ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 3 2008
Richard N. Winn
Abstract To address the need for improved approaches to study mutations transmitted to progeny from mutagen-exposed parents, we evaluated , transgenic medaka, a small fish that carries the cII mutation target gene, as a new model for germ cell mutagenesis. Mutations in the cII gene in progeny derived from ethyl-nitrosourea (ENU)-exposed males were readily detected. Frequencies of mutant offspring, proportions of mosaic or whole body mutant offspring, and mutational spectra differed according to germ cell stage exposed to ENU. Postmeiotic germ cells (spermatozoa/late spermatids) generated a higher frequency of mutant offspring (11%) compared to premeiotic germ cells (3.5%). Individuals with cII mutant frequencies (MF) elevated more than threefold above the spontaneous MF (3 × 10,5) in the range of 10,4 to 10,3 were mosaic mutant offspring, whereas those with MFs approaching 1 × 10,2 were whole body mutant offspring. Mosaic mutant offspring comprised the majority of mutant offspring derived from postmeiotic germ cells, and unexpectedly, from spermatogonial stem cells. Mutational spectra comprised of two different mutations, but at identical sites were unusual and characteristic of delayed mutations, in which fixation of a second mutation was delayed following fertilization. Delayed mutations and prevalence of mosaic mutant offspring add to growing evidence that implicates germ cells in mediating processes postfertilization that contribute to genomic instability in progeny. This model provides an efficient and sensitive approach to assess germ cell mutations, expands opportunities to increase understanding of fundamental mechanisms of mutagenesis, and provides a means for improved assessment of potential genetic health risks. Environ. Mol. Mutagen., 2008. © 2008 Wiley-Liss, Inc. [source]

Transgenic and knock-out mouse pups: the growing need for behavioral analysis

GENES, BRAIN AND BEHAVIOR, Issue 3 2002
I. Branchi
Few laboratories working with transgenic and knock-out mice analyze the neurobehavioral consequences of genetic manipulation in early ontogeny. However, the study of behavioral endpoints during the early postnatal period in genetically modified mice is important not only to assess possible developmental abnormalities, but also to better understand and disentangle the effects of genetic manipulations in adulthood. We propose that the assessment of neurobehavioral development represents an appropriate strategy to identify possible compensatory and/or unexpected effects. Nowadays, a large number of experimental protocols that take into account the practical constraints imposed by the peculiar physiological and behavioral responses of an immature subject are available to assess the neurobehavioral profile of developing mice. While this knowledge should be applied to the field of transgenic and knock-out mice in general, it should be recommended, in particular, for the study of mouse models of neurodevelopmental disorders. [source]

Role of endogenous regucalcin in transgenic rats: Suppression of kidney cortex cytosolic protein phosphatase activity and enhancement of heart muscle microsomal Ca2+ -ATPase activity

JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2002
Masayoshi Yamaguchi
Abstract Rats were generated by pronuclear injection of the transgene with a cDNA construct encoding rat regucalcin that is a regulatory protein of Ca2+ signaling. Transgenic (TG) founders were fertile, transmitted the transgene at the expected frequency, and bred to homozygote. Western analysis of the cytosol prepared from the tissue of TG female rats (5-week-old) showed a remarkable expression of regucalcin (3.3 kDa) protein in the liver, kidney cortex, heart, lung, stomach, brain, spleen, muscle, colon, and duodenum. Regucalcin expression of TG male rats was seen in the liver, kidney cortex, heart, and lung. In wild-type (wt) male and female rats, regucalcin was mainly present in the liver and kidney cortex. Regucalcin inhibited protein phosphatase activity in rat kidney cortex cytosol and activated Ca2+ -ATPase activity in rat heart muscle microsomes. The suppressive effect of regucalcin on protein phosphatase activity was significantly enhanced in the cytosol of kidney cortex of TG male and female rats as compared with those of wt rats. Likewise, heart muscle microsomal Ca2+ -ATPase activity was significantly enhanced in TG rats. The changes in their enzyme's activities in TG rats were completely abolished in the presence of anti-regucalcin monoclonal antibody (100 ng/ml) in the enzyme reaction mixture. Moreover, the body weight of TG female rats was significantly lowered as compared with that of wt rats. Serum inorganic phosphorus concentration was significantly increased in TG male and female rats, while serum calcium, glucose, triglyceride, free cholesterol, albumin, and urea nitrogen concentrations were not significantly altered in TG rats. Regucalcin TG rats should be a useful model to define a regulatory role of endogenous regucalcin in the tissues in vivo. J. Cell. Biochem. 86: 520,529, 2002. © 2002 Wiley-Liss, Inc. [source]

Purification and characterization of recombinant human erythropoietin from milk of transgenic pigs

JOURNAL OF CHEMICAL TECHNOLOGY & BIOTECHNOLOGY, Issue 5 2009
Eun Gyo Lee
Abstract BACKGROUND: Human erythropoietin (hEPO), a hydrophobic acidic glycoprotein responsible for the regulation of red blood cell production in mammals, is used for the treatment of anemia. In general, the purification of transgenic animal-derived therapeutic proteins is not easy due to their low titer concentrations and abundant contaminant proteins. For the first time, here the purification and characterization of rhEPO from the milk of transgenic pigs are described. RESULTS: The rhEPO was purified by heparin chromatography, reverse-phase chromatography, and gel filtration chromatography, resulting in a 16.5% yield and > 98% purity. The rhEPO purified from the milk of transgenic pigs contained less acidic isoforms and was underglycosylated in contrast to CHO-derived rhEPO. Cell proliferation of the F-36/EPO-dependent cell line was proportional to the dose of transgenic pig-derived rhEPO. CONCLUSION: Transgenic pig-derived rhEPO with high purity was achieved after three-step chromatography following two-step precipitation. The transgenic pig-derived rhEPO was demonstrated to have comparable potency with CHO-derived rhEPO. Transgenic pig-derived rhEPO may not be therapeutically feasible because of different glycosylation, and thus further studies are required to elucidate the effect of this aberrant glycosylation on the biological activity and stability in vivo. Copyright © 2008 Society of Chemical Industry [source]

Ex ante analysis of the benefits of transgenic drought tolerance research on cereal crops in low-income countries

AGRICULTURAL ECONOMICS, Issue 4 2009
Genti Kostandini
Drought tolerance; Transgenic; Research benefits; Intellectual property rights Abstract This article develops a framework to examine the ex ante benefits of transgenic research on drought in eight low-income countries, including the benefits to producers and consumers from farm income stabilization and the potential magnitude of private sector profits from intellectual property rights (IPRs). The framework employs country-specific agroecological,drought risk zones and considers both yield increases and yield variance reductions when estimating producer and consumer benefits from research. Benefits from yield variance reductions are shown to be an important component of aggregate drought research benefits, representing 40% of total benefits across the eight countries. Further, estimated annual benefits of US$178 million to the private sector suggest that significant incentives exist for participation in transgenic drought tolerance research. [source]

Analyses of Sexual Reproductive Success in Transgenic and/or Mutant Plants

JOURNAL OF INTEGRATIVE PLANT BIOLOGY, Issue 8 2009
Cristiane P. G. Calixto
The pistil, the female reproductive organ of plants, is a key player in the success of sexual plant reproduction. Ultimately, the production of fruits and seeds depends on the proper pistil development and function. Therefore, the identification and characterization of pistil expressed genes is essential for a better understanding and manipulation of the plant reproduction process. For studying the function of pistil expressed genes, transgenic and/or mutant plants for the genes of interest are used. The present article provides a review of methods already exploited to analyze sexual reproductive success. We intend to supply useful information and to guide future experiments in the study of genes affecting pistil development and function. [source]

A viral PAMP double-stranded RNA induces allergen-specific Th17 cell response in the airways which is dependent on VEGF and IL-6

ALLERGY, Issue 10 2010
J.-P. Choi
To cite this article: Choi J-P, Kim Y-S, Tae Y-M, Choi E-J, Hong B-S, Jeon SG, Gho YS, Zhu Z, Kim Y-K. A viral PAMP double-stranded RNA induces allergen-specific Th17 cell response in the airways which is dependent on VEGF and IL-6. Allergy 2010; 65: 1322,1330. Abstract Background:, Innate immune response by a viral pathogen-associated molecular pattern dsRNA modulates the subsequent development of adaptive immune responses. Although virus-associated asthma is characterized by noneosinophilic inflammation, the role of Th17 cell response in the development of virus-associated asthma is still unknown. Objective:, To evaluate the role of the Th17 cell response and its underlying polarizing mechanisms in the development of an experimental virus-associated asthma. Methods:, An experimental virus-associated asthma was created via airway sensitization with ovalbumin (OVA, 75 ,g) and a low (0.1 ,g) or a high (10 ,g) doses of synthetic dsRNA [polyinosine,polycytidylic acid; poly(I:C)]. Transgenic (IL-17-, IL-6-deficient mice) and pharmacologic [a vascular endothelial growth factor receptor (VEGFR) inhibitor] approaches were used to evaluate the roles of Th17 cell responses. Results:, After cosensitization with OVA and low-dose poly(I:C), but not with high-dose poly(I:C), inflammation scores after allergen challenge were lower in IL-17-deficient mice than in wild-type (WT) mice. Moreover, inflammation enhanced by low-dose poly(I:C), but not by high-dose poly(I:C), was impaired in IL-6-deficient mice; this phenotype was accompanied by the down-regulation of IL-17 production from T cells from both lymph nodes and lung tissues. Airway exposure of low-dose poly(I:C) enhanced the production of VEGF and IL-6, and the production of IL-6 was blocked by treatment with a VEGFR inhibitor (SU5416). Moreover, the allergen-specific Th17 cell response and subsequent inflammation in the low-dose poly(I:C) model were impaired by the VEGFR inhibitor treatment during sensitization. Conclusions:, Airway exposure of low-level dsRNA induces an allergen-specific Th17 cell response, which is mainly dependent on VEGF and IL-6. [source]

Stable expression of AtGA2ox1 in a low-input turfgrass (Paspalum notatum Flugge) reduces bioactive gibberellin levels and improves turf quality under field conditions

PLANT BIOTECHNOLOGY JOURNAL, Issue 6 2007
Mrinalini Agharkar
Summary Bahiagrass (Paspalum notatum Flugge) is a prime candidate for molecular improvement of turf quality. Its persistence and low input characteristics made it the dominant utility turfgrass along highways in the south-eastern USA. However, the comparatively poor turf quality due to reduced turf density and prolific production of unsightly inflorescences currently limits the widespread use of bahiagrass as residential turf. Alteration of endogenous gibberellin (GA) levels by application of growth regulators or transgenic strategies has modified plant architecture in several crops. GA catabolizing AtGA2ox1 was subcloned under the control of the constitutive maize ubiquitin promoter and Nos 3'UTR. A minimal AtGA2ox1 expression cassette lacking vector backbone sequences was stably introduced into apomictic bahiagrass by biolistic gene transfer as confirmed by Southern blot analysis. Expression of AtGA2ox1 in bahiagrass as indicated by reverse transcription,polymerase chain reaction and Northern blot analysis resulted in a significant reduction of endogenous bioactive GA1 levels compared to wild type. Interestingly, transgenic plants displayed an increased number of vegetative tillers which correlated with the level of AtGA2ox1 expression and enhanced turf density under field conditions. This indicates that GAs contribute to signalling the outgrowth of axillary buds in this perennial grass. Transgenic plants also showed decreased stem length and delayed flowering under controlled environment and field conditions. Consequently, turf quality following weekly mowing was improved in transgenic bahiagrass. Transgene expression and phenotype were transmitted to seed progeny. Argentine bahiagrass produces seeds asexually by apomixis, which reduces the risk of unintended transgene dispersal by pollen and results in uniform progeny. [source]

Ultrastructural Morphometry of Mammary Gland in Transgenic and Non-transgenic Rabbits

ANATOMIA, HISTOLOGIA, EMBRYOLOGIA, Issue 6 2006
S. Dragin
Summary The mammary gland of transgenic animals has been used for the production of recombinant proteins of therapeutic and nutraceutical use. The objective of this study was to compare the ultrastructure of transgenic and non-transgenic rabbit mammary gland tissue. New Zealand White transgenic rabbits were obtained by breeding non-transgenic rabbits with transgenic founder rabbits containing a whey acidic protein-human factor VIII (WAP-hFVIII) transgene integrated into their genome. Samples of mammary gland tissue from lactating rabbit females were isolated by surgical procedures. These samples were examined by optical and electron microscopy and photographs were taken. Measurements of ultrastructural organelles were made from digital images of the mammary cells. No differences were found in the cellular structure of mammary tissue, but significant differences t(0.001) in the relative volume of mitochondria and vacuoles between transgenic and non-transgenic mammary gland epithelium were observed. [source]

Human prion strain selection in transgenic mice,

ANNALS OF NEUROLOGY, Issue 2 2010
Kurt Giles DPhil
Objective: Transgenic (Tg) mice expressing chimeras of mouse and human prion proteins (PrPs) have shorter incubation periods for Creutzfeldt-Jakob disease (CJD) prions than mice expressing full-length human PrP. Increasing the sequence similarity of the chimeric PrP to mouse PrP, by reverting human residues to mouse, resulted in a Tg line, denoted Tg22372, which was susceptible to sporadic (s) CJD prions in ,110 days. Methods: Mice expressing chimeric mouse/human PrP transgenes were produced. The mice were inoculated intracerebrally with extracts prepared from the brains of patients who died of CJD. Onset of neurological dysfunction marked the end of the incubation time. After sacrifice of the Tg mice, their brains were analyzed for PrPSc and neuropathological changes. Results: Reversion of 1 additional residue (M111V) resulted in a new Tg line, termed Tg1014, susceptible to sCJD prions in ,75 days. Tg1014 mice also have shorter incubation periods for variant (v) CJD prions, providing a more tractable model for studying this prion strain. Transmission of vCJD prions to Tg1014 mice resulted in 2 different strains, determined by neuropathology and biochemical analysis, which correlated with the length of the incubation time. One strain had the biochemical, neuropathological, and transmission characteristics, including longer incubation times, of the inoculated vCJD strain; the second strain produced a phenotype resembling that of sCJD prions including relatively shorter incubation periods. Mice with intermediate incubation periods for vCJD prions had a mixture of the 2 strains. Both strains were serially transmitted in Tg1014 mice, which led to further reduction in incubation periods. Conversion of vCJD-like to sCJD-like strains was favored in Tg1014 mice more than in the Tg22372 line. The single amino acid difference therefore appears to offer selective pressure for propagation of the sCJD-like strain. Interpretation: These 2 Tg mouse lines provide relatively rapid models to study human prion diseases as well as the evolution of human prion strains. ANN NEUROL 2010;68:151,161 [source]

A murine model of mixed connective tissue disease induced with U1 small nuclear RNP autoantigen

ARTHRITIS & RHEUMATISM, Issue 2 2006
Eric L. Greidinger
Objective To test whether immunizing mice with autoantigens closely linked to mixed connective tissue disease (MCTD) could induce an MCTD-like clinical syndrome distinguishable from systemic lupus erythematosus (SLE). Methods Transgenic and knockout C57BL/6-derived mice were immunized subcutaneously at age 8,12 weeks with U1,70-kd small nuclear RNP (70K) fusion protein along with either Freund's complete adjuvant (CFA) or U1 RNA. After 2 months, mice were killed and analyzed histologically and serologically. Results Immunization of C57BL/6-derived mice transgenic for human HLA,DR4 with 70K and either CFA or U1 RNA led to anti-70K antibodies in 62% of mice (21 of 34), and diversified anti-RNP immune responses. MCTD-like lung disease also developed in 50% of immunized mice (17 of 34), and anti-70K antibodies were strongly correlated with lung disease. CFA and U1 RNA were comparably able to induce this syndrome. Mice deficient in Toll-like receptor 3 (TLR-3) also developed this same syndrome when immunized with 70K and CFA. However, TLR-3,/, mice failed to develop MCTD-like lung disease when treated with 70K and U1 RNA. Rather, TLR-3,/, mice immunized with 70K and U1 RNA developed an autoimmune syndrome characterized by glomerulonephritis typical of SLE. Conclusion Exposure to 70K in an appropriate context is sufficient to induce autoimmunity and target organ injury consistent with MCTD. This system represents a new model of autoimmune interstitial lung disease, and establishes a closer link between anti-70K immunity and MCTD-like lung disease. Of note, changes in innate immune signaling can cause the same trigger to lead to the development of SLE-like nephritis rather than MCTD-like lung disease. [source]

Insights into the Pathogenesis of Hydrocephalus from Transgenic and Experimental Animal Models

BRAIN PATHOLOGY, Issue 3 2004
Leslie Crews
Hydrocephalus is a progressive brain disorder characterized by abnormalities in the flow of cerebrospinal fluid (CSF) and ventricular dilatation that leads to cerebral atrophy, and if left untreated, can be fatal. Genetic mutations, congenital malformations, infectious diseases, intracerebral hemorrhages and tumors are common conditions resulting in hydrocephalus. Although the causes of obstructive hydrocephalus are better understood, the mechanisms resulting in chronic, progressive communicating congenital and acquired hydrocephalus are less well understood. In this regard, recent studies in transgenic (tg) mice suggest that increased expression of cytokines such as TGF-,1 might play an important role by disrupting the vascular extracellular matrix (ECM) remodeling, promoting hemorrhages, and altering the reabsorption of CSF. In this context, the main objective of this manuscript is to provide an overview on the cellular and molecular mechanisms of hydrocephalus based on studies derived from tg and experimental animal models. [source]

Fibre types in skeletal muscle: a personal account

ACTA PHYSIOLOGICA, Issue 4 2010
S. Schiaffino
Abstract Muscle performance is in part dictated by muscle fibre composition and a precise understanding of the genetic and acquired factors that determine the fibre type profile is important in sport science, but is also relevant to neuromuscular diseases and to metabolic diseases, such as type 2 diabetes. The dissection of the signalling pathways that determine or modulate the muscle fibre phenotype has thus potential clinical significance. In this brief review, I examine the evolution of the notion of muscle fibre types, discuss some aspects related to species differences, point at problems in the interpretation of transgenic and knockout models and show how in vivo transfection can be used to identify regulatory factors involved in fibre type diversification, focusing on the calcineurin-nuclear factor of activated T cells (NFAT) pathway. [source]

Designing mouse behavioral tasks relevant to autistic-like behaviors,

DEVELOPMENTAL DISABILITIES RESEARCH REVIEW, Issue 4 2004
Jacqueline N. Crawley
Abstract The importance of genetic factors in autism has prompted the development of mutant mouse models to advance our understanding of biological mechanisms underlying autistic behaviors. Mouse models of human neuropsychiatric diseases are designed to optimize (1) face validity, i.e., resemblance to the human symptoms; (2) construct validity, i.e., similarity to the underlying causes of the disease; and (3) predictive validity, i.e., expected responses to treatments that are effective in the human disease. There is a growing need for mouse behavioral tasks with all three types of validity for modeling the symptoms of autism. We are in the process of designing a set of tasks with face validity for the defining features of autism: deficits in appropriate reciprocal social interactions, deficits in verbal social communication, and high levels of ritualistic repetitive behaviors. Social approach is tested in an automated three-chambered apparatus that offers the subject a choice between a familiar environment, a novel environment, and a novel environment containing a stranger mouse. Preference for social novelty is tested in the same apparatus, with a choice between the start chamber, the chamber containing a familiar mouse, and the chamber containing a stranger mouse. Social communication is evaluated by measuring the ultrasonic distress vocalizations emitted by infant mouse pups and the parental response of retrieving the pup to the nest. Resistance to change in ritualistic repetitive behaviors is modeled by forcing a change in habit, including reversal of the spatial location of a reinforcer in a T-maze task and in the Morris water maze. Mouse behavioral tasks that may model additional features of autism are discussed, including tasks relevant to anxiety, seizures, sleep disturbances, and sensory hypersensitivity. Applications of these tests include (1) behavioral phenotyping of transgenic and knockout mice with mutations in genes relevant to autism, (2) characterization of mutant mice derived from random chemical mutagenesis, (3) DNA microarray analyses of genes in inbred strains of mice that differ in social interaction, social communication and resistance to change in habit, and (4) evaluation of proposed therapeutics for the treatment of autism. Published 2004 Wiley-Liss, Inc. MRDD Research Reviews 2004;10:248,258. [source]

Promoter analysis of ventricular myosin heavy chain (vmhc) in zebrafish embryos

DEVELOPMENTAL DYNAMICS, Issue 7 2009
Daqing Jin
Abstract In zebrafish, ventricular myosin heavy chain (vmhc) gene is initially expressed at the anterior lateral mesoderm and thereafter its expression is restricted to the cardiac ventricle. The transcriptional control mechanisms in regulating chamber-specific expression of myosin heavy chains are not well defined. We isolated and analyzed zebrafish vmhc upstream region to examine the spatial and temporal regulation of vmhc using transgenic and transient expression techniques. Promoter deletion analyses defined a basal promoter region sufficient to drive vmhc expression in the ventricle and an upstream fragment necessary for repressing ectopic vmhc expression in the atrium. The transcriptional mechanism that prevents vmhc expression in the atrium is mediated through Nkx2.5 binding elements (NKE). We have further discovered that paired-related homeobox transcriptional factor 2 (Prx2/S8)-like binding elements are required for promoting vmhc expression, and Prrx1b, a Prx-related homeobox protein, participates in the regulation of vmhc expression with other transcriptional factors. Developmental Dynamics 238:1760,1767, 2009. © 2009 Wiley-Liss, Inc. [source]

Frontal nasal prominence expression driven by Tcfap2a relies on a conserved binding site for STAT proteins

DEVELOPMENTAL DYNAMICS, Issue 5 2006
Amy L. Donner
Abstract The AP-2 transcription factor family is linked with development of the head and limbs in both vertebrate and invertebrate species. Recent evidence has also implicated this gene family in the evolution of the neural crest in chordates, a critical step that allowed the development and elaboration of the vertebrate craniofacial skeleton. In mice, the inappropriate embryonic expression of one particular AP-2 gene, Tcfap2a, encoding AP-2,, results in multiple developmental abnormalities, including craniofacial and limb defects. Thus, Tcfap2a provides a valuable genetic resource to analyze the regulatory hierarchy responsible for the evolution and development of the face and limbs. Previous studies have identified a 2-kilobase intronic region of both the mouse and human AP-2, locus that directs expression of a linked LacZ transgene to the facial processes and the distal mesenchyme of the limb bud in transgenic mice. Further analysis identified two highly conserved regions of ,200,400 bp within this tissue-specific enhancer. We have now initiated a transgenic and biochemical analysis of the most important of these highly conserved regions. Our analysis indicates that although the sequences regulating face and limb expression have been integrated into a single enhancer, different cis -acting sequences ultimately control these two expression domains. Moreover, these studies demonstrate that a conserved STAT binding site provides a major contribution to the expression of Tcfap2a in the facial prominences. Developmental Dynamics 235:1358,1370, 2006. © 2006 Wiley-Liss, Inc. [source]

Loss of the Tg737 protein results in skeletal patterning defects

DEVELOPMENTAL DYNAMICS, Issue 1 2003
Qihong Zhang
Abstract Tg737 mutant mice exhibit pathologic conditions in numerous tissues along with skeletal patterning defects. Herein, we characterize the skeletal pathologic conditions and confirm a role for Tg737 in skeletal patterning through transgenic rescue. Analyses were conducted in both the hypomorphic Tg737orpk allele that results in duplication of digit one and in the null Tg737,2-3,Gal allele that is an embryonic lethal mutation exhibiting eight digits per limb. In early limb buds, Tg737 expression is detected throughout the mesenchyme becoming concentrated in precartilage condensations at later stages. In situ analyses indicate that the Tg737orpk mutant limb defects are not associated with changes in expression of Shh, Ihh, HoxD11,13, Patched, BMPs, or Glis. Likewise, in Tg737,2-3,Gal mutant embryos, there was no change in Shh expression. However, in both alleles, Fgf4 was ectopically expressed on the anterior apical ectodermal ridge. Collectively, the data argue for a dosage effect of Tg737 on the limb phenotypes and that the polydactyly is independent of Shh misexpression. Developmental Dynamics 227:78,90, 2003. © 2003 Wiley-Liss, Inc. [source]

Drosophila RSK negatively regulates bouton number at the neuromuscular junction

DEVELOPMENTAL NEUROBIOLOGY, Issue 4 2009
Matthias Fischer
Abstract Ribosomal S6 kinases (RSKs) are growth factor-regulated serine-threonine kinases participating in the RAS-ERK signaling pathway. RSKs have been implicated in memory formation in mammals and flies. To characterize the function of RSK at the synapse level, we investigated the effect of mutations in the rsk gene on the neuromuscular junction (NMJ) in Drosophila larvae. Immunostaining revealed transgenic expressed RSK in presynaptic regions. In mutants with a full deletion or an N-terminal partial deletion of rsk, an increased bouton number was found. Restoring the wild-type rsk function in the null mutant with a genomic rescue construct reverted the synaptic phenotype, and overexpression of the rsk -cDNA in motoneurons reduced bouton numbers. Based on previous observations that RSK interacts with the Drosophila ERK homologue Rolled, genetic epistasis experiments were performed with loss- and gain-of-function mutations in Rolled. These experiments provided evidence that RSK mediates its negative effect on bouton formation at the Drosophila NMJ by inhibition of ERK signaling. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009 [source]

Characterization and differentiation of diverse transgenic and nontransgenic soybean varieties from CE protein profiles

ELECTROPHORESIS, Issue 13 2007
Carmen García-Ruiz
Abstract Nowadays, soybeans are commercialized in a wide variety of colors and tones. Moreover, some pigmented seeds are being commercialized as soybeans while, on other occasions, these seeds are labeled as mung beans, azuki beans or soybean frijoles generating confusion on their identity. In this work, CE has been applied for the first time for the characterization and differentiation of different pigmented beans commercialized as soybeans. Other seeds commercialized as azuki, mung green soybeans or soybean frijoles were also analyzed. Borate buffer (at pH,8.5) containing 20% v/v ACN was used as the separation media and solution containing ACN/water (75:25 v/v) with 0.3% v/v acetic acid was used to solubilize the proteins from the samples. A 50,cm bare fused-silica capillary was employed for obtaining adequate separations in about 12,min. The CE protein pattern observed for yellow soybeans was different from that corresponding to green and red soybeans. The seeds commercialized as black soybean presented electropherograms identical or similar to those yielded by the yellow seeds with the exception of the sample labeled as black soybeans frijoles that presented a totally different pattern. In addition, CE protein profiles obtained for azuki and mung green soybeans were very similar to those corresponding to red soybeans and green soybeans, respectively. Finally, the CE method was also applied to differentiate transgenic and nontransgenic soybean varieties. Discriminant analysis, using several protein peak areas as variable, was used to successfully classify these samples. [source]

Life history of the bird cherry-oat aphid, Rhopalosiphum padi, on transgenic and non-transformed wheat challenged with Wheat streak mosaic virus

ENTOMOLOGIA EXPERIMENTALIS ET APPLICATA, Issue 1 2009
Edgardo S. Jiménez-Martínez
Abstract The life history of the bird cherry-oat aphid, Rhopalosiphum padi (L.) (Hemiptera: Aphididae), was studied via laboratory assays on Wheat streak mosaic virus (WSMV)-infected and non-infected transgenic and non-transformed wheat [Triticum aestivum L. (Poaceae)]. Although R. padi is not a WSMV vector, it is known to colonize WSMV-infected wheat plants. Two transgenic soft white winter wheat genotypes, 366-D03 and 366-D8, that express the WSMV coat protein gene, and the WSMV-susceptible non-transformed cultivar Daws were tested. All genotypes showed disease symptoms when infected with WSMV. Whereas plant height was significantly reduced on virus-infected compared to non-infected plants of all genotypes, virus-infected transgenic plants exhibited lower virus titer and lower disease rating scores than Daws. No significant effects of WSMV infection or genotypes were observed on the length of R. padi nymphal development period, nor on their pre-, and post-reproductive periods. Rhopalosiphum padi reproductive period was significantly longer on Daws infected with WSMV than on non-infected plants of this cultivar. In contrast, there were no significant differences in length of R. padi reproductive period between virus-infected and non-infected transgenic plants within a genotype. Rhopalosiphum padi daily fecundity was significantly lower and adult longevity significantly longer on virus-infected than on non-infected plants of all genotypes. Total aphid fecundity and intrinsic rate of increase were not significantly different among treatments. The percentage of winged aphids that developed was greater on WSMV-infected compared to non-infected plants within a genotype. Results indicate that both virus infection status of plants and wheat genotype influence the life history of R. padi. [source]

Further characterization and validation of gpt delta transgenic mice for quantifying somatic mutations in vivo

ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 4 2001
Roy R. Swiger
Abstract The utility of any mutation assay depends on its characteristics, which are best discovered using model mutagens. To this end, we report further on the characteristics of the lambda-based gpt delta transgenic assay first described by Nohmi et al. ([1996]: Environ Mol Mutagen 28:465,470). Our studies show that the gpt transgene responds similarly to other transgenic loci, specifically lacZ and cII, after treatment with acute doses of N -ethyl- N -nitrosourea (ENU). Because genetic neutrality is an important factor in the design of treatment protocols for mutagenicity testing, as well as for valid comparisons between different tissues and treatments, a time-course study was conducted. The results indicate that the gpt transgene, like cII and lacZ, is genetically neutral in vivo. The sensitivities of the loci are also equivalent, as evidenced by spontaneous mutant frequency data and dose, response curves after acute treatment with 50, 150, or 250 mg/kg ENU. The results are interesting in light of transgenic target size and location and of host genetic background differences. Based on these studies, protocols developed for other transgenic assays should be suitable for the gpt delta. Additionally, a comparison of the gpt and an endogenous locus, Dlb-1, within the small intestine of chronically treated animals (94 ,g/mL ENU in drinking water daily) shows differential accumulation of mutations at the loci during chronic exposure. The results further support the existence of preferential repair at endogenous, expressed genes relative to transgenes. Environ. Mol. Mutagen. 37:297,303, 2001 © 2001 Wiley-Liss, Inc. [source]

Persistence and degradation of maize-expressed vaccine protein, Escherichia coli heat-labile enterotoxin subunit B, in soil and water,

ENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 6 2008
Hirofumi Kosaki
Abstract Transgenic plants represent an innovative platform for the cost-effective large-scale production of various pharmaceutical proteins. The eventual open-field production of plant-made pharmaceuticals (PMPs) requires risk assessment to determine the potential for harm to the surrounding ecosystem. In the present study, the environmental persistence of a transgenic maize-expressed antigen, Escherichia coli heat-labile enterotoxin subunit B (LTB), was studied under laboratory conditions. To semiquantitatively monitor the persistence of LTB in soil, extraction with a high-salt, high-pH extraction buffer was optimized using the closely homologous Vibrio cholerae enterotoxin subunit B (CTB) as a test substance. The time to dissipation of 50% (DT50) of the extractable fraction of maize-expressed LTB was 4 to 15 d in pond water and 35 to 90 d in soils. Both extraction efficacy and persistence were strongly affected by the matrix type and incubation conditions. In contrast with maize-expressed LTB, the DT50 for bacterially produced LTB and CTB was less than 4 d both in pond water and soil. Although maize-expressed LTB was more stable than bacterially produced analogue, its dissipation was governed by an initial lag, which could be attributed to release from the plant material, followed by rapid decline. [source]

Early growth response 2 regulates the survival of thymocytes during positive selection

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 1 2010
Victoria J. Lawson
Abstract The early growth response (Egr) transcription factor family regulates multiple steps during T-cell development. We examine here the role played by Egr2 in positive selection. In double-positive cells, Egr2 is upregulated immediately following TCR ligation, and its expression requires both the MAPK and calcineurin signaling pathways. Inducible transgenic and knockout mice were generated to cause gain- or loss-of-function of Egr2 in double-positive cells, and had reciprocal effects; more mature single-positive cells were made when Egr2 was overexpressed, and fewer when Egr2 was absent. These defects were associated with changes in the survival of positively selected cells rather than perturbation of positive selection or immediate post-selection signaling. The survival function of Egr2 at least partly depends upon its ability to activate the cytokine-mediated survival pathway, likely through negative regulation of both the IL-7R and suppressor of cytokine signaling 1 (Socs1), the molecular switch whose downregulation normally results in restored responsiveness to cytokine signaling following selection. While gain of Egr2 caused a decrease in Socs1 mRNA, loss of Egr2 resulted in downregulation of IL-7R, upregulation of Socs1, and inhibition of Stat5 phosphorylation and IL-7-mediated survival post-selection. Therefore, expression of Egr2 following positive selection links the initial TCR signaling event to subsequent survival of signaled cells. [source]

MHC-restricted T cell receptor signaling is required for ,,,TCR replacement of the pre T cell receptor

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2008
Andrew L. Croxford
Abstract A developmental block is imposed on CD25+CD44, thymocytes at the ,-selection checkpoint in the absence of the pre T cell receptor (preTCR) ,-chain, pT,. Early surface expression of a transgenic ,,,TCR has been shown to partially circumvent this block, such that thymocytes progress to the CD4+CD8+ double-positive stage. We wanted to analyze whether a restricting MHC element is required for ,,,TCR-expressing double-negative (DN) thymocytes to overcome the developmental block in pT,-deficient animals. We used the HY-I knock-in model that endows thymocytes with ,,,TCR expression in the DN compartment but has the advantage of physiological expression levels, in contrast to conventional TCR transgenes. On a pT,-deficient background, this HY-I TCR transgene ,rescued' CD25+CD44, thymocytes from apoptosis and enabled progression to later differentiation stages. On a non-selecting MHC background, however, pT,-deficient HY-I mice presented a pronounced reduction in numbers of splenocytes and thymocytes when compared to animals of selecting MHC genotype, showing that MHC restriction is necessary to drive HY-TCR-mediated rescue of pT,-deficient thymocytes. [source]

Development of nephritis but not sialadenitis in autoimmune-prone BAFF transgenic mice lacking marginal zone B cells

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 9 2006
Carrie
Abstract B cell-activating factor belonging to the TNF family (BAFF) is a B cell survival factor required for B cell maturation. BAFF transgenic (Tg) mice develop autoimmune disorders characterized by autoantibody production, which leads to nephritis and salivary gland destruction (sialadenitis), features reminiscent of systemic lupus erythematosus and Sjögren's syndrome (SS), respectively. Disease in BAFF Tg mice correlates with the expansion of the marginal zone (MZ) B cell compartment and the abnormal presence of MZ-like B cells in the blood, LN and inflamed salivary glands, suggesting a role for these cells in BAFF-induced autoimmunity. Lymphotoxin-, (LT,)-deficient mice show disrupted splenic architecture, lack MZ B cells and some peripheral LN, and are unable to mount T cell-dependent immune responses. BAFF Tg mice lacking LT, (LT,,-BTg) retained these defects, yet still developed nephritis associated with the presence of B-1 B cells in the kidneys. However, in contrast to old BAFF Tg mice, aging LT,,-BTg mice no longer developed sialadenitis. Thus, autoimmune disorders in BAFF Tg mice are possibly events coordinated by MZ and B-1 B cells at separate anatomical sites. [source]

CCL17 transgenic mice show an enhanced Th2-type response to both allergic and non-allergic stimuli

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 8 2006
Yuichiro Tsunemi Dr.
Abstract CC chemokine ligand (CCL)17 is implicated in the pathogenesis of atopic dermatitis (AD). To study the effect of CCL17 produced by keratinocytes (KC) during inflammation, we created transgenic (Tg) mice in which CCL17 is overexpressed in KC. Th2-type contact hypersensitivity (CHS) was enhanced and Th1-type CHS was suppressed in these mice. Increased numbers of CC chemokine receptor (CCR)4+ cells and mast cells infiltrated in Tg mice. Levels of IL-4 mRNA were higher and those of IFN-, mRNA were lower in both acute and chronic CHS. Higher levels of serum IgE were observed after CHS. Numbers of CCR4+ cells among PBMC were increased in Tg mice challenged acutely on the trunk. Chronic irritation with croton oil induced dermatitis and an elevation of serum IgE levels. Tg mice showed enhanced ear swelling after tape stripping. CCL17 was thought to modify the inflammation caused by sensitizing reagents as well as irritant reagents by attracting CCR4+ cells into the lesional skin and creating a Th2-dominant condition. AD-like conditions such as increased number of mast cells and elevated levels of serum IgE were observed. Thus, CCL17 may participate in the pathogenesis of skin diseases such as AD by regulating both allergic and irritant inflammation. [source]

Copolymer effects on microglia and T,cells in the central nervous system of humanized mice

EUROPEAN JOURNAL OF IMMUNOLOGY, Issue 12 2005
Zsolt Illes
The random amino acid copolymers FYAK and VWAK ameliorate EAE in a humanized mouse model expressing both a human transgenic myelin basic protein (MBP)85,99-specific T,cell receptor and HLA-DR2. Here we show that microglia isolated from the central nervous system (CNS) of humanized mice with EAE induced by MBP85,99 and treated with these copolymers had reduced expression of HLA-DR, and thus reduced capacity to present MBP85,99 and activate transgenic T,cells. In vitro microglia up-regulated empty HLA-DR2 upon activation with GM-CSF with or without LPS or IFN-,, but not with IL-4 or IL-10. Correspondingly, gene chip arrays showed that the CNS of untreated and YFAK-treated mice differentially expressed pro- and anti-inflammatory molecules during MBP85,99-induced EAE. Interestingly, microglia expressed the full-length ,,,and ,,,subunits of the tetrameric adaptor protein complexes AP-1 and AP-2 respectively, but after treatment with GM-CSF these complexes were cleaved, as had been found in immature dendritic cells derived from bone marrow. Strikingly, in vivo the perivascular lymphocyte infiltration seen in untreated mice immunized with MBP85,99 was composed of equal numbers of hV,2+ MPB85,99-specific transgenic and hV,2, endogenous T,cells, while the much smaller infiltration seen after treatment with YFAK was composed predominantly of hV,2, endogenous T,cells. [source]

Mice transgenic for exon 1 of the Huntington's disease gene display reduced striatal sensitivity to neurotoxicity induced by dopamine and 6-hydroxydopamine

EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2001
Åsa Petersén
Abstract Huntington's disease is an autosomal dominant hereditary neurodegenerative disorder characterized by severe striatal cell loss. Dopamine (DA) has been suggested to play a role in the pathogenesis of the disease. We have previously reported that transgenic mice expressing exon 1 of the human Huntington gene (R6 lines) are resistant to quinolinic acid-induced striatal toxicity. In this study we show that with increasing age, R6/1 and R6/2 mice develop partial resistance to DA- and 6-hydroxydopamine-mediated toxicity in the striatum. Using electron microscopy, we found that the resistance is localized to the cell bodies and not to the neuropil. The reduction of dopamine and cAMP regulated phosphoprotein of a molecular weight of 32 kDa (DARPP-32) in R6/2 mice does not provide the resistance, as DA-induced striatal lesions are not reduced in size in DARPP-32 knockout mice. Neither DA receptor antagonists nor a N -methyl- d -aspartate (NMDA) receptor blocker reduce the size of DA-induced striatal lesions, suggesting that DA toxicity is not dependent upon DA- or NMDA receptor-mediated pathways. Moreover, superoxide dismutase-1 overexpression, monoamine oxidase inhibition and the treatment with the free radical scavenging spin-trap agent phenyl-butyl-tert-nitrone (PBN) also did not block DA toxicity. Levels of the antioxidant molecules, glutathione and ascorbate were not increased in R6/1 mice. Because damage to striatal neurons following intrastriatal injection of 6-hydroxydopamine was also reduced in R6 mice, a yet-to-be identified antioxidant mechanism may provide neuroprotection in these animals. We conclude that striatal neurons of R6 mice develop resistance to DA-induced toxicity with age. [source]

Increased glucose metabolism and ATP level in brain tissue of Huntington's disease transgenic mice

FEBS JOURNAL, Issue 19 2008
Judit Oláh
Huntington's disease (HD) is a progressive neurodegenerative disorder characterized by multifarious dysfunctional alterations including mitochondrial impairment. In the present study, the formation of inclusions caused by the mutation of huntingtin protein and its relationship with changes in energy metabolism and with pathological alterations were investigated both in transgenic and 3-nitropropionic acid-treated mouse models for HD. The HD and normal mice were characterized clinically; the affected brain regions were identified by immunohistochemistry and used for biochemical analysis of the ATP-producing systems in the cytosolic and the mitochondrial compartments. In both HD models, the activities of some glycolytic enzymes were somewhat higher. By contrast, the activity of glyceraldehyde-3-phosphate dehydrogenase was much lower in the affected region of the brain compared to that of the control. Paradoxically, at the system level, glucose conversion into lactate was enhanced in cytosolic extracts from the HD brain tissue, and the level of ATP was higher in the tissue itself. The paradox could be resolved by taking all the observed changes in glycolytic enzymes into account, ensuing an experiment-based detailed mathematical model of the glycolytic pathway. The mathematical modelling using the experimentally determined kinetic parameters of the individual enzymes and the well-established rate equations predicted the measured flux and concentrations in the case of the control. The same mathematical model with the experimentally determined altered Vmax values of the enzymes did account for an increase of glycolytic flux in the HD sample, although the extent of the increase was not predicted quantitatively. This suggested a somewhat altered regulation of this major metabolic pathway in HD tissue. We then used the mathematical model to develop a hypothesis for a new regulatory interaction that might account for the observed changes; in HD, glyceraldehyde-3-phosphate dehydrogenase may be in closer proximity (perhaps because of the binding of glyceraldehyde-3-phosphate dehydrogenase to huntingtin) with aldolase and engage in channelling for glyceraldehyde-3-phosphate. By contrast to most of the speculation in the literature, our results suggest that the neuronal damage in HD tissue may be associated with increased energy metabolism at the tissue level leading to modified levels of various intermediary metabolites with pathological consequences. [source]