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1.
Adenoviral vectors expressing siRNAs for discovery and validation of gene function 总被引:5,自引:0,他引:5 下载免费PDF全文
Arts GJ Langemeijer E Tissingh R Ma L Pavliska H Dokic K Dooijes R Mesić E Clasen R Michiels F van der Schueren J Lambrecht M Herman S Brys R Thys K Hoffmann M Tomme P van Es H 《Genome research》2003,13(10):2325-2332
RNA interference is a powerful tool for studying gene function and for drug target discovery in diverse organisms and cell types. In mammalian systems, small interfering RNAs (siRNAs), or DNA plasmids expressing these siRNAs, have been used to down-modulate gene expression. However, inefficient transfection protocols, in particular, for primary cell types, have hampered the use of these tools in disease-relevant cellular assays. To be able to use this technology for genome-wide function screening, a more robust transduction protocol, resulting in a longer duration of the knock-down effect, is required. Here, we describe the validation of adenoviral vectors that express hairpin RNAs that are further processed to siRNAs. Infection of cell lines, or primary human cells, with these viruses leads to an efficient, sequence-specific, and prolonged reduction of the corresponding target mRNA, resulting in a reduction of the encoded protein level in the cell. For knock-down of one of the targets, GalphaS, we have measured inhibition of ligand-dependent, G-protein-coupled signaling. It is expected that this technology will prove to be of great value in target validation and target discovery efforts. 相似文献
2.
Brink AA Wiegant JC Szuhai K Tanke HJ Kenter GG Fleuren GJ Schuuring E Raap AK 《Cancer Genetics and Cytogenetics》2002,134(2):145-150
Infection with high-risk type human papillomavirus (HPV) is a necessary causal factor in the pathogenesis of cervical carcinoma. In most invasive cervical cancers, HPV is integrated in the host cell genome, and additional genetic aberrations are observed among which are chromosomal aberrations. To analyze in detail such often complex chromosomal changes and simultaneously map HPV integration sites, we extended the multiplicity of the combined binary ratio labeling fluorescence in situ hybridization (COBRA-FISH) technique to 49 by inclusion of a large Stokes' shift fluorochrome as the third binary label. The technique allows mapping of the integrated HPV genome in the context of p- and q-arm COBRA-FISH, with a sensitivity of one copy of the HPV genome as tested for HPV 16 in SiHa cells. We investigated the molecular karyotypes and integration patterns of HPV types 16 and 18 in metaphase spreads from short-term cultures of primary cervical carcinomas (n=5). Of the tested cervical carcinomas, two contained integrated HPV at 8q24, one of which in addition harbored the integrated virus near a translocation breakpoint. Two carcinomas had integrated HPV at 17q21 through 23 in a morphologically normal chromosome 17. One carcinoma contained HPV at 1q42 in a morphologically normal chromosome 1. Our data illustrate the efficacy of 49-color COBRA-FISH to resolve complex karyotypes and simultaneously map specific sequences in metaphases obtained from short-term solid tumor cultures. 相似文献
3.
Hofstra RM Mulder IM Vossen R de Koning-Gans PA Kraak M Ginjaar IB van der Hout AH Bakker E Buys CH van Ommen GJ van Essen AJ den Dunnen JT 《Human mutation》2004,23(1):57-66
Duchenne and Becker muscular dystrophy (DMD and BMD) are caused by mutations in the dystrophin gene. Large rearrangements in the gene are found in about two-thirds of DMD patients, with approximately 60% carrying deletions and 5-10% carrying duplications. Most of the remaining 30-35% of patients are expected to have small nucleotide substitutions, insertions, or deletions. To detect these subtle changes within the coding and splice site determining sequences of the dystrophin gene, we established a semiautomated denaturing gradient gel electrophoresis (DGGE) mutation scanning system. The DGGE scan covers the dystrophin gene with 95 amplicons, PCRed either individually or in a multiplex setup. PCR and pooling were performed semiautomatically, using a pipetting robot and 384-well plates, enabling concurrent amplification of DNA of four patients in one run. Amplification of individual fragments was performed using one PCR program. The products were pooled just before gel loading; DGGE requires only a single gel condition. Validation was performed using DNA samples harboring 39 known DMD variants, all of which could be readily detected. DGGE mutation scanning was applied to analyze 135 DMD/BMD patients and potential DMD carriers without large deletions or duplications. In DNA from 25 out of 44 DMD patients (57%) and from 5 out of 39 BMD patients (13%), we identified clear pathogenic changes. All mutations were different, with the exception of one DMD mutation, which occurred twice. In DNA from 10 out of 44 potential DMD carriers, including four obligate carriers, we detected causative changes, including one pathogenic change in every obligate carrier. In addition to these pathogenic changes, we detected 15 unique unclassified variants, i.e., changes for which a pathogenic nature is uncertain. 相似文献
4.
Jantine Posthuma De Boer Pim W. van Egmond Marco N. Helder Renée X. de Menezes Anne-Marie Cleton-Jansen Jeroen A.M. Beli?n Henk M. W. Verheul Barend J. van Royen Gert-Jan J.L. Kaspers Victor W. van Beusechem 《Oncotarget》2012,3(10):1169-1181
Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents. Despite aggressive therapy, survival outcomes remain unsatisfactory, especially for patients with metastatic disease or patients with a poor chemotherapy response. Chemoresistance contributes to treatment failure. To increase the efficacy of conventional chemotherapy, essential survival pathways should be targeted concomitantly. Here, we performed a loss-of-function siRNA screen of the human kinome in SaOS-2 cells to identify critical survival kinases after doxorubicin treatment. Gene silencing of JNK-interacting-protein-1 (JIP1) elicited the most potent sensitisation to doxorubicin. This candidate was further explored as potential target for chemosensitisation in OS. A panel of OS cell lines and human primary osteoblasts was examined for sensitisation to doxorubicin using small molecule JIP1-inhibitor BI-78D3. JIP1 expression and JIP1-inhibitor effects on JNK-signalling were investigated by Western blot analysis. JIP1 expression in human OS tumours was assessed by immunohistochemistry on tissue micro arrays. BI-78D3 blocked JNK-signalling and sensitised three out of four tested OS cell lines, but not healthy osteoblasts, to treatment with doxorubicin. Combination treatment increased the induction of apoptosis. JIP1 was found to be expressed in two-thirds of human primary OS tissue samples. Patients with JIP1 positive tumours showed a trend to inferior overall survival. Collectively, JIP1 appears a clinically relevant novel target in OS to enhance the efficacy of doxorubicin treatment by means of RNA interference or pharmacological inhibition. 相似文献
5.
L.H. ten Kate G.J. Kleinrensink M.A. van Veelen R.H.M. Goossens 《Minimally invasive therapy & allied technologies》2013,22(3):138-141
Removing the gallbladder is a relatively simple laparoscopic operation. Tissue trauma, caused by laparoscopic cholecystectomy, is usually minimal. Misidentifying the cystic duct and artery and the common hepatic duct represents the most severe complication. However, the Critical View of Safety (CVS) technique reduces the risk of trauma, by accomplishing a safe 360° identification of the cystic duct origin at the gallbladder neck [5]. This technique is employed and trained at the Erasmus Medical Centre (EMC), Rotterdam, The Netherlands.?This study presents the potential value of a training device which residents can use during practice. This training device must cover both user friendliness and information supply. The current information supply was studied subjectively; the CVS protocol and the users' experience were studied both objectively and subjectively. The results of these studies show that the present information supply is not satisfactory, that there is a CVS protocol which can be easily used in a training device, and that most actions defining the cystic duct during operation revert on users' experience. Therefore, it is desirable to design a new training device according to the experience of the target group and the protocols, and taking into account an optimal human‐product interaction. 相似文献
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van Deursen D Botma GJ Jansen H Verhoeven AJ 《Molecular and cellular endocrinology》2008,294(1-2):37-44
Expression of hepatic lipase (HL) in the liver is reduced during prolonged fasting. This effect is mainly mediated via catecholamines, which signal through elevation of Ca(i)(2+) as well as cAMP. We have studied the effect of cAMP on HL expression in cell culture. Overnight incubation of HepG2 cells with 10-300microM 8-bromo-cyclic AMP resulted in a dose-dependent, up to 50% reduction in secretion of HL, but had no effect on secretion of alpha(1)-antitrypsin or overall protein synthesis. HL mRNA levels were decreased 1.5 fold, as determined by semi-quantitative and real-time RT-PCR. In HepG2 cells transiently transfected with human HL (-685/+13) or rat HL (-446/+9) promoter-reporter constructs, cAMP induced a similar dose-dependent suppression of HL promoter activity. cAMP responsiveness in HepG2 cells was mediated by a conserved 10-bp response element at -45/-36, that represents a potential binding site for CCAAT/enhancer-binding protein beta (C/EBPbeta). cAMP reduced expression of the 45kDa C/EBPbeta protein and binding of C/EBPbeta to the proximal promoter region of the human HL gene by 50%, as determined by immunoblotting and chromatin immunoprecipitation assay, respectively. In human H295R adrenocortical cells, cAMP failed to suppress HL promoter activity, and only slightly reduced C/EBPbeta expression. We conclude that the fall in HL expression during prolonged fasting may be mediated through elevation of cAMP and lowering of C/EBPbeta expression. 相似文献
9.
Geertje M. de Boer Gert-Jan Braunstahl Esmee K. van der Ploeg Cathelijne M. van Zelst Alie van Bruggen Guido Epping Menno van Nimwegen Gert Verhoeven Erwin Birnie Bianca M. Boxma-de Klerk Marjolein J. W. de Bruijn Ralph Stadhouders Rudi W. Hendriks Gerdien A. Tramper-Stranders 《Clinical and experimental allergy》2021,51(9):1172-1184
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