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EGFP Expression (egfp + expression)
Selected AbstractsLiposome-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] Identification of brain neurons expressing the dopamine D4 receptor gene using BAC transgenic miceEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 9 2006Daniela Noaín Abstract The dopamine D4 receptor (D4R) has received considerable interest because of its higher affinity for atypical antipsychotics, the extremely polymorphic nature of the human gene and the genetic association with attention deficit and hyperactivity disorder (ADHD). Several efforts have been undertaken to determine the D4R expression pattern in the brain using immunohistochemistry, binding autoradiography and in situ hybridization, but the overall published results present large discrepancies. Here, we have explored an alternative genetic approach by studying bacterial artificial chromosome (BAC) transgenic mice that express enhanced green fluorescent protein (EGFP) under the transcriptional control of the mouse dopamine D4 receptor gene (Drd4). Immunohistochemical analysis performed in brain sections of Drd4 -EGFP transgenic mice using an anti-EGFP polyclonal antibody showed that transgenic expression was predominant in deep layer neurons of the prefrontal cortex, particularly in the orbital, prelimbic, cingulate and rostral agranular portions. In addition, discrete groups of Drd4 -EGFP labelled neurons were observed in the anterior olfactory nucleus, ventral pallidum, and lateral parabrachial nucleus. EGFP was not detected in the striatum, hippocampus or midbrain as described using other techniques. Given the fine specificity of EGFP expression in BAC transgenic mice and the high sensitivity of the EGFP antibody used in this study, our results indicate that Drd4 expression in the adult mouse brain is limited to a more restricted number of areas than previously reported. Its leading expression in the prefrontal cortex supports the importance of the D4R in complex behaviours depending on cortical dopamine (DA) transmission and its possible role in the etiopathophysiology of ADHD. [source] Dynamics of a Transgene Expression in Acute Rat Brain Slices Transfected with Adenoviral VectorsEXPERIMENTAL PHYSIOLOGY, Issue 4 2003C. E. L. Stokes We present a quantitative account of the expression dynamics of a transgene (enhanced green fluorescent protein, EGFP) in acute brain slices transfected with an adenoviral vector (AVV) under control of the human cytomegalovirus (HCMV) promoter. Micromolar concentrations of EGFP could be detected in brainstem and hippocampal slices as early as 7 h after in vitro transfection with a viral titre of 4.4 × 109 plaque-forming units (pfu) ml,1. Although initially EGFP appeared mainly in glia, it could be detected in neurones with longer incubation times of 10-12 h. However, fluorescence was never detected within some populations of neurones, such as hippocampal pyramidal cells, or within the hypoglossal motor nucleus. The density of cells expressing EGFP peaked at 10 h and then decreased, possibly suggesting that high concentrations of EGFP are toxic. The age of the animal significantly affected the speed of EGFP accumulation: after 10 h of incubation in 30-day-old rats only 4.88 ± 0.51 cells/10 000 ,m2 were fluorescent compared to 7.28 ± 0.39 cells/10 000 ,m2 in 12-day-old rats (P < 0.05). HCMV promoter-driven transgene expression depended on the activity of protein kinase A, and was depressed with a cAMP/protein kinase A antagonist (20 ,M Rp-cAMPS; P < 0.0005). This indicates that expression of HCMV-driven constructs is likely to be skewed towards cellular populations where cAMP-dependent signalling pathways are active. We conclude that acute transfection of brain slices with AVVs within hours causes EGFP expression in micromolar concentrations and that such transfected cells may remain viable for use in physiological experiments. [source] Heterogeneity of Kir4.1 channel expression in glia revealed by mouse transgenesisGLIA, Issue 16 2009Xiaofang Tang Abstract The weakly inwardly rectifying K+ channel Kir4.1 is found in many glial cells including astrocytes. However, questions remain regarding the relative contribution of Kir4.1 to the resting K+ conductance of mature astrocytes in situ. We employed a bacterial artificial chromosome transgenic approach in mice to visualize Kir4.1 expression in vivo. These mice (Kir4.1-EGFP) express enhanced green fluorescent protein (EGFP) under the transcriptional control of the Kir4.1 promoter. The brains of adult Kir4.1-EGFP transgenic mice showed co-expression of EGFP and Kir4.1 in astrocytes. In addition, weaker expression of EGFP was detected in NG2+ glial cells when compared with EGFP expression in GFAP+ glial cells. Whole-cell voltage clamp recordings of EGFP+ glial cells in the CA1 area of the adult mouse hippocampus indicated astrocytes displaying properties consistent with both the "passive" and "complex" subpopulations. EGFP+ cells with bright fluorescence had the linear current,voltage (I,V) relationships and extensive gap junctional coupling characteristic of passive astrocytes. However, EGFP+ glia with weaker fluorescence displayed properties associated with complex astrocytes including nonlinear I,V relationships and lack of intercellular gap junctional coupling. Pharmacological blockade of inward currents implied that Kir4.1 channels constitute the dominant resting K+ conductance in both glial cell types and are more highly expressed in passive astrocytes. These results suggest differential expression of Kir4.1 in glia and that this channel likely underlies the resting K+ conductance in passive and complex astrocytes. © 2009 Wiley-Liss, Inc. [source] Validation of novel promoter sequences derived from two endogenous ubiquitin genes in transgenic Aedes aegyptiINSECT MOLECULAR BIOLOGY, Issue 4 2010M. A. E. Anderson Abstract To date, only a limited number of promoter sequences have been described to drive transgene expression in the disease vector Aedes aegypti. We sought to increase this repertoire by characterizing the ability of upstream sequences derived from the Ae. aegypti UbL40 and polyubiquitin genes to drive the expression of marker proteins. Both genomic fragments were able to drive robust expression of luciferase in cultured mosquito cells. Following Mos1-transformation, the UbL40 promoter drove strong expression of a fluorescent marker in early larvae and in ovaries, while the polyubiquitin promoter drove robust EGFP expression in all stages of development, including constitutive expression throughout the midgut. These promoter fragments provide two new expression profiles for future Ae. aegypti genetic experiments. [source] Tissue- and agonist-specific regulation of human and murine plasminogen activator inhibitor-1 promoters in transgenic miceJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 11 2003M. Eren Summary., Numerous studies have described regulatory factors and sequences that control transcriptional responses in vitro. However, there is a paucity of information on the qualitative and quantitative regulation of heterologous promoters using transgenic strategies. In order to investigate the physiological regulation of human plasminogen activator inhibitor type-1 (hPAI-1) expression in vivo compared to murine PAI-1 (mPAI-1) and to test the physiological relevance of regulatory mechanisms described in vitro, we generated transgenic mice expressing enhanced green fluorescent protein (EGFP) driven by the proximal ,2.9 kb of the hPAI-1 promoter. Transgenic animals were treated with Ang II, TGF-,1 and lipopolysaccharide (LPS) to compare the relative activation of the human and murine PAI-1 promoters. Ang II increased EGFP expression most effectively in brain, kidney and spleen, while mPAI-1 expression was quantitatively enhanced most prominently in heart and spleen. TGF-,1 failed to induce activation of the hPAI-1 promoter but potently stimulated mPAI-1 in kidney and spleen. LPS administration triggered robust expression of mPAI-1 in liver, kidney, pancreas, spleen and lung, while EGFP was induced only modestly in heart and kidney. These results indicate that the transcriptional response of the endogenous mPAI-1 promoter varies widely in terms of location and magnitude of response to specific stimuli. Moreover, the physiological regulation of PAI-1 expression likely involves a complex interaction of transcription factors and DNA sequences that are not adequately replicated by in vitro functional studies focused on the proximal ,2.9 kb promoter. [source] Transgenic sperm produced by electrotransfection and allogeneic transplantation of chicken fetal spermatogonial stem cellsMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2010Fei Yu To study self-renewal, genetic modification, and differentiation of avian spermatogonial stem cells (SSCs), we isolated chicken SSCs from fetal testes on the 16th hatching day via enzyme digestion, and then cultured the SSCs over 2 months after purification in vitro. SSCs were identified by alkaline phosphatase staining and SSEA-1 fluorescence. The EGFP gene was transfected into SSCs by three different methods: electroporation, liposome transfer and calcium acid phosphate precipitation. The transfection rate and cell survival rate using electroporation were higher than when using liposomes or calcium acid phosphate (20.52% vs. 9.75% and 5.61%; 69.86% vs. 65.00% and 51.16%, respectively). After selection with G418 for 8 days, the transgenic SSCs were transplanted into the testes of cocks treated with busulfan. Twenty-five days after transplantation, the recipients' semen was light ivory in color, and the density of spermatozoa was 3.87 (×107/ml), with 4.25% expressing EGFP. By 85 days after transplantation, the number of spermatozoa increased to 32.7 (×107/ml) and the rate of EGFP expression was 16.25%. Frozen sections of the recipients' testes showed that transgenic SSCs were located on the basal membrane of the seminiferous tubules and differentiated into spermatogenic cells at different stages. The EGFP gene was successfully amplified from the DNA of all recipients' semen samples. Mol. Reprod. Dev. 77: 340,347, 2010. © 2010 Wiley-Liss, Inc. [source] The proximal promoter governs germ cell-specific expression of the mouse glutathione transferase mGstm5 geneMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2009Hironari Dehari To explain the tissue-selective expression patterns of a distinct subclass of glutathione S -transferase (GST), transgenic mice expressing EGFP under control of a 2 kb promoter sequence in the 5,-flanking region of the mGstm5 gene were produced. The intent of the study was to establish whether the promoter itself or whether posttranscriptional mechanisms, particularly at the levels of mRNA translation and stability or protein targeting, based on unique properties of mGSTM5, determine the restricted expression pattern. Indeed, the transgene expression was limited to testis as the reporter was not detected in somatic tissues such as brain, kidney or liver, indicating that the mGstm5 proximal promoter is sufficient to target testis-specific expression of the gene. EGFP expression was also more restricted vis-a-vis the natural mGstm5 gene and exclusively found in germ but not in somatic cells. Real-time quantitative PCR (qPCR) data were consistent with alternate transcription start sites in which the promoter region of the natural mGstm5 gene in somatic cells is part of exon 1 of the germ cell transcript. Thus, the primary transcription start site for mGstm5 is upstream of a TATA box in testis and downstream of this motif in somatic cells. The 5, flanking sequence of the mGstm5 gene imparts germ cell-specific transcription. Mol. Reprod. Dev. 76: 379,388, 2009. © 2008 Wiley-Liss, Inc. [source] Restricted transgene persistence after lentiviral vector-mediated fetal gene transfer in the pregnant rabbit modelTHE JOURNAL OF GENE MEDICINE, Issue 9 2008Rafael Moreno Abstract Background Prenatal gene transfer may enable early causal intervention for the treatment or prevention of many devastating diseases. Nevertheless, permanent correction of most inherited disorders requires a sustained level of expression from the therapeutic transgene, which could theoretically be achieved with integrating vectors. Methods Rabbit fetuses received 8.5 × 106 HIV-based recombinant lentivirus particles containing the enhanced green fluorescent protein (EGFP) transgene by intrahepatic, intra-amniotic or intraperitoneal injection at 22 days of gestation. Provirus presence and transgene expression in rabbit tissues were evaluated at both 1.5 and 16 weeks post- in utero intervention by polymerase chain reaction (PCR) and reverse transcriptase-PCR, respectively. Moreover, we assessed persistence of EGFP by immunohistochemistry. Enzyme-linked immunosorbent assays confirmed the development of antibodies specific against both the viral vector and the reporter protein. Results Regardless of the route of administration employed, lentiviral vector-based in utero gene transfer was safe and reached 85% of the intervened fetuses at birth. However, the integrated provirus frequency was significantly reduced to 50% of that in young rabbits at 16 weeks post-treatment. In these animals, EGFP expression was evident in many tissues, including cytokeratin 5-rich basal cells from stratified and pseudostratified epithelia, suggesting that the lentiviral vector might have reached progenitor cells. Conversely, we identified the presence of immune-inflammatory infiltrates in several EGFP-expressing tissues. Moreover, almost 70% of the lentiviral vector-treated rabbits elicited a humoral immune response against the viral envelope and/or the EGFP. Conclusions At two-thirds gestational age, the adaptive immune system of the rabbit appears a relevant factor limiting transgene persistence and expression following lentiviral vector-mediated in utero gene transfer. Copyright © 2008 John Wiley & Sons, Ltd. [source] Sequence-specific gene silencing in murine muscle induced by electroporation-mediated transfer of short interfering RNATHE JOURNAL OF GENE MEDICINE, Issue 1 2004Tsunao Kishida Abstract Background Post-genomic biomedical research requires efficient techniques for functional analyses of poorly characterized genes in living organisms. Sequence-specific gene silencing in mammalian organs may provide valuable information on the physiological and pathological roles of predicted genes in mammalian systems. Here, we attempted targeted gene knockdown in vivo in murine skeletal muscle through the electroporation-mediated transfer of short interfering RNA (siRNA). Methods siRNA duplexes corresponding to the firefly luciferase (Luc), green fluorescent protein (GFP), or glyceraldehyde-3-phosphate dehydrogenase (GAPD) genes were delivered by electroporation into the tibial muscle of normal or enhanced GFP (EGFP) transgenic mice. Plasmid vectors carrying the Luc, hRluc or ,-galactosidase (,-gal) reporter genes were also delivered. The Luc and hRluc activities in the muscle lysates were assayed. The EGFP and GAPD expression was detected by fluorescence microscopic observation and RT-PCR, respectively. Results When Luc-specific siRNA was co-delivered with the Luc expression vector into the tibial muscle, the reporter gene expression was markedly suppressed (less than 1% of the control level) for 5 days. As little as 0.05 µg of siRNA almost completely blocked the reporter gene expression from 10 µg of the plasmid. To examine whether siRNA can also suppress expression of an endogenous gene, transgenic mice carrying the EGFP gene received intramuscular transfection of a mixture of ,-gal plasmid and GFP-specific siRNA. ,-Gal-positive cells failed to express detectable levels of EGFP, while EGFP expression was not inhibited in control mice that received nonspecific siRNA. Expression of GAPD was also suppressed by the specific siRNA. Conclusions The present system may provide a useful means of phenotypic analysis of genetic information in mammalian organs for basic research as well as therapeutic molecular targeting in the post-genomic era. Copyright © 2003 John Wiley & Sons, Ltd. [source] Evaluation of Production Parameters with the Vaccinia Virus Expression System Using Microcarrier Attached HeLa CellsBIOTECHNOLOGY PROGRESS, Issue 2 2005Nicole A. Bleckwenn Parameters that affect production of the recombinant reporter protein, EGFP, in the T7 promoter based VOTE vaccinia virus-HeLa cell expression system were examined. Length of infection phase, inducer concentration, and timing of its addition relative to infection were evaluated in 6-well plate monolayer cultures. One hour infection with 1.0 mM IPTG added at the time of infection provided a robust process. For larger scale experiments, anchorage-dependent HeLa cells were grown on 5 g/L Cytodex 3 microcarriers. The change to this dynamic culture environment, with cell-covered microcarriers suspended in culture medium in spinner flasks, suggested a re-examination of the multiplicity of infection (MOI) for this culture type that indicated a need for an increase in the number of virus particles per cell to 5.0, higher than that needed for complete infection in monolayer tissue flask culture. Additionally, dissolved oxygen level and temperature during the protein production phase were evaluated for their effect on EGFP expression in microcarrier spinner flask culture. Both increased dissolved oxygen, based on surface area to volume (SA/V) adjustments, and decreased temperature from 37 to 31 °C showed increases in EGFP production over the course of the production phase. The level of production achieved with this system reached approximately 17 ,g EGFP/106 infected cells. [source] Retrovirus-mediated transfer of the herpes simplex virus thymidine kinase and enhanced green fluorescence protein genes in primary T lymphocytesBRITISH JOURNAL OF HAEMATOLOGY, Issue 4 2000Sabrina Di Florio The EGFP-tk retroviral vector, encoding enhanced green fluorescent protein (EGFP) and the herpes simplex virus thymidine kinase (HSV-tk) packaged in a Phoenix amphotropic cell line, was used to transduce healthy donor T lymphocytes. Infection yielded a mean of 41·8 ± 9·3% SD (range 31·1,48·4%) EGFP-positive cells and a mean of 92 ± 2% SD (range 90,94%) after cell sorting. EGFP expression remained stable for 30 d after infection. The entire gene transfer procedure had no significant effect on lymphocyte subsets and slightly reduced clonogenicity. Ganciclovir (gcv) treatment (1 µg/ml × 10 d) killed all EGFP-positive cells in the transduced and transduced/sorted populations, but had no effect on untransduced controls. Our results show that primary T lymphocytes can be transduced using an EGFP-tk vector that yields a homogeneous infected population without affecting lymphocyte subsets, function and clonogenicity. [source] FLUORESCENCE ACTIVATED CELL SORTING OF TRANSIENTLY TRANSFECTED As4.1 CELLS SHOWS RENIN ENHANCER DIRECTS ON/OFF SWITCHING OF RENIN PROMOTER IN VITROCLINICAL AND EXPERIMENTAL PHARMACOLOGY AND PHYSIOLOGY, Issue 4 2008Brian J MorrisArticle first published online: 27 FEB 200 SUMMARY 1The proximal promoter of the renin gene is weak and its activity is influenced by a strong, far-upstream enhancer. This and the ability of renin expression in renal afferent arteriolar cells to be ,recruited' under chronic stimulation is consistent with the on/off switching (variegation) model of gene expression. If true, this would provide an example in which variegation controls a physiologically regulable gene. 2The present study tested the hypothesis that renin promoter activity may accord with the variegation model, at least in individual juxtaglomerular (mouse As4.1) cells in vitro. 3As4.1 cells were transiently transfected with constructs containing the mouse renin (Ren-1c) enhancer adjacent to the Ren-1c promoter and a linked reporter gene encoding enhanced green fluorescent protein (EGFP). The EGFP signal from individual cells was monitored by fluorescence activated cell sorting. 4In the presence of the renin enhancer there was 10-fold higher EGFP expression in transfected cells compared with cells transfected with EGFP constructs containing the promoter alone. There was, moreover, an 8-fold increase in the number of EGFP expressing cells. However, EGFP expression in individual transfected cells was similar in the presence or absence of the enhancer. 5Results from the in vitro system used suggest that the Ren-1c enhancer does not regulate the rate of promoter activity, but rather increases the probability of achieving an active transcriptional state. Limitations of these findings are discussed. [source] Zebrafish sp7:EGFP: A transgenic for studying otic vesicle formation, skeletogenesis, and bone regenerationGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 8 2010April DeLaurier Adult transgenic zebrafish expressing eGFP under the control of the zinc finger transcription factor Sp7 gene, which is expressed in osteoblasts but not chondrocytes. In this line, eGFP expression recapitulates the endogenous gene pattern of expression in the otic placode, otic vesicle and developing skeletal structures. GFP-positive cells are also observed in adult skeletal structures and in regenerating fins. This transgenic line will be a very useful tool for studying otic development and the development and regeneration of the skeleton. See the article by DeLaurier et al. in this issue. [source] | |||||||||||||||||||||||||