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GFP Reporter (gfp + reporter)
Selected AbstractsThe acyltransferase gene bus-1 exhibits conserved and specific expression in nematode rectal cells and reveals pathogen-induced cell swellingDEVELOPMENTAL DYNAMICS, Issue 12 2008Maria J. Gravato-Nobre Abstract Susceptibility to the rectal pathogen Microbacterium nematophilum provides a means of examining hindgut differentiation in C. elegans. Mutants of bus - 1 are resistant to infection with this pathogen. We show here that bus - 1 encodes a predicted acyltransferase expressed in rectal epithelial cells (K, F, and U), suggesting its involvement in regional surface modification. bus - 1 reporter genes were used to show spatial regulation by hindgut developmental control genes: egl - 38, mab - 9, and mab - 23. A bus - 1::GFP reporter reveals the conspicuous rectal epithelial swelling induced by M. nematophilum. The C. briggsae ortholog of bus - 1 exhibits conserved function and rectal expression, but it is expressed in vulval as well as rectal cells, correlated with pathogen adhesion to both vulval and rectal cells in this species. Another acyltransferase affecting bacterial adhesion, bus - 18/acl - 10, was also identified, which also shows strong rectal expression, but it is expressed in additional epithelial tissues and is required for general surface integrity. Developmental Dynamics 237:3762,3776, 2008. © 2008 Wiley-Liss, Inc. [source] Isolation and characterization of neural precursor cells from the Sox1,GFP reporter mouseEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 7 2005Perrine Barraud Abstract We have made use of a reporter mouse line in which enhanced green fluorescence protein (GFP) is inserted into the Sox1 locus. We show that the GFP reporter is coexpressed with the Sox1 protein as well as with other known markers for neural stem and progenitor cells, and can be used to identify and isolate these cells by fluorescence-activated cell sorting (FACS) from the developing or adult brain and from neurosphere cultures. All neurosphere-forming cells with the capacity for multipotency and self-renewal reside in the Sox1,GFP-expressing population. Thus, the Sox1,GFP reporter system is highly useful for identification, isolation and characterization of neural stem and progenitor cells, as well as for the validation of alternative means for isolating neural stem and progenitor cells. Further, transplantation experiments show that Sox1,GFP cells isolated from the foetal brain give rise to neurons and glia in vivo, and that many of the neurons display phenotypic characteristics appropriate for the developing brain region from which the Sox1,GFP precursors were derived. On the other hand, Sox1,GFP cells isolated from the adult subventricular zone or expanded neurosphere cultures gave rise almost exclusively to glial cells following transplantation. Thus, not all Sox1,GFP cells possess the same capacity for neuronal differentiation in vivo. [source] Toxicity of short-chain alcohols to the nematode Caenorhabditis elegans: A comparison of endpointsJOURNAL OF BIOCHEMICAL AND MOLECULAR TOXICOLOGY, Issue 2 2005Grace Thompson Abstract The toxicities of 4 short-chain alcohols,namely methanol, ethanol, iso -propanol and iso -butanol,were compared in the nematode Caenorhabditis elegans using several different ecotoxicological endpoints. Range-finding tests were conducted using transgenic PC161 worms carrying a double reporter construct (GFP plus lacZ) linked to the stress-inducible hsp16-1 promoter. These tests showed little response from the GFP reporter, but gave good dose,response curves for the lacZ reporter,showing clear induction at 0.5% v/v ethanol in an overnight assay, but only at 4% in a shorter 6-h assay. Comparison of the short-term dose,response curves shows a confusing pattern of differences between the four alcohols tested, although dose-dependence is evident across at least part of the concentration range. Feeding inhibition assays are somewhat inconclusive with regard to alcohol type, although iso -butanol and iso -propanol appear more toxic than ethanol, while methanol is least toxic. To resolve some of the remaining ambiguities, we also used a fecundity assay to show that iso -propanol is more toxic than ethanol, and a lethality assay to show that iso -butanol is more toxic than iso -propanol. Most of the endpoints studied are consistent with the following order of toxicity: iso -butanol > iso -propanol > ethanol , methanol. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 19:87,95, 2005; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20060 [source] Identification of the Tctex-1 regulatory element that directs expression to neural stem/progenitor cells in developing and adult brainTHE JOURNAL OF COMPARATIVE NEUROLOGY, Issue 16 2010Yun-Yu Tseng Abstract Previous studies showed that Tctex-1 immunoreactivity is selectively enriched in the germinal zones of adult brain. In this report we identify a regulatory region of the Tctex-1 gene that is capable of directing transgenic expression of green fluorescent protein (GFP) reporter that recapitulates the spatial and temporal expression pattern of endogenous Tctex-1. This construct specifically targeted expression to the nestin+/Pax6+/GLAST+ radial glial cells and Tbr2+ intermediate progenitors when the reporter construct was delivered to developing mouse neocortex via in utero electroporation. Characterization of mice transgenically expressing GFP under the same regulatory element showed that the GFP expression is faithful to endogenous Tctex-1 at the subgranular zone (SGZ) of dentate gyrus, ventricular/subventricular zone of lateral ventricles, and ependymal layer of 3rd ventricle of adult brains. Immunolocalization and bromodeoxyuridine incorporation studies of adult SGZ in four independent mouse lines showed that Tctex-1:GFP reporter selectively marks nestin+/GFAP+/Sox2+ neural stem-like cells in two mouse lines (4 and 13). In two other mouse lines (17 and 18), Tctex-1:GFP is selectively expressed in Type-2 and Type-3 transient amplifying progenitors and a small subset of young neuronal progeny. The P/E-Tctex-1 reporter mouse studies independently confirmed the specific enrichment of Tctex-1 at adult SGZ stem/progenitor cells. Furthermore, these studies supported the notion that an analogous transcriptional program may be used to regulate neurogenesis in embryonic cerebral cortex and adult hippocampus. Finally, the genomic sequences and the reporter mouse lines described here provide useful experimental tools to advance adult neural stem cell research. J. Comp. Neurol. 518:3327,3342, 2010. © 2010 Wiley-Liss, Inc. [source] The effect of Bcl-2, YAMA, and XIAP over-expression on apoptosis and adenovirus production in HEK293 cell lineBIOTECHNOLOGY & BIOENGINEERING, Issue 4 2009Kalbinder Singh Sandhu Abstract Many viruses induce cell death and lysis as part of their replication and dissemination strategy, and in many cases features of apoptosis are observed. Attempts have been made to further increase productivity by prolonging cell survival via the over-expression of anti-apoptotic genes. Here, we extend the study to investigate the association between virus replication and apoptosis, pertinent to large-scale vector production for gene therapy. Infection of an HEK293 cell line with a replication defective type-5-adenovirus expressing a GFP reporter (Ad5GFP) resulted in rapid decline in viability associated with increased virus titer. The over-expression of bcl-2 resulted in improved cell resistance to apoptosis and prolonged culture duration, but reduced virus specific and total productivity. In contrast, the over-expression of pro-caspase-3 (Yama/CPP32/apopain) resulted in reduced cell survival but increased virus productivity. The treatment of infected cells with caspase inhibitors support the preposition that caspase-3 dependent apoptosis, and to a lesser degree caspase-9 dependent apoptosis, represent important steps in virus production, thus implicating the intrinsic apoptosis pathway in the production of adenovirus from HEK293 cells. The suppression of apoptosis by the over-expression of XIAP (inhibitors of caspase family cell death proteases) further shows that caspase-mediated activation plays an important role in virus infection and maturation. Biotechnol. Bioeng. 2009; 104: 752,765 © 2009 Wiley Periodicals, Inc. [source] Sigma factor selectivity in Borrelia burgdorferi: RpoS recognition of the ospE/ospF/elp promoters is dependent on the sequence of the ,10 regionMOLECULAR MICROBIOLOGY, Issue 6 2006Christian H. Eggers Summary Members of the ospE/ospF/elp lipoprotein gene families of Borrelia burgdorferi, the Lyme disease agent, are transcriptionally upregulated in response to the influx of blood into the midgut of an infected tick. We recently have demonstrated that despite the high degree of similarity between the promoters of the ospF (PospF) and ospE (PospE) genes of B. burgdorferi strain 297, the differential expression of ospF is RpoS-dependent, while ospE is controlled by ,70. Herein we used wild-type and RpoS-deficient strains of B. burgdorferi and Escherichia coli to analyse transcriptional reporters consisting of a green fluorescent protein (gfp) gene fused to PospF, PospE, or two hybrid promoters in which the ,10 regions of PospF and PospE were switched [PospF (E , 10) and PospE,(F , 10) respectively]. We found that the PospF,10 region is both necessary and sufficient for RpoS-dependent recognition in B. burgdorferi, while ,70 specificity for PospE is dependent on elements outside of the ,10 region. In E. coli, sigma factor selectivity for these promoters was much more permissive, with expression of each being primarily due to ,70. Alignment of the sequences upstream of each of the ospE/ospF/elp genes from B. burgdorferi strains 297 and B31 revealed that two B31 ospF paralogues [erpK (BBM38) and erpL (BBO39)] have ,10 regions virtually identical to that of PospF. Correspondingly, expression of gfp reporters based on the erpK and erpL promoters was RpoS-dependent. Thus, the sequence of the PospF,10 region appears to serve as a motif for RpoS recognition, the first described for any B. burgdorferi promoter. Taken together, our data support the notion that B. burgdorferi utilizes sequence differences at the ,10 region as one mechanism for maintaining the transcriptional integrity of RpoS-dependent and -independent genes activated at the onset of tick feeding. [source] | ||||||||||