition in the cortex and hippocampus of male and female Alzheimer's disease mice between the ages of 3 to 15 months were similar in the presence and absence of cholesterol 24-hydroxylase. A modest but statistically significant decline in insoluble Aβ42 peptide levels was detected in the hippocampus of 12-month-old knockout/Alzheimer's disease males. The levels of insoluble Aβ40 and Aβ42 peptides in 15-month-old knockout/ Alzheimer's disease females were also reduced slightly. Although amyloid plaque accumulation did not affect brain sterol or fatty acid synthesis rates in 24-hydroxylase WT or knockout mice, loss of one or both cholesterol 24-hydroxylase alleles increased longevity in Alzheimer's disease mice. These studies suggest that reducing de novo cholesterol synthesis in the brain will not substantially alter the course of Alzheimer's disease, but may confer a survival advantage. [ABSTRACT FROM AUTHOR]

From a large-scale screen using splicing microarrays and RT-PCR, we identified 63 alternative splicing (AS) events that are coordinated in 3 distinct temporal patterns during mouse heart development. More than half of these splicing transitions are evolutionarily conserved between mouse and chicken. Computational analysis of the introns flanking these splicing events identified enriched and conserved motifs including binding sites for CUGBP and ETR-3-like factors (CELF), muscleblind-like (MBNL) and Fox proteins. We show that CELF proteins are down-regulated >10-fold during heart development, and MBNL1 protein is concomitantly up-regulated nearly 4-fold. Using transgenic and knockout mice, we show that reproducing the embryonic expression patterns for CUGBP1 and MBNL1 in adult heart induces the embryonic splicing patterns for more than half of the developmentally regulated AS transitions. These findings indicate that CELF and MBNL proteins are determinative for a large subset of splicing transitions that occur during postnatal heart development. [ABSTRACT FROM AUTHOR]

Currently, tools to generate loss-of-function mutations in rats are limited. Therefore, we have developed a lentiviral single-vector system for the temporal control of ubiquitous shRNA expression. Here, we report transgenic rats carrying an insulin receptor-specific shRNA transcribed from a regulatable promoter and identified by concomitant EGFP expression. In the absence of the inducer doxycycline (Dox), we observed no siRNA expression. However, Dox treatment at very low concentrations led to a rapid induction of the siRNA and ablation of INSR protein expression. As anticipated, blood glucose levels increased, whereas insulin signaling and glucose regulation were impaired. Importantly, this phenotype was reversible (i.e., discontinuation of Dox treatment led to INSR re-expression and remission of diabetes symptoms). The lentiviral system offers a simple tool for reversible gene ablation in the rat and can be used for other species that cannot be manipulated by conventional recombination techniques [ABSTRACT FROM AUTHOR]

Mutation of the RB-i and p53 tumor suppressors is associated with the development of human osteosarcoma. With the goal of generating a mouse model of this disease, we used conditional and transgenic mouse strains to inactivate Rb and/or p53 specifically in osteoblast precursors. The resulting Rb;p53 double mutant (DKO) animals are viable but develop early onset osteosarcomas with complete penetrance. These tumors display many of the characteristics of human osteosarcomas, including being highly metastatic. We established cell lines from the DKO osteosarcomas to further investigate their properties. These immortalized cell lines are highly proliferative and they retain their tumorigenic potential, as judged by their ability to form metastatic tumors in immunocompromised mice. Moreover, they can be induced to differentiate and, depending on the inductive signal, will adopt either the osteogenic or adipogenic fate. Consistent with this multipotency, a significant portion of these tumor cells express Sca-1, a marker that is typically associated with stem cells/uncommitted progenitors. By assaying sorted cells in transplant assays, we demonstrate that the tumorigenicity of the osteosarcoma cell lines correlates with the presence of the Sca-1 marker. Finally, we show that loss of Rb and p53 in Sca-1-positive mesenchymal stem/progenitor cells is sufficient to yield transformed cells that can initiate osteosarcoma formation in vivo. o [ABSTRACT FROM AUTHOR]

To determine whether adipocyte storage capacity influences the onset and severity of type 2 diabetes and other components of the metabolic syndrome, we made normal and db/db mice resistant to obesity by overexpressing leptin receptor-b on the aP2-Lepr-b promoter. On a 4% diet, these mice have no phenotype, but on a 60% fat diet, they resist diet-induced obesity because constitutive adipocyte-specific overexpression of Lepr-b prevents obesity via the antilipogenic autocrine/paracrine action of leptin on adipocytes. After 8 months on the same 60% fat diet, body fat of transgenic mice was 70% below WT controls. Cardiac and liver fat was elevated in the transgenics, and their hyperinsulinemia was more marked, suggesting greater insulin resistance. The aP2- Lepr-b transgene also prevented obesity in db/db mice; at 10 weeks of age their body fat was half that of the db/db mice. This lack of obesity was attributable to reduced expression of sterol regulatory element binding proteinic and its target lipogenic enzymes in adipose tissue and a 6-fold increase in Pref-1 mRNA. Severe diabetes was present in transgenics at 4 weeks of age, 10 weeks before db/db controls. Echocardiographic evidence of cardiomyopathy appeared at 10 weeks, weeks before the db/db mice. Histologically, loss of β cells and myocardial fibrosis was present in the transgenic group at least 6 weeks before the db/db mice. These results suggest that the expression level of genes that regulate the adipogenic response to overnutrition profoundly influences the age of onset and severity of diet-induced type 2 diabetes and co-morbidities. [ABSTRACT FROM AUTHOR]

Kuru provides our principal experience of an epidemic human prion disease and primarily affected the Fore linguistic group of the Eastern Highlands of Papua New Guinea. Kuru was transmitted by the practice of consuming dead relatives as a mark of respect and mourning (transumption). To date, detailed information of the prion strain type propagated in kuru has been lacking. Here, we directly compare the transmission properties of kuru prions with sporadic, iatrogenic, and variant Creutzfeldt-Jakob disease (CJD) prions in Prnp-null transgenic mice expressing human prion protein and in wild-type mice. Molecular and neuropathological data from these transmissions show that kuru prions are distinct from variant CJD and have transmission properties equivalent to those of classical (sporadic) CJD prions. These findings are consistent with the hypothesis that kuru originated from chance consumption of an individual with sporadic CJD. [ABSTRACT FROM AUTHOR]

Transient receptor potential (TRP) channels found in animals, protists, and fungi are primary chemo-, thermo-, or mechanosensors. Current research emphasizes the characteristics of individual channels in each animal TRP subfamily but not the mechanisms common across sub-families. A forward genetic screen of the TrpY1, the yeast TRP channel, recovered gain-of-function (GOF) mutations with phenotype in vivo and in vitro. Single-channel patch-clamp analyses of these GOF-mutant channels show prominent aberrations in open probability and channel kinetics. These mutations revealed functionally important aromatic amino acid residues in four locations: at the intracellular end of the fifth transmembrane helix (TM5), at both ends of TM6, and at the immediate extension of TM6. These aromatics have counterparts in most TRP subfamilies. The one in TM5 (F380L) aligns precisely with an exceptional Drosophila mutant allele (F550l) that causes constitutive activity in the canonical TRP channel, resulting in rapid and severe retinal degeneration beyond mere loss of photo-taxis. Thus, this phenylalanine maintains the balance of various functional states (conformations) of a channel for insect phototransduction as well as one for fungal mechanotransduction. This residue is among a small cluster of phenylalanines found in all known subfamilies of TRP channels. This unique case illustrates that GOF mutations can reveal structure-function principles that can be generalized across different TRP subfamilies. It appears that the con- served aromatics in the four locations have conserved functions in most TRP channels. The possible mechanistic roles of these aromatics and the further use of yeast genetics to dissect TRP channels are discussed. [ABSTRACT FROM AUTHOR]

FoxP3+CD4+ regulatory T cells (Tregs) play a key role in the maintenance of peripheral self-tolerance, and it has been suggested that diabetes-susceptible nonobese diabetic (NOD) mice are defective in the generation and numbers of Tregs. We found thymic selection of Tregs to be under genetic control. Fetal thymic organ cultures on the NOD background required 3-to 10-fold more antigen than corresponding cultures on the B6 background for optimal induction of Tregs, but once the threshold for induction was reached the NOD background yielded close to 10-fold more Tregs. This increased selection of Tregs was also found in non- transgenic NOD mice in fetal through adult stages. This trait did not map to the MHC, idd3, or the chromosome 3 (Chr3) regions that control clonal deletion, but mainly to two regions on Chrl and Chril. Thus, NOD mice do not have a global defect in the generation or maintenance of Tregs; if anything, they show the opposite. [ABSTRACT FROM AUTHOR]

The existence of vast regulatory networks mediated by microRNAs (miRNAs) suggests broad potential for miRNA dysfunction to contribute to disease. However, relatively few miRNA-target interactions are likely to make detectable contributions to phenotype, and effective strategies to identify these few interactions are currently wanting. We hypothesized that signaling cascades represent critical points of susceptibility to miRNA dysfunction, and we developed a strategy to test this theory by using quantitative cell-based screens. Here we report a screen for miRNAs that affect the Wingless (Wg) pathway, a conserved pathway that regulates growth and tissue specification. This process identified ectopic miR-315 as a potent and specific activator of Wg signaling, an activity that we corroborated in transgenic animals. This miR-315 activity was mediated by direct inhibition of Axin and Notum, which encode essential, negatively acting components of the Wg pathway. Genetic interaction tests substantiated both of these genes as key functional targets of miR-315. The ability of ectopic miR-315 to activate Wg signaling was not a trivial consequence of predicted miRNA-target relationships because other mIRNA5 with conserved sites in the Axin 3′ UTR neither activated Wg outputs nor inhibited an Axin sensor. In summary, activity-based screening can selectively identify miRNAs whose deregulation can lead to interpretable phenotypic consequences. [ABSTRACT FROM AUTHOR]

Huntington's disease (HD) is a progressive neurodegenerative disease caused by a glutamine expansion within huntingtin protein. The exact pathological mechanisms determining disease onset and progression remain unclear. However, aggregates of insoluble mutant huntingtin (mhtt), a hallmark of HD, are readily detected within neurons in HD brain. Although aggregated polyglutamines may not be inherently toxic, they constitute a biomarker for mutant huntingtin useful for developing therapeutics. We previously reported that the small molecule, C2-8, inhibits polyglutamine aggregation in cell culture and brain slices and rescues degeneration of photoreceptors in a Drosophila model of HD. In this study, we assessed the therapeutic potential of C2-8 in the R6/2 mouse model of HD, which has been used to provide proof-of-concept data in considering whether to advance therapies to human HD. We show that, at nontoxic doses, C2-8 penetrates the blood-brain barrier and is present in brain at a high concentration. C2-8-treated mice showed improved motor performance and reduced neuronal atrophy and had smaller huntingtin aggregates. There have been no prior drug-like, non-toxic, brain-penetrable aggregation inhibitors to arise from cell-based high-throughput screens for reducing huntingtin aggregation that is efficacious in preclinical in vivo models. C2-8 provides an essential tool to help elucidate mechanisms of neurodegeneration in HD and a therapeutic lead for further optimization and development. [ABSTRACT FROM AUTHOR]

Although the role of p21Waf1/Cip1 gene expression is well documented in various cell culture studies, its in vivo roles are poorly understood. To gain further insight into the role of p21Waf1/Cip1 gene expression in vivo, we attempted to visualize the dynamics of p21Waf1/Cip1 gene expression in living animals. In this study, we established a transgenic mice line (p21-p-luc) expressing the firefly luciferase under the control of the p21Waf1/Cip1 gene promoter. In conjunction with a noninvasive bioluminescent imaging technique, p21-p-luc mice enabled us to monitor the endogenous p21Waf1/Cip1 gene expression in vivo. By monitoring and quantifying the p21Waf1/Cip1 gene expression repeatedly in the same mouse throughout its entire lifespan, we were able to unveil the dynamics of p21Waf1/Cip1 gene expression in the aging process. We also applied this system to chemically induced skin carcinogenesis and found that the levels of p21Waf1/Cip1 gene expression rise dramatically in benign skin papillomas, suggesting that p21Waf1/Cip1 plays a preventative role(s) in skin tumor formation. Surprisingly, moreover, we found that the level of p21Waf1/Cip1 expression strikingly increased in the hair bulb and oscillated with a 3-week period correlating with hair follicle cycle progression. Notably, this was accompanied by the expression of p63 but not p53. This approach, together with the analysis of p21Waf1/Cip1 knockout mice, has uncovered a novel role for the p21Waf1/Cip1 gene in hair development. These data illustrate the unique utility of bioluminescence imaging in advancing our understanding of the timing and, hence, likely roles of specific gene expression in higher eukaryotes. [ABSTRACT FROM AUTHOR]

The factors involved in the differentiation of memory CD4 T cells from naïve precursors are poorly understood. We developed a system to examine the effect of increased competition for antigen by CD4 T cells on the generation of memory in response to infection with a recombinant vesicular stomatitis virus. Competition was initially regulated by increasing the precursor frequency of adoptively transferred naïve T cell antigen receptor transgenic CD4 T cells. Despite robust proliferation at high precursor frequencies, memory CD4 T cells did not develop, whereas decreasing the input number of naïve CD4 T cells promoted memory development after infection. The lack of memory development was linked to reduced blastogenesis and poor effector cell induction, but not to initial recruitment or proliferation of antigen-specific CD4 T cells. To prove that availability of antigen alone could regulate memory CD4 T cell development, we used treatment with an mAb specific for the epitope recognized by the transferred CD4 T cells. At high doses, this mAb effectively inhibited the antigen-specific CD4 T cell response. However, at a very low dose of mAb, primary CD4 T cell expansion was unaffected, although memory development was dramatically reduced. Moreover, the induction of effector function was concomitantly inhibited. Thus, competition for antigen during CD4 T cell priming is a major contributing factor to the development of the memory CD4 T cell pool. [ABSTRACT FROM AUTHOR]

Dangerous organophosphorus (OP) compounds have been used as insecticides in agriculture and in chemical warfare. Because exposure to OP could create a danger for humans in the future, butyrylcholinesterase (BChE) has been developed for prophylaxis to these chemicals. Because it is impractical to obtain sufficient quantities of plasma BChE to treat humans exposed to OP agents, the production of recombinant BChE (rBChE) in milk of transgenic animals was investigated. Transgenic mice and goats were generated with human BChE cDNA under control of the goat β-casein promoter. Milk from transgenic animals contained 0.1-5 g/liter of active rBChE. The plasma half-life of PEGylated, goat-derived, purified rBChE in guinea pigs was 7-fold longer than non-PEGylated dimers. The rBChE from transgenic mice was inhibited by nerve agents at a 1:1 molar ratio. Transgenic goats produced active rBChE in milk sufficient for prophylaxis of humans at risk for exposure to OP agents. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is an age-dependent neurodegenerative disease that causes progressive cognitive impairment. The initiation and progression of AD has been linked to cholesterol metabolism and inflammation, processes that can be modulated by liver × receptors (LXRs). We show here that endogenous LXR signaling impacts the development of AD-related pathology. Genetic loss of either Lxrα or Lxrβ in APP/PS1 transgenic mice results in increased amyloid plaque load. LXRs regulate basal and inducible expression of key cholesterol homeostatic genes in the brain and act as potent inhibitors of inflammatory gene expression. Ligand activation of LXRs attenuates the inflammatory response of primary mixed glial cultures to fibrillar amyloid β peptide (fAβ) in a receptor-dependent manner. Furthermore, LXRs promote the capacity of microglia to maintain fAβ-stimulated phagocytosis in the setting of inflammation. These results identify endogenous LXR signaling as an important determinant of AD pathogenesis in mice. We propose that LXRs may be tractable targets for the treatment of AD due to their ability to modulate both lipid metabolic and inflammatory gene expression in the brain. [ABSTRACT FROM AUTHOR]

Many therapeutic leads fail to advance clinically because of bioavailability, selectivity, and formulation problems. Molecular transporters can be used to address these problems. Molecular transporter conjugates of otherwise poorly soluble or poorly bioavailable drugs or probes exhibit excellent solubility in water and biological fluids and at the same time an enhanced ability to enter tissues and cells and with modification to do so selectively. For many conjugates, however, it is necessary to release the drug/probe cargo from the transporter after uptake to achieve activity. Here, we describe an imaging method that provides quantification of transporter conjugate uptake and cargo release in real-time in animal models. This method uses transgenic (luciferase) reporter mice and whole-body imaging, allowing noninvasive quantification of transporter conjugate uptake and probe (luciferin) release in real time. This process effectively emulates drug-conjugate delivery, drug release, and drug turnover by an intracellular target, providing a facile method to evaluate comparative uptake of new transporters and efficacy and selectivity of linker release as required for fundamental studies and therapeutic applications. [ABSTRACT FROM AUTHOR]

Mutants in the Big Blue transgenic mouse system show spontaneous clustered multiple mutations with unexpectedly high frequency. consistent with chronocoordinate events. We tested the prediction that the multiple mutations seen within the lad mutation target sometimes occur in the context of chronocoordinate multiple mutations spanning multiple kilobases (mutation showers). Additional sequencing of mutants was performed in regions immediately flanking the lad region (total of 10.7 kb). Nineteen additional mutations were found outside the lad region ("ectomutations") from 10 mutants containing two or more lad mutations, whereas only one ectomutation was found in 130 mutants with a single mutation (P < 0.0001). The mutation showers had an average of approximately one mutation per 3 kb. Four mutants showed closely spaced double mutations in the new sequence, and analysis of the spacing between these mutations revealed significant clustering (P = 0.0098). To determine the extent of the mutation showers, regions (8.5 kb total) remote from the lad region (≈16-17 kb away) were sequenced. Only two additional ectomutations were found in these remote regions, consistent with mutation showers that generally do not extend more than ≈30 kb. We conclude that mutation showers exist and that they constitute at least 0.2% and possibly 1% or more of mutational events observed in this system. The existence of mutation showers has implications for oncogenesis and evolution, raising the possibilities of "cancer in an instant" and "introns as sponges to reduce the deleterious impact of mutation showers." [ABSTRACT FROM AUTHOR]

The R120G mutation in the small heat shock-like protein αB-crystallin (CryABR120G) causes desmin-related myopathy (DRM), which is characterized by the formation of desmin- and CryAB-containing aggregates within muscle fibers. Mice with cardiac-specific overexpression of CryABR120G develop cardiomyopathy at 3 months and die at 6–7 months from heart failure (HF). Previous studies showed that overexpression of CryABR120G results in accumulation of preamyloid oligomer (PAO). PAO is considered to be the cytotoxic entity in many of the protein misfolding-based neurodegenerative diseases. On the basis of data from mouse models of neurodegenerative diseases showing that exercise or environmental enrichment reduces the amyloid oligomer level and improves cognitive ability, we hypothesized that CryABR120G-induced DRM would also respond favorably to prolonged voluntary exercise, reducing HF symptoms and rescuing the mice from premature death. Six months of voluntary exercise in CryABR120G animals resulted in 100% survival at a time when all unexercised mice had died. After 22 weeks of exercise, PAO levels were decreased by 47% compared with the unexercised CryABR120G control mice (P = 0.00001). Although CryABR120G expression led to decreased levels of the metallomembrane endopeptidase neprilysin, normal levels were maintained in the exercised CryABR120G mice, and in vitro loss-of-function and gain-of-function experiments using adenovirus-infected cardiomyocytes confirmed the importance of neprilysin in ameliorating PAO accumulation. The data demonstrate that voluntary exercise slows the progression to HF in the CryABRR120G DRM model and that PAO accumulation is mediated, at least in part, by decreased neprilysin activity. [ABSTRACT FROM AUTHOR]

Assisted reproductive technologies (ARTs) have now contributed to the birth of >3 million babies worldwide, but concerns remain regarding the safety of these methods. We have used a transgenic mouse model to examine the effects of ARTs on the frequency and spectrum of point mutations in midgestation mouse fetuses produced by either natural reproduction or various methods of ART, including preimplantation culture, embryo transfer, in vitro fertilization. intracytoplasmic sperm injection, and round spermatid injection. Our results show that there is no significant difference in the frequency or spectrum of de novo point mutations found in naturally conceived fetuses and fetuses produced by in vitro fertilization, intracytoplasmic sperm injection, or round spermatid injection. These results, based on analyses of a transgenic mouse system, indicate that with respect to maintenance of genetic integrity, ARTs appear to be safe. [ABSTRACT FROM AUTHOR]

Production of transgenic animals has raised concern regarding their potential ecological impact should they escape or be released to the natural environment. This concern has arisen mainly from research on laboratory-reared animals and theoretical modeling exercises. In this study, we used biocontained naturalized stream environments and conventional hatchery environments to show that differences in phenotype between transgenic and wild genotypes depend on rearing conditions and, critically, that such genotype-by-environment interactions may influence subsequent ecological effects in nature. Genetically wild and growth hormone transgenic coho salmon (Oncorhynchus kisutch) were reared from the fry stage under either standard hatchery conditions or under naturalized stream conditions. When reared under standard hatchery conditions, the transgenic fish grew almost three times longer than wild conspecifics and had (under simulated natural conditions) stronger predation effects on prey than wild genotypes (even after compensation for size differences). In contrast, when fish were reared under naturalized stream conditions, transgenic fish were only 20% longer than the wild fish, and the magnitude of difference in relative predation effects was much reduced. These data show that genotype-by-environment interactions can influence the relative phenotype of transgenic and wild-type organisms and that extrapolations of ecological consequences from phenotypes developed in the unnatural laboratory environment may lead to an overestimation or underestimation of ecological risk. Thus, for transgenic organisms that may not be released to nature, the establishment of a range of highly naturalized environments will be critical for acquiring reliable experimental data to be used in risk assessments. [ABSTRACT FROM AUTHOR]

Gene transcription can be regulated by promoter competition when multiple promoters are available for a single enhancer. The converse, however, is largely undescribed. Here we report an occurrence of several enhancers competing for one promoter (enhancer interference) and propose its underlying mechanism. The promoter targeting sequence from the Abdominal-B locus of the Drosophila bithorax complex overcomes the enhancer-blocking activity of insulators in transgenic embryos. It may facilitate the long-range enhancer-promoter communication in Abdominal-B by bypassing insulator elements such as Frontabdominal-7 and Front- abdominal-8. In transgenic embryos, the anti-insulator activity allowed both an insulator-blocked enhancer and a nonblocked enhancer to contact the same promoter. We found that these two enhancers exhibited mutual inhibition or mutual exclusion in cells where both are transcriptionally active. This enhancer interference occurred at the level of enhancer-promoter communication. It occurred only between enhancers on opposite sides of an insulator and depended on enhancer activity. We hypothesize that enhancer interference limits the interaction of the Abdominal-B promoter to the enhancer(s) from only one regulatory domain in a specific abdominal segment. [ABSTRACT FROM AUTHOR]