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Reporter Vectors (reporter + vector)
Selected AbstractsA new inducible adenoviral expression system that responds to inflammatory stimuli in vivoTHE JOURNAL OF GENE MEDICINE, Issue 12 2006Gang Cai Abstract Background Gene transfer using inducible promoters, which control expression of transgenic proteins in response to physiological conditions, may have significant advantages. In this study, we tried to achieve an inducible adenoviral expression system for physiologically responsive gene therapy of autoimmune or inflammatory diseases. Methods A luciferase reporter vector with a hybrid promoter containing the human IL-1, enhancer region (,3690 to , 2720) and the human CIITA promoter IV (,399 to + 2) was constructed. A replication-deficient adenovirus was engineered with luciferase controlled by the IL1,/CIITApIV promoter (Ad-IL1,/CIITApIV-Luc). The reporter vector or adenovirus was transfected to C57Bl/6 myeloid dendritic cells (DCs), RAW264.7, and Hep G2 to study the in vitro characteristics of this hybrid promoter. An inflammation model was prepared by injecting lipopolysaccharide (LPS) into Balb/c mice intraperitoneally (i.p.), and infected with Ad-IL1,/CIITApIV-Luc or Ad-CMV-Luc to study the in vivo characteristics of the IL1,/CIITApIV promoter. Results The IL1,/CIITApIV hybrid promoter has pronounced promoter activity, broad-range responsiveness to cytokines or LPS, and can be rechallenged after first induction. In the inflammation model, IL1,/CIITApIV could drive hepatic luciferase expression increasedly rapidly after LPS challenge and in a LPS dose-dependent manner. Conclusions Using the IL1,/CIITApIV hybrid promoter in gene transfer vectors may make it possible to produce transgenic proteins in vivo in direct relationship with the intensity and duration of an individual's status. By providing endogenously controlled production of transgenic proteins, this approach might limit the severity of autoimmune or inflammatory response without interfering with the beneficial components of host defense and immunity. Copyright © 2006 John Wiley & Sons, Ltd. [source] Reproducible fashion of the HSP70B' promoter-induced cytotoxic response on a live cell-based biosensor by cell cycle synchronizationBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2010Satoshi Migita Abstract Live cell-based sensors potentially provide functional information about the cytotoxic effect of reagents on various signaling cascades. Cells transfected with a reporter vector derived from a cytotoxic response promoter can be used as intelligent cytotoxicity sensors (i.e., sensor cells). We have combined sensor cells and a microfluidic cell culture system that can achieve several laminar flows, resulting in a reliable high-throughput cytotoxicity detection system. These sensor cells can also be applied to single cell arrays. However, it is difficult to detect a cellular response in a single cell array, due to the heterogeneous response of sensor cells. The objective of this study was cell homogenization with cell cycle synchronization to enhance the response of cell-based biosensors. Our previously established stable sensor cells were brought into cell cycle synchronization under serum-starved conditions and we then investigated the cadmium chloride-induced cytotoxic response at the single cell level. The GFP positive rate of synchronized cells was approximately twice as high as that of the control cells, suggesting that cell homogenization is an important step when using cell-based biosensors with microdevices, such as a single cell array. Biotechnol. Bioeng. 2010;107: 561,565. © 2010 Wiley Periodicals, Inc. [source] Anti-miR-21 oligonucleotide sensitizes leukemic K562 cells to arsenic trioxide by inducing apoptosisCANCER SCIENCE, Issue 4 2010Yumin Li Arsenic trioxide (ATO), an ancient traditional Chinese medicine, has been successfully used as a therapeutic agent for leukemia. Drug resistance and toxicity are major concerns with the treatment. MicroRNAs (miRNAs) are endogenous small non-coding RNA molecules that might modulate cellular sensitivity to anticancer drugs. miRNA-21 (miR-21) is one of the most prominent miRNAs involved in various aspects of human cancers. However, miR-21 has been rarely characterized in chronic myelogenous leukemia (CML). Here, we used a specific anti-miR-21 oligonucleotide (AMO-miR-21) to sensitize K562 cells to ATO by degradation of miR-21. The results showed that both AMO-miR-21 and ATO caused growth inhibition, apoptosis, and G1-phase arrest in K562 cells. Meanwhile, AMO-miR-21 significantly promoted ATO-mediated growth inhibition and apotosis without affecting the G1 phase. Apoptotic cells were confirmed morphologically with Giemsa's staining. Furthermore, dual-luciferase reporter vector, containing two tandem miR-21 binding sites from PDCD4 3,UTR, validated that PDCD4 was directly regulated by miR-21. Therefore, AMO-miR-21 sensitized leukemic K562 cells to ATO by inducing apoptosis partially due to its up-regulation of PDCD4 protein level. The combination of ATO and AMO-miR-21 present therapeutic potential for CML. (Cancer Sci 2010; 101: 948,954) [source] Misregulation of gene expression in the redox-sensitive NF-,b-dependent limb outgrowth pathway by thalidomideDEVELOPMENTAL DYNAMICS, Issue 2 2002Jason M. Hansen Abstract Thalidomide is known to induce oxidative stress, but mechanisms have not been described through which oxidative stress could contribute to thalidomide-induced terata. Oxidative stress modulates intracellular glutathione (GSH) and redox status and can perturb redox-sensitive processes, such as transcription factor activation and/or binding. Nuclear factor-kappa B (NF-,B), a redox-sensitive transcription factor involved in limb outgrowth, may be modulated by thalidomide-induced redox shifts. Thalidomide-resistant Sprague-Dawley rat embryos (gestation day [GD] 13) treated with thalidomide in utero showed no changes in GSH distribution in the limb but thalidomide-sensitive New Zealand White rabbit embryos (GD 12) showed selective GSH depletion in the limb bud progress zone (PZ). NF-,B and regulatory genes that initiate and maintain limb outgrowth and development, such as Twist and Fgf-10, are selectively expressed in the PZ. Green fluorescent protein (GFP) reporter vectors containing NF-,B binding promoter sites were transfected into both rat and rabbit limb bud cells (LBCs). Treatment with thalidomide caused a preferential decrease in GFP expression in rabbit LBCs but not in rat LBCs. N-acetylcysteine and ,-N-t-phenylbutyl nitrone (PBN), a free radical trapping agent, rescued GFP expression in thalidomide-treated cultures compared with cultures that received thalidomide only. In situ hybridization showed a preferential decrease in Twist, Fgf-8, and Fgf-10 expression after thalidomide treatment (400 mg/kg per day) in rabbit embryos. Expression in rat embryos was not affected. Intravenous cotreatment with PBN and thalidomide (gavage) in rabbits restored normal patterns and localization of Twist, Fgf-8, and Fgf-10 expression. These findings show that NF-,B binding is diminished due to selective thalidomide-induced redox changes in the rabbit, resulting in the significant attenuation of expression of genes necessary for limb outgrowth. © 2002 Wiley-Liss, Inc. [source] New hemocyte-specific enhancer-reporter transgenes for the analysis of hematopoiesis in DrosophilaGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 11 2009Tsuyoshi Tokusumi Abstract Based on environmental challenges or altered genetic composition, Drosophila larvae can produce up to three types of blood cells that express genetic programs essential for their distinct functions. Using transcriptional enhancers for genes expressed exclusively in plasmatocytes, crystal cells, or lamellocytes, several new hemocyte-specific enhancer-reporter transgenes were generated to facilitate the analysis of Drosophila hematopoiesis. This approach took advantage of fluorescent variants of insulated P-element reporter vectors for multilabeling cell analyses; two additional color variants were generated in these studies. These vectors were successfully used to produce transgenic fly lines that label specific hemocyte lineages with separate colors. Combining three transgene reporters allowed for the unambiguous identification of plasmatocytes, crystal cells, and lamellocytes within a complex hemocyte population. While this work focused on the hematopoietic process, these new vectors can be used to mark multiple cell types or trace complex cell lineages during any chosen aspect of Drosophila development. genesis 47:771,774, 2009. © 2009 Wiley-Liss, Inc. [source] A haplotype of the catalase gene confers an increased risk of essential hypertension in Chinese Han,HUMAN MUTATION, Issue 3 2010Zhimin Wang Abstract Our previous study in an isolated population showed an association between a genetic variant in the catalase gene (CAT) and essential hypertension (EH). This study indicates that three variants in the promoter and 5,-UTR region of CAT are predominant in Chinese Han, and they form two major haplotypes. A case,control study showed that the CATH2 haplotype confers susceptibility to EH (Pgenotype=0.0017, and Pallilc=0.00078). Subjects bearing CATH1/CATH2 and CATH2/CATH2 genotypes demonstrated a higher susceptibility to EH than CATH1/CATH1 homozygotes, with odds ratios of 1.474 and 1.625, respectively. Also, CATH1/CATH1 individuals had a later-onset age (P=0.015). Expression analysis using luciferase reporter vectors indicated that the CATH1 haplotype showed a lower transcriptional activity than the haplotype CATH2 (P<0.05 in all four cell lines), and we observed similar results in the endogenous allelic expression ratios of CATH1/CATH2 in cell lines. In contrast, most CATH1 haplotypes showed a higher transcription level than CATH2 haplotypes (10 out of 11 or 90.9%) in blood from normal individuals (P<0.01). We therefore hypothesize that CATH1 and CATH2 may play alternating roles at different level of oxidative stress. Hum Mutat 31:272,278, 2010. © 2009 Wiley-Liss, Inc. [source] Retroviral vector silencing during iPS cell induction: An epigenetic beacon that signals distinct pluripotent statesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2008Akitsu Hotta Abstract Retroviral vectors are transcriptionally silent in pluripotent stem cells. This feature has been potently applied in studies that reprogram somatic cells into induced pluripotent stem (iPS) cells. By delivering the four Yamanaka factors in retroviral vectors, high expression is obtained in fibroblasts to induce the pluripotent state. Partial reprogramming generates Class I iPS cells that express the viral transgenes and endogenous pluripotency genes. Full-reprogramming in Class II iPS cells silences the vectors as the endogenous genes maintain the pluripotent state. Thus, retroviral vector silencing serves as a beacon marking the fully reprogrammed pluripotent state. Here we review known silencer elements, and the histone modifying and DNA methylation pathways, that silence retroviral and lentiviral vectors in pluripotent stem cells. Both retroviral and lentiviral vectors are influenced by position effects and often exhibit variegated expression. The best vector designs facilitate full-reprogramming and subsequent retroviral silencing, which is required for directed-differentiation. Current retroviral reprogramming methods can be immediately applied to create patient-specific iPS cell models of human disease, however, future clinical applications will require novel chemical or other reprogramming methods that reduce or eliminate the integrated vector copy number load. Nevertheless, retroviral vectors will continue to play an important role in genetically correcting patient iPS cell models. We anticipate that novel pluripotent-specific reporter vectors will select for isolation of high quality human iPS cell lines, and select against undifferentiated pluripotent cells during regenerative medicine to prevent teratoma formation after transplantation. J. Cell. Biochem. 105: 940,948, 2008. © 2008 Wiley-Liss, Inc. [source] CSRP2, TIMP-1, and SM22, promoter fragments direct hepatic stellate cell-specific transgene expression in vitro, but not in vivoLIVER INTERNATIONAL, Issue 1 2004Jens Herrmann Abstract: Background/Aims: The activation of hepatic stellate cells (HSC) and their transdifferentiation into myofibroblasts (MFB) is the key step for development of liver fibrosis. Over the past several years, significant progress has been made in the understanding of the critical pathways involved incells undergoing activation. Cellular activation in the course of transdifferentiation involves, among other biochemical modifications, functionally relevant changes in the control of gene expression. These include the up-regulation of transcription factors, different extracellular matrix proteins, cell adhesion molecules, smooth muscle specific genes, and proteins involved in matrix remodelling, or cytoskeletal organization. The corresponding regulatory elements of these genes have afforded us the opportunity to express transgenes with antifibrotic potential in a cell type- and/or transdifferentiation-dependent manner. Methods: In the present study, we have tested several promoters for their ability to mediate cell-specific expression, including those for CSRP2, SM22,, and TIMP-1 (CSRP2, gene encoding the LIM domain protein CRP2; SM22,, smooth muscle-specific gene encoding a 22-kDa protein; TIMP-1, gene encoding the tissue inhibitor of metalloproteinases-1), which in liver are specifically expressed in HSC or become strongly activated during the acute remodelling into MFB. We constructed adenoviral reporter vectors in which relevant portions of the promoters were fused to the green fluorescent protein. Results and Conclusion: Our experiments demonstrate that each of these promoters is sufficient to achieve strong or partially selective expression in vitro but none is able to direct a specific or inducible expression of transgenes in HSC/MFB in vivo. [source] Identification and Characterization of a DNase Hypersensitive Region of the Human Tyrosinase GenePIGMENT CELL & MELANOMA RESEARCH, Issue 6 2003James P. Fryer Mutations of the tyrosinase gene produce oculocutaneous albinism type 1 (OCA1). Most affected individuals are compound heterozygotes with different maternal and paternal mutations, but a substantial number of presumed tyrosinase alleles in these individuals have no identifiable mutation in the coding or proximal promoter region of the gene. This suggests that mutations in other regions of the gene, such as regulatory regions that are removed from the direct proximity of the coding sequence, may account for these currently unidentifiable mutations. The mouse tyrosinase gene has a distal enhancer or locus control region (LCR) that provides position-independent stimulation of gene expression, and a homologous regulatory region (HR) of the human gene could be the site of some of these mutations. We report a region 9 kb upstream of the human tyrosinase transcriptional start site that may be involved in regulation of this gene. Analysis of this region shows DNase I hypersensitivity in a cell lineage-specific pattern, a pattern indicative of regulatory regions of a gene. This region also has significant enhancer function when reporter vectors containing it are transfected into either human or mouse melanocyte cell lines, and elimination of specific sequences with homology to the mouse core enhancer in this region extinguishes the enhancer function. We believe that this region of homology contains sequences critical in the regulation of the human tyrosinase gene and is a candidate for the location of OCA1 mutations. [source] A comparative analysis of constitutive and cell-specific promoters in the adult mouse hippocampus using lentivirus vector-mediated gene transferTHE JOURNAL OF GENE MEDICINE, Issue 11 2008Hitoshi Kuroda Abstract Background Viral vectors provide powerful tools for transgene delivery to the mammalian brain to assess the effects of therapeutic proteins, antisense RNAs or small interfering RNAs. A key advantage of such approaches is that specific brain regions implicated in a particular disease can be independently targeted. Methods To optimize transgene expression in sub-regions of the mouse hippocampus and with a view towards devising gene therapy strategies for Alzheimer's disease, we designed lentivirus-based reporter vectors bearing various promoters, including constitutive and cell-specific promoters. Furthermore, we devised methods allowing a side-by-side comparison of transgene expression levels in neural cells both in vitro and in vivo. Results Following stereotaxic injection into the adult mouse hippocampus, titer-adjusted lentiviral vectors bearing constitutive promoters resulted in robust and sub-region-specific transgene expression. Our results show that the human CMV-IE promoter resulted in efficient transgene expression in the entire hippocampus whereas transgene expression mediated by the hybrid hEF1,/HTLV promoter was limited mainly in the dentate gyrus and the CA2/3 region. Finally, the neuron-specific human synapsin I promoter was particularly effective in the dentate gyrus. Conclusions These findings indicate that subregion-specific transgene expression in the hippocampus can be achieved following lentivirus vector-mediated gene transfer. Copyright © 2008 John Wiley & Sons, Ltd. [source] Lipid-based transfection as a method for gene delivery in zebrafish (Danio rerio) embryosAQUACULTURE RESEARCH, Issue 12 2007Vanesa Robles Abstract A major challenge to the widespread production of transgenic, knockout and knockdown zebrafish has been the absence of a simple and effective procedure for introducing macromolecules into the fertilized egg. None of the existing techniques for gene transfer in fish embryos has proven to be a major advance over cytoplasm microinjection, which is a technically demanding and time-consuming procedure. This report addresses this need, considering that the development of protocols for lipid-based transfection with fish embryos would considerably simplify gene transfer in this complex biological model. In this study, lipid-based transfection with two different reporter vectors was carried out in zebrafish embryos at different developmental stages. The parameters tested included different plasmid/transfection reagent ratios as well as the influence of an added transfection enhancer reagent. When embryos were transfected in the blastula stage with a pEGFP-N1 vector, more than 35% successfully incorporated the plasmid and expressed the fluorescent protein 24 h after transfection. The transfection enhancer did not show any significant effect in our experiments. This work presents an approach to implement this technique as a faster, cheaper and more practical alternative than microinjection. [source] | |||||||||||||