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Transgene
Kinds of Transgene Terms modified by Transgene Selected AbstractsActivin/nodal signaling modulates XPAPC expression during Xenopus gastrulationDEVELOPMENTAL DYNAMICS, Issue 3 2008Xin Lou Abstract Gastrulation is the first obligatory morphogenesis during vertebrate development, by which the body plan is established. Nodal signaling is a key player in many developmental processes, including gastrulation. XPAPC has been found to exert its biological function through modifying the adhesion property of cells and interacting with other several important molecules in embryos. In this report, we show that nodal signaling is necessary and sufficient for XPAPC expression during Xenopus gastrulation. Furthermore, we isolated 4.8 kb upstream DNA sequence of Xenopus XPAPC, and proved that this 4.8-kb genomic contig is sufficient to recapitulate the expression pattern of XPAPC from gastrula to tail bud stage. Transgene and ChIP assays indicate that Activin/nodal signaling participates in regulation of XPAPC expression through a Smad binding element within the XPAPC promoter. Concomitant investigation suggests that the canonical Wnt pathway-activated XPAPC expression requires nodal signaling. Developmental Dynamics 237:683,691, 2008. © 2008 Wiley-Liss, Inc. [source] Complementation of Physiological and Behavioral Defects by a Slowpoke Ca2+ -Activated K+ Channel TransgeneJOURNAL OF NEUROCHEMISTRY, Issue 3 2000Robert Brenner Abstract: The Drosophila slowpoke gene encodes a large conductance calcium-activated potassium channel used in neurons, muscle, and some epithelial cells. Tissue-specific transcriptional promoters and alternative mRNA splicing generate a large array of transcripts. These distinct transcripts are thought to tailor the properties of the channel to the requirements of the cell. Presumably, a single splice variant cannot satisfy the specific needs of all cell types. To test this, we examined whether a single slowpoke splice variant was capable of complementing all slowpoke behavioral phenotypes. Null mutations in slowpoke cause animals to be semiflightless and to manifest an inducible "sticky-feet" phenotype. The well-characterized slowpoke transcriptional control region was used to direct the expression of a single slowpoke splice variant (cDNA H13) in transgenic flies. The endogenous gene in these flies had been inactivated by the slo4 mutation. Action-potential recordings and voltage-clamp recordings demonstrated the production of functional channels from the transgene. The transgene completely complemented the flight defect, but not the sticky-feet phenotype. We conclude that distinct slowpoke channel isoforms, produced by alternative splicing, are not interchangeable and are required for proper function of different cell types. [source] BIOCENTURY, outrunner of China's biological technology in agriculture Biocentury Transgene (China) Co., Ltd.BIOTECHNOLOGY JOURNAL, Issue 11 2006Hongzhi Cui No abstract is available for this article. [source] RNA interference by expressing short hairpin RNA in the Ciona intestinalis embryoDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 6 2008Aya Nishiyama We carried out RNA interference by expressing short hairpin RNA (shRNA) in the Ciona intestinalis embryo. For this purpose, we identified a gene encoding U6 small nuclear RNA (snRNA) in the C. intestinalis genome. The 1-kb sequence upstream of the U6 snRNA gene was sufficient for directing transcription of short RNA as revealed by Northern blot hybridization. An shRNA-expressing plasmid vector was constructed, in which shRNA-encoding oligonucleotides are inserted downstream of the U6 promoter. An shRNA that contained a sequence homologous to the C. intestinalis tyrosinase gene (Ci-tyrosinase) suppressed melanization of pigment cells in the brain of morphologically normal tailbud embryos. An shRNA that perfectly matched the translated sequence of enhanced green fluorescent protein (EGFP) (a mutant type of Aequorea victoria green fluorescent protein) suppressed the expression of the coelectroporated EGFP transgene. These results suggest that the expression of shRNA interferes with functions of both endogenous and exogenous genes. The shRNA-expressing plasmid constructed in the present study provides an easy and inexpensive alternative for the functional analysis of genes in ascidian embryos. [source] Retinoic acid controls expression of tissue remodeling genes Hmgn1 and Fgf18 at the digit,interdigit junctionDEVELOPMENTAL DYNAMICS, Issue 2 2010Xianling Zhao Abstract Previous studies on retinoic acid receptor (RAR) mutants suggested that retinoic acid (RA) is required for loss of interdigital mesenchyme during digit formation. Here, we report that the RA-generating enzyme retinaldehyde dehydrogenase-2 (Raldh2) is expressed in the interdigital mesenchyme whereas Cyp26b1, controlling RA degradation, is expressed in digits, limiting autopodal RA action to the interdigital zones. Embryonic day 13.5 Raldh2,/, mouse embryos lose expression of the RARE-lacZ RA-reporter transgene and matrix metalloproteinase-11 (Mmp11) throughout the interdigital mesenchyme, while expression of RARb, Fgf18, and high mobility group N1 (Hmgn1) is lost at the digit,interdigit junction. Raldh2,/, autopods exhibit reduced interdigital apoptosis associated with loss of Bmp7 expression, but Bmp2, Bmp4, Msx2, and Fgf8 were unaffected. Although interdigital expression of Hmgn1 was greatly down-regulated in Raldh2,/, autopods, complementary expression of Sox9 in digit cartilage was unaffected. Regulation of Hmgn1 and Fgf18 at the digit,interdigit junction suggests RA controls tissue remodeling as well as apoptosis. Developmental Dynamics 239:665,671, 2010. © 2009 Wiley-Liss, Inc. [source] gfap and nestin reporter lines reveal characteristics of neural progenitors in the adult zebrafish brainDEVELOPMENTAL DYNAMICS, Issue 2 2009Chen Sok Lam Abstract Adult neurogenesis arises from niches that harbor neural stem cells (NSC). Although holding great promise for regenerative medicine, the identity of NSC remains elusive. In mammals, a key attribute of NSC is the expression of the filamentous proteins glial fibrillary acidic protein (GFAP) and NESTIN. To assess whether these two markers are relevant in the fish model, two transgenic zebrafish lines for gfap and nestin were generated. Analysis of adult brains showed that the fusion GFAP,green fluorescent protein closely mimics endogenous GFAP, while the nestin transgene recapitulates nestin at the ventricular zones. Cells expressing the two reporters display radial glial morphology, colocalize with the NSC marker Sox2, undergo proliferation, and are capable of self-renewal within the matrix of distinct thickness in the telencephalon. Together, these two transgenic lines reveal a conserved feature of putative NSC in the adult zebrafish brain and provide a means for the identification and manipulation of these cells in vivo. Developmental Dynamics 238:475,486, 2009. © 2009 Wiley-Liss, Inc. [source] Analysis of the IKK,/NF-,B signaling pathway during embryonic angiogenesisDEVELOPMENTAL DYNAMICS, Issue 10 2008Yanjun Hou Abstract The nuclear factor-,B (NF-,B) signaling pathway regulates cellular growth, survival, differentiation and development. In this study, the functions of I,B kinase (IKK), in angiogenesis during mouse development were examined. Conditional disruption of the Ikk, locus in endothelial cells using the well-characterized Tie2-Cre transgene resulted in embryonic lethality between embryonic day (E) 13.5 and E15.5. Examination of the mutant embryos revealed that while deletion of Ikk, occurred in endothelial cells throughout the embryo, only the vascular network in the fetal liver was affected. Disruption of the fetal liver vasculature was accompanied by decreased cell proliferation and increased apoptosis of hepatocytes, but hematopoiesis was not affected. Increased apoptosis was not observed outside of fetal liver in the mutant embryos. These results indicate that the IKK,/NF-,B pathway plays a previously unappreciated role in development of the sinusoidal vasculature in the fetal liver and additionally that this pathway is critical in the crosstalk between endothelial cells and hepatocytes during mouse development. Developmental Dynamics 237:2926,2935, 2008. © 2008 Wiley-Liss, Inc. [source] The Tol2kit: A multisite gateway-based construction kit for Tol2 transposon transgenesis constructsDEVELOPMENTAL DYNAMICS, Issue 11 2007Kristen M. Kwan Abstract Transgenesis is an important tool for assessing gene function. In zebrafish, transgenesis has suffered from three problems: the labor of building complex expression constructs using conventional subcloning; low transgenesis efficiency, leading to mosaicism in transient transgenics and infrequent germline incorporation; and difficulty in identifying germline integrations unless using a fluorescent marker transgene. The Tol2kit system uses site-specific recombination-based cloning (multisite Gateway technology) to allow quick, modular assembly of [promoter],[coding sequence],[3, tag] constructs in a Tol2 transposon backbone. It includes a destination vector with a cmlc2:EGFP (enhanced green fluorescent protein) transgenesis marker and a variety of widely useful entry clones, including hsp70 and beta-actin promoters; cytoplasmic, nuclear, and membrane-localized fluorescent proteins; and internal ribosome entry sequence,driven EGFP cassettes for bicistronic expression. The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large-scale projects testing the functions of libraries of regulatory or coding sequences. Developmental Dynamics 236:3088,3099, 2007. © 2007 Wiley-Liss, Inc. [source] Frontal nasal prominence expression driven by Tcfap2a relies on a conserved binding site for STAT proteinsDEVELOPMENTAL DYNAMICS, Issue 5 2006Amy 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] Human and pig SRY 5, flanking sequences can direct reporter transgene expression to the genital ridge and to migrating neural crest cellsDEVELOPMENTAL DYNAMICS, Issue 3 2006Alexandre Boyer Abstract Mechanisms for sex determination vary greatly between animal groups, and include chromosome dosage and haploid,diploid mechanisms as seen in insects, temperature and environmental cues as seen in fish and reptiles, and gene-based mechanisms as seen in birds and mammals. In eutherian mammals, sex determination is genetic, and SRY is the Y chromosome located gene representing the dominant testes determining factor. How SRY took over this function from ancestral mechanisms is not known, nor is it known what those ancestral mechanisms were. What is known is that SRY is haploid and thus poorly protected from mutations, and consequently is poorly conserved between mammalian species. To functionally compare SRY promoter sequences, we have generated transgenic mice with fluorescent reporter genes under the control of various lengths of human and pig SRY 5, flanking sequences. Human SRY 5, flanking sequences (5 Kb) supported reporter transgene expression within the genital ridge of male embryos at the time of sex determination and also supported expression within migrating truncal neural crest cells of both male and female embryos. The 4.6 Kb of pig SRY 5, flanking sequences supported reporter transgene expression within the male genital ridge but not within the neural crest; however, 2.6 Kb and 1.6 Kb of pig SRY 5, flanking sequences retained male genital ridge expression and now supported extensive expression within cells of the neural crest in embryos of both sexes. When 2 Kb of mouse SRY 5, flanking sequences (,3 to ,1 Kb) were placed in front of the 1.6 Kb of pig SRY 5, flanking sequences and this transgene was introduced into mice, reporter transgene expression within the male genital ridge was retained but neural crest expression was lost. These observations suggest that SRY 5, flanking sequences from at least two mammalian species contain elements that can support transgene expression within cells of the migrating neural crest and that additional SRY 5, flanking sequences can extinguish this expression. Developmental Dynamics 235:623,632, 2006. © 2006 Wiley-Liss, Inc. [source] Conditional expression of a myocardium-specific transgene in zebrafish transgenic linesDEVELOPMENTAL DYNAMICS, Issue 4 2005Chiu-Ju Huang Abstract To develop the first heart-specific tetracycline (Tet)-On system in zebrafish, we constructed plasmids in which the cardiac myosin light chain 2 promoter of zebrafish was used to drive the reverse Tet-controlled transactivator (rtTA) and the green fluorescent protein (GFP) reporter gene was preceded by an rtTA-responsive element. In the zebrafish fibroblast cell-line, rtTA-M2, one of rtTA's derivatives, demonstrated the highest increase in luciferase activity upon doxycycline (Dox) induction. We then generated two germ lines of transgenic zebrafish: line T03 was derived from microinjection of a plasmid containing rtTA-M2 and a plasmid containing a responsive reporter gene, whereas line T21 was derived from microinjection of a single dual plasmid. Results showed that line T21 was superior to line T03 in terms of greater GFP intensity after induction and with of minimal leakiness before induction. The photographic images of induced GFP in the heart of F2 larvae showed that the fluorescent level of GFP was dose-responsive. The level of GFP expressed in the F3 3 days postfertilization larvae that were treated with Dox for 1 hr decreased gradually after the withdrawal of the inducer; and the fluorescent signal disappeared after 5 days. The GFP induction and reduction were also tightly controlled by Dox in the F3 adult fish from line T21. This Tet-On system developed in zebrafish shows much promise for the study of the gene function in a specific tissue at the later developmental stage. Developmental Dynamics 233:1294,1303, 2005. © 2005 Wiley-Liss, Inc. [source] Hoxb3 vagal neural crest-specific enhancer element for controlling enteric nervous system developmentDEVELOPMENTAL DYNAMICS, Issue 2 2005Kwok Keung Chan Abstract The neural and glial cells of the intrinsic ganglia of the enteric nervous system (ENS) are derived from the hindbrain neural crest at the vagal level. The Hoxb3 gene is expressed in the vagal neural crest and in the enteric ganglia of the developing gut during embryogenesis. We have identified a cis -acting enhancer element b3IIIa in the Hoxb3 gene locus. In this study, by transgenic mice analysis, we examined the tissue specificity of the b3IIIa enhancer element using the lacZ reporter gene, with emphasis on the vagal neural crest cells and their derivatives in the developing gut. We found that the b3IIIa-lacZ transgene marks only the vagal region and not the trunk or sacral region. Using cellular markers, we showed that the b3IIIa-lacZ transgene was expressed in a subset of enteric neuroblasts during early development of the gut, and the expression was maintained in differentiated neurons of the myenteric plexus at later stages. The specificity of the b3IIIa enhancer in directing gene expression in the developing ENS was further supported by genetic analysis using the Dom mutant, a spontaneous mouse model of Hirschsprung's disease characterized by the absence of enteric ganglia in the distal gut. The colonization of lacZ -expressing cells in the large intestine was incomplete in all the Dom/b3IIIa-lacZ hybrid mutants we examined. To our knowledge, this is the only vagal neural crest-specific genetic regulatory element identified to date. This element could be used for a variety of genetic manipulations and in establishing transgenic mouse models for studying the development of the ENS. Developmental Dynamics 233:473,483, 2005. © 2005 Wiley-Liss, Inc. [source] Functional analysis in Drosophila indicates that the NBCCS/PTCH1 mutation G509V results in activation of smoothened through a dominant-negative mechanismDEVELOPMENTAL DYNAMICS, Issue 4 2004Gary R. Hime Abstract Mutations in the human homolog of the patched gene are associated with the developmental (and cancer predisposition) condition Nevoid Basal Cell Carcinoma Syndrome (NBCCS), as well as with sporadic basal cell carcinomas. Most mutations that have been identified in the germline of NBCCS patients are truncating or frameshift mutations, with amino acid substitutions rarely found. We show that a missense mutation in the sterol-sensing domain G509V acts as a dominant negative when assayed in vivo in Drosophila. Ectopic expression of a Drosophila patched transgene, carrying the analogous mutation to G509V, causes ectopic activation of Hedgehog target genes and ectopic membrane stabilisation of Smoothened. The G509V transgene behaves in a manner similar, except in its subcellular distribution, to a C-terminal truncation that has been characterised previously as a dominant-negative protein. G509V exhibits vesicular localisation identical to the wild-type protein, but the C-terminal truncated Patched molecule is localised predominantly to the plasma membrane. This finding suggests that dominant-negative function can be conferred by interruption of different aspects of Patched protein behaviour. Another mutation at the same residue, G509R, did not exhibit dominant-negative activity, suggesting that simple removal of the glycine at 509 is not sufficient to impart dominant-negative function. Developmental Dynamics 229:780,790, 2004. © 2004 Wiley-Liss, Inc. [source] Endothelium-specific Cre recombinase activity in flk-1-Cre transgenic miceDEVELOPMENTAL DYNAMICS, Issue 2 2004Alexander H. Licht Abstract The use of the Cre-loxP recombination system allows the conditional inactivation of genes in mice. The availability of transgenic mice in which the Cre recombinase expression is highly cell type specific is a prerequisite to successfully use this system. We previously have characterized regulatory regions of the mouse flk-1 gene sufficient for endothelial cell-specific expression of the LacZ reporter gene in transgenic mice. These regions were fused to the Cre recombinase gene, and transgenic mouse lines were generated. In the resulting flk-1-Cre transgenic mice, specificity of Cre activity was determined by cross-breeding with the reporter mouse lines Rosa26R or CAG-CAT-LacZ. We examined double-transgenic mice at different stages of embryonic development (E9.5,E16.5) and organs of adult animals by LacZ staining. Strong endothelium-specific staining of most vascular beds was observed in embryos older than E11.5 in one or E13.5 in a second line. In addition, the neovasculature of experimental BFS-1 tumors expressed the transgene. These lines will be valuable for the conditional inactivation of floxed target genes in endothelial cells of the embryonic vascular system. Developmental Dynamics 229:312,318, 2004. © 2004 Wiley-Liss, Inc. [source] Expression of a dominant negative form of Daxx in vivo rescues motoneurons from Fas (CD95)-induced cell deathDEVELOPMENTAL NEUROBIOLOGY, Issue 2 2005Cedric Raoul Abstract Fas-induced death of motoneurons in vitro has been shown to involve two signaling cascades that act together to execute the death program: a Fas-Daxx-ASK-1-p38 kinase-nNOS branch, which controls transcriptional and post-translational events, and the second classical Fas-FADD-caspase-8 branch. To analyze the role of Daxx in the developmental motoneuron cell death, we studied Fas-dependent cell death in motoneurons from transgenic mice that overexpress a dominant-negative form of Daxx. Motoneurons purified from these transgenic mice are resistant to Fas-induced death. This protective effect is specific to Fas because ultraviolet irradiation-triggered death is not affected by the transgene. The Daxx and the FADD pathways work in parallel because only Daxx, but not FADD, is involved in the transcriptional control of neuronal nitric oxide synthase and nitric oxide production. Nevertheless, we do not observe involvement of Daxx in developmental motoneuronal cell death, as the pattern of naturally occurring programmed cell death in vivo is normal in transgenic mice overexpressing the dominant negative form of Daxx, suggesting that Daxx-independent pathways are used during development. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005 [source] Loss of steroidogenic factor 1 alters cellular topography in the mouse ventromedial nucleus of the hypothalamusDEVELOPMENTAL NEUROBIOLOGY, Issue 4 2004Aline M. Davis Abstract Knockout (KO) mice lacking the orphan nuclear receptor steroidogenic factor 1 (SF-1) exhibit marked structural abnormalities of the ventromedial nucleus of the hypothalamus (VMH). In this study, we sought to determine the molecular mechanisms underlying the VMH abnormalities. To trace SF-1-expressing neurons, we used a SF-1/enhanced green fluorescent protein (eGFP) transgene. Although the total numbers of eGFP-positive cells in wild-type (WT) and SF-1 KO mice were indistinguishable, cells that normally localize precisely within the VMH were scattered more diffusely in adjacent regions in SF-1 KO mice. This abnormal distribution is likely due to the loss of SF-1 expression in VMH neurons rather than secondary effects of deficient steroidogenesis, as redistribution also was seen in mice with a CNS-specific KO of SF-1. Thus, the absence of SF-1 alters the distribution of cells that normally form the VMH within the mediobasal hypothalamus. Consistent with this model, the hypothalamic expression patterns of the transcription factors islet-1 and nkx2.1 also were displaced in SF-1 KO mice. Independent of gene expression, birthdate analyses further suggested that cells with earlier birthdates were affected more severely by the loss of SF-1 than were later born cells. We conclude that the absence of SF-1 causes major changes in cellular arrangement within and around the developing VMH that result from altered cell migration. © 2004 Wiley Periodicals, Inc. J Neurobiol 60: 424,436, 2004 [source] Conditional ablation of neurones in transgenic miceDEVELOPMENTAL NEUROBIOLOGY, Issue 3 2001Anthony R. Isles Abstract Conditional targeted ablation of specific cell populations in living transgenic animals is a very powerful strategy to determine cell functions in vivo. This approach would be of particular value to study the functions of distinct neuronal populations; however, the transgene of choice for conditional cell ablation studies in mice, the herpes simplex virus thymidine kinase gene, cannot be used to ablate neurones as its principal mode of action relies on cell proliferation. Here we report that expression of the E.coli nitroreductase gene (Ntr) and metabolism of the prodrug CB1954 (5-aziridin-1-yl-2-4-dinitrobenzamide) to its cytotoxic derivative can be used to conditionally and acutely ablate specific neuronal populations in vivo. As proof of principal, we have ablated olfactory and vomeronasal receptor neurones by expressing Ntr under the control of the olfactory marker protein (OMP) gene promoter. We demonstrate that following CB1954 administration, olfactory and vomeronasal receptor neurones expressing the transgene were selectively eliminated from the olfactory epithelium (OE), and projections to the olfactory bulb (OB) were lost. The functional efficacy of cell ablation was demonstrated using a highly sensitive behavioural test to show that ablated mice had lost the olfactory ability to discriminate distinct odors and were consequently rendered anosmic. Targeted expression of Ntr to specific neuronal populations using conventional transgenes, as described here, or by "knock-in" gene targeting using embryonic stem cells may be of significant value to address the functions of distinct neuronal populations in vivo. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 183,193, 2001 [source] Assessing environmental risks of transgenic plantsECOLOGY LETTERS, Issue 2 2006D. A. Andow Abstract By the end of the 1980s, a broad consensus had developed that there were potential environmental risks of transgenic plants requiring assessment and that this assessment must be done on a case-by-case basis, taking into account the transgene, recipient organism, intended environment of release, and the frequency and scale of the intended introduction. Since 1990, there have been gradual but substantial changes in the environmental risk assessment process. In this review, we focus on changes in the assessment of risks associated with non-target species and biodiversity, gene flow, and the evolution of resistance. Non-target risk assessment now focuses on risks of transgenic plants to the intended local environment of release. Measurements of gene flow indicate that it occurs at higher rates than believed in the early 1990s, mathematical theory is beginning to clarify expectations of risks associated with gene flow, and management methods are being developed to reduce gene flow and possibly mitigate its effects. Insect pest resistance risks are now managed using a high-dose/refuge or a refuge-only strategy, and the present research focuses on monitoring for resistance and encouraging compliance to requirements. We synthesize previous models for tiering risk assessment and propose a general model for tiering. Future transgenic crops are likely to pose greater challenges for risk assessment, and meeting these challenges will be crucial in developing a scientifically coherent risk assessment framework. Scientific understanding of the factors affecting environmental risk is still nascent, and environmental scientists need to help improve environmental risk assessment. [source] Mutation spectrum in UVB-exposed skin epidermis of Xpa -knockout mice: Frequent recovery of triplet mutationsENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 1 2007Hironobu Ikehata Abstract Knockout mutations in both alleles of the Xpa gene give rise to a complete deficiency in nucleotide excision repair (NER) in mammalian cells. We used transgenic mice harboring the ,-phage-based lacZ mutational reporter gene to study the effect of Xpa null mutation (Xpa,/,) on damage induction, repair, and mutagenesis in mouse skin epidermis after UVB irradiation. UVB induced equal amounts of cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts (64PPs) in mouse skin epidermis of Xpa,/, and wild-type mice. Neither photolesion was removed in the Xpa,/, epidermis by 12 hr after irradiation whereas removal of 64PPs was observed in the epidermis of wild-type mice. Irradiation with 200 and 300 J/m2 UVB increased the lacZ mutant frequency in the epidermis of Xpa,/, mice, but the induced mutant frequencies were not significantly different from those previously determined for wild-type mice. One-hundred lacZ mutants isolated from the UVB-exposed epidermis of Xpa,/, mice were analyzed and compared with mutant sequences previously determined for irradiated wild-type mice. The distribution of the mutations along the lacZ transgene and the preferred dipyrimidine context of the UV-specific mutations were similar in mutants from the Xpa,/, and wild-type mice. The spectra of the mutations in the two genotypes were both highly UV-specific and similar in a dominance of C , T transitions at dipyrimidine sites; however, Xpa,/, mice had a higher frequency than wild-type mice of two-base tandem substitutions, including CC , TT mutations, three-base tandem mutations and double base substitutions that were separated by one unchanged base in a three-base sequence (alternating mutations). These tandem/alternating mutations included a remarkably large number of triplet mutations, a recently reported, novel type of UV-specific mutation, characterized by multiple base substitutions or frameshifts within a three-nucleotide sequence containing a dipyrimidine. We conclude that the triplet mutation is a UV-specific mutation that preferably occurs in NER-deficient genetic backgrounds. Environ. Mol. Mutagen., 2007. © 2006 Wiley-Liss, Inc. [source] Evaluation of mutant frequencies of chemically induced tumors and normal tissues in ,/cII transgenic miceENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 1 2005Jon C. Mirsalis Abstract Genomic instability has been implicated as an important component in tumor progression. Evaluation of mutant frequencies (MFs) in tumors of transgenic mice containing nontranscribed marker genes should be useful for quantitating mutation rates in tumors as the physiologically inactive transgene provides neither a positive nor a negative selective pressure on the tumor. We have conducted long-term carcinogenicity studies in ,/cII transgenic B6C3F1 mice using a variety of genotoxic and nongenotoxic test agents and have evaluated the mutant frequencies in both tumors and normal tissues from these animals. Mice were administered diethylnitrosamine (DEN) as three intraperitoneal injections of 15 mg/kg; phenobarbital (PB) or oxazepam (OXP) provided ad libitum at 0.1% or 0.25% in the diet, respectively; DEN initiation plus PB in the diet; or urethane (UTH) provided ad libitum at 0.2% in the drinking water. Normal tissues and tumors were isolated at various times over a 2-year period and half of each tissue/tumor was evaluated histopathologically and the other half was evaluated for MF in the cII transgene. Approximately 20 mutants from each of 166 individual tissues (tumor and nontumor) were sequenced to determine whether increases in MF represented unique mutations or were due to clonal expansion. UTH produced significant increases in MF in normal liver and lung. DEN either with or without PB promotion produced significant increases in MF in liver and correction of MF for clonality produced little change in the overall MF in these groups. PB produced a twofold increase in liver MF over controls after 27 weeks of treatment, but a similar increase was not observed with longer dosing times; at later time points, the MF in the PB groups was lower than that of the control group, suggesting that PB is not producing direct DNA damage in the liver. OXP failed to produce an increase in MF over controls, even after 78 weeks of treatment. Selected cases of genomic instability were observed in tumors from all treatments except OXP, with individual liver tumors showing very high MF values even after clonal correction. One rare and interesting finding was noted in a single mouse treated with UTH, where a mammary metastasis had an MF approximately 10-fold greater than the parent tumor, with 75% of the mutations independent, providing strong evidence of genomic instability. There was no clear correlation between tumor phenotype and MF except that pulmonary adenomas generally had higher MFs than normal lung in both genotoxic and nongenotoxic treatment groups. Likewise, there was no correlation between tumor size and MF after correction for clonality. The results presented here demonstrate that individual tumors can show significant genomic instability, with very significant increases in MF that are not attributed to clonal expansion of a single mutant cell. Environ. Mol. Mutagen., 2005. © 2004 Wiley-Liss, Inc. [source] Just how does the cII selection system work in MutaÔMouse?ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 4 2001Roy R. Swiger Abstract The lambda CII protein is an essential component in the lytic vs. lysogeny decision a bacteriophage makes upon infection of a host at low temperatures. The protein interacts with numerous phage promoters modulating the expression of the CI repressor, thus providing the mechanism for lysogenization soon after infection. The Big Blue® and MutaÔMouse are two widely used in vivo mutational model systems. The assays rely on retrievable lambda-based transgenes housing mutational targets (lacI or lacZ, respectively). The transgenes provide an elegant vehicle for the quantification of mutations sustained in virtually any tissue of the rodent. The use of the bacteriophage cII locus as an alternative, or additional mutational target for use with the Big Blue® rodent system was first reported by Jakubczak et al. ([1996]: Proc Natl Acad Sci USA 93:9073,9078). More recently, this selection assay has been applied successfully to the MutaÔMouse (Swiger et al. [1999]: Environ Mol Mutagen 33:201,207). The use of an Hfl bacterial strain and low temperature allows the determination of mutations sustained at the cII locus in either system, with high fidelity. The cII selection assay in the Big Blue® relies on the presence of the lambda repressor protein CI. In contrast, the recombinant construct used to make the MutaÔMouse transgene lacks functional CI protein. Nevertheless, we report an excellent system for quantifying mutations at the cII locus in MutaÔMouse. Just how does cII selection work in the MutaÔMouse? Written in the context of lambda recombinant genetics, this paper explores the question further. Environ. Mol. Mutagen. 37:290,296, 2001 © 2001 Wiley-Liss, Inc. [source] Further characterization and validation of gpt delta transgenic mice for quantifying somatic mutations in vivoENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 4 2001Roy 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] Transgenic strains of the nematode Caenorhabditis elegans as biomonitors of metal contaminationENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 8 2000L. K. Cioci Abstract Transition metal contamination poses a serious environmental and human health threat. The bioavailability of transition metals in environmental samples can best be assessed with living organisms. A transgenic strain of the free-living soil nematode Caenorhabditis elegans has been engineered for monitoring the bioavailability of metals. A reporter transgene consisting of a fragment of the promoter from the C. elegans metallothionein-2 gene (mtl-2) that controls the transcription of a ,-galactosidase reporter (lacZ) has been integrated into the genome of this organism. By using these transgenic C. elegans, the toxicological response to metals in samples can be quickly measured with a simple histochemical staining assay. The C. elegans that contain the mtl-2:lacZ transgene provide a more sensitive assay of exposure to cadmium, mercury, zinc, and nickel than 24-h LC50 assays or those using nematodes with heat-shock protein,based reporter transgenes. This study demonstrates that C. elegans that contain mtl-2:lacZ transgenes can function as sensitive toxicological indicators of metals. [source] Gene therapy in epilepsyEPILEPSIA, Issue 1 2009Véronique Riban Summary Results from animal models suggest gene therapy is a promising new approach for the treatment of epilepsy. Several candidate genes such as neuropeptide Y and galanin have been demonstrated in preclinical studies to have a positive effect on seizure activity. For a successful gene therapy-based treatment, efficient delivery of a transgene to target neurons is also essential. To this end, advances have been made in the areas of cell transplantation and in the development of recombinant viral vectors for gene delivery. Recombinant adeno-associated viral (rAAV) vectors in particular show promise for gene therapy of neurological disorders due to their neuronal tropism, lack of toxicity, and stable persistence in neurons, which results in robust, long-term expression of the transgene. rAAV vectors have been recently used in phase I clinical trials of Parkinson's disease with an excellent safety profile. Prior to commencement of phase I trials for gene therapy of epilepsy, further preclinical studies are ongoing including evaluation of the therapeutic benefit in chronic models of epileptogenesis, as well as assessment of safety in toxicological studies. [source] Liposome-mediated uptake of exogenous DNA by equine spermatozoa and applications in sperm-mediated gene transferEQUINE VETERINARY JOURNAL, Issue 1 2008B. A. BALL Summary Reasons for performing study: Sperm-mediated gene transfer has been reported as a method for production of transgenic animals in a variety of species, and this technique represents a possible method for production of transgenic equids. Objectives: To evaluate the uptake of exogenous DNA (enhanced green fluorescent protein; pEGFP) by equine spermatozoa and to assess the ability of transfected spermatozoa to introduce this transgene into early equine embryos. Methods: To evaluate incorporation of pEGFP into equine spermatozoa, washed spermatozoa were incubated with 32P-pEGFP, with or without lipofection. Spermatozoa were also transfected with fluorescently-labelled DNA (Alexa647 -pEGFP) and changes in sperm viability and DNA uptake were assessed. Mares were inseminated with pEGFP-transfected spermatozoa and embryos recovered. Expression of pEGFP was assessed by epifluorescence microscopy of embryos, and the presence of pEGFP DNA and mRNA was assessed by PCR and RT-PCR, respectively. Results: Liposome-mediated transfection increased the incorporation of 32P-pEGFP into spermatozoa compared to controls. Flow cytometric evaluation of spermatozoa after transfection with Alexa647 -pEGFP revealed a linear increase in the proportion of live, Alexa647+ spermatozoa with increasing DNA concentrations. After insemination with transfected spermatozoa, 8 embryos were recovered. There was no evidence of EGFP expression in the recovered embryos; however, PCR analysis revealed evidence of the pEGFP transgene in 2 of 5 embryos analysed. Conclusions: The incorporation of exogenous DNA by equine spermatozoa was enhanced by liposome-mediated transfection and this did not adversely affect sperm viability, acrosomal integrity or fertility. Although the EGFP transgene was detected in a proportion of Day 7,10 embryos, there was no evidence of expression of EGFP in these embryos. Potential relevance: Sperm-mediated gene transfer offers a potential technique for the generation of transgenic equids. [source] MHC-restricted T cell receptor signaling is required for ,,,TCR replacement of the pre T cell receptorEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 2 2008Andrew 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] The tyrosine kinase Syk is required for light chain isotype exclusion but dispensable for the negative selection of B,cellsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 4 2004Josephine Meade Abstract In this study we set out to test whether Syk was required for negative selection of immature B,cells. B,cells expressing a B,cell antigen receptor (BCR) transgene (3,83, anti-H-2Kk) underwent negative selection independently of Syk in both fetal liver organ culture and radiation chimera models. Furthermore, Syk-independent negative selection was not reversed by transgenic overexpression of Bcl-2. Receptor editing was not apparent in Syk-deficient B,cells, presumably as a consequence of the failure of mature edited B,cells to develop in the absence of Syk. Interestingly, light chain isotype exclusion by the BCR transgene failed in the absence of Syk. We observed a dramatic reduction in the overall BCR-mediated tyrosine phosphorylation of cellular proteins in Syk-deficient immature B,cells. However, the tyrosine phosphorylation of a number of substrates including phospholipase,C,2, although reduced, was not completely abrogated. BCR ligation triggered an increase in calcium flux in the absence of Syk. Thus signaling events that mediate negative selection can still occur in the absence of Syk. This may be due to redundancy with zeta-associated protein,70 (ZAP-70), which we demonstrate to be expressed in immature B,cells. [source] A hVIPR transgene as a novel tool for the analysis of circadian function in the mouse suprachiasmatic nucleusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2003V. M. King Abstract A mouse bearing a novel transgene encoding the human VPAC2 receptor (hVIPR; Shen et al. (2000) PNAS, 97, 11575,11580) was used to investigate circadian function in the hypothalamic suprachiasmatic nuclei (SCN). Neurons expressing hVPAC2R, detected by a beta-galactosidase (,-GAL) tag, have a distinct distribution within the SCN, closely matching that of neurophysin (NP) neurons and extending into the region of peptide histidine isoleucine (PHI) cells. In common with NP and PHI cells, neurons expressing hVPAC2R are circadian in nature, as revealed by synchronous rhythmic expression of mPERIOD (mPER) proteins. A population of SCN cells not expressing PHI, NP or hVPAC2R exhibited circadian PER expression antiphasic with the rest of the SCN. Nocturnal light exposure induced mPER1 in the ventral SCN and mPER2 widely across the nucleus. Induction of nuclear mPER2 in hVPAC2R cells confirmed their photic responsiveness. Having established their circadian properties, we tested the utility of SCN neurons expressing the hVIPR transgene as functionally and anatomically explicit markers for SCN tissue grafts. Prenatal SCN tissue from hVIPR transgenic pups survived transplantation into adult CD1 mice, and expressed ,-GAL, PER and PHI. Over a series of studies, hVIPR transgenic SCN grafts restored circadian activity rhythms to 17 of 72 arrhythmic SCN lesioned recipients (23.6%). By using heterozygous hVIPR transgenic grafts on a heterozygous Clock mutant background we confirmed that restored activity rhythms were conferred by the donor tissue. We conclude that the hVIPR transgene is a powerful and flexible tool for examination of circadian function in the mouse SCN. [source] A hVIPR transgene as a novel tool for the analysis of circadian function in the mouse suprachiasmatic nucleusEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2003V.M. King No abstract is available for this article. [source] A metapopulation model for the introgression from genetically modified plants into their wild relativesEVOLUTIONARY APPLICATIONS (ELECTRONIC), Issue 2 2009Patrick G. Meirmans Abstract Most models on introgression from genetically modified (GM) plants have focused on small spatial scales, modelling gene flow from a field containing GM plants into a single adjacent population of a wild relative. Here, we present a model to study the effect of introgression from multiple plantations into the whole metapopulation of the wild relative. The most important result of the model is that even very low levels of introgression and selection can lead to a high probability that the transgene goes to fixation in the metapopulation. Furthermore, the overall frequency of the transgene in the metapopulation, after a certain number of generations of introgression, depends on the population dynamics. If there is a high rate of migration or a high rate of population turnover, the overall transgene frequency is much higher than with lower rates. However, under an island model of population structure, this increased frequency has only a very small effect on the probability of fixation of the transgene. Considering these results, studies on the potential ecological risks of introgression from GM plants should look not only at the rate of introgression and selection acting on the transgene, but also at the metapopulation dynamics of the wild relative. [source] | ||||||||||||||||||||||||||||||||||||||||||||||