TY - JOUR. T1 - RNA interference technology used for the study of aquatic virus infections. AU - Reshi, Mohammad Latif. AU - Wu, Jen Leih. AU - Wang, Hao Ven. AU - Hong, Jiann Ruey. N1 - Funding Information: This work was supported by grants NSC 97-2313-B-006-004-MY3 and NCS 102-3011-P-006-002 , awarded to Dr. Jiann-Ruey Hong from the National Science Council, Taiwan, Republic of China.. PY - 2014/9. Y1 - 2014/9. N2 - Aquaculture is one of the most important economic activities in Asia and is presently the fastest growing sector of food production in the world. Explosive increases in global fish farming have been accompanied by an increase in viral diseases. Viral infections are responsible for huge economic losses in fish farming, and control of these viral diseases in aquaculture remains a serious challenge. Recent advances in biotechnology have had a significant impact on disease reduction in aquaculture. RNAi is one of the most important technological breakthroughs in modern biology, ...
Author Summary RNA interference is a gene regulatory system in which small RNA molecules turn off genes that have similar sequences to the small RNAs. This has become a powerful tool because a researcher can use RNA interference to turn off any gene of interest in order to test its function. There is great interest in identifying the genes required for the RNA interference pathway, and one approach to identifying such genes has been to use RNA interference to turn off potential RNA interference genes and to ask whether RNA interference still functions when these genes are turned off. The goal of our report is to ask how it is possible for RNA interference to turn itself off, using a mathematical model of the system. The results show that RNA interference cannot turn itself off if the RNA interference pathway is too effective to start with, so that experiments in which RNA interference acts on itself will only work in systems having a low efficiency. The results of our model suggest possible ways to
Home , Papers , [EXPRESS] RNA interference-based functional knockdown of the voltage gated potassium channel Kv7.2 in dorsal root ganglion neurons after in vitro and in vivo gene transfer by adeno-associated virus (AAV) vectors. ...
The African trypanosome, Trypanosoma brucei possesses a large and unique intraflagellar structure called the paraflagellar rod (PFR). The PFR is composed of 2 major proteins, PFRA and PFRC. We have generated an inducible mutant trypanosome cell line (snl-2) that expresses linked inverted copies of a PFRA gene, capable of forming a PFRA double-stranded (ds) RNA. When expression of this dsRNA was induced, new PFRA RNA and PFRA protein quickly disappeared and PFR construction was affected, resulting in cell paralysis. This inducible RNA interference (RNAi) effect was fast-acting, heritable and reversible. It allowed us to demonstrate that PFR proteins are able to enter both mature and growing flagella but appear to concentrate differentially in new flagella because of the construction process. The PFR is constructed by a polar assembly process at the distal end of the flagellum resulting in a stable cytoskeletal structure with low turn-over. The inducible RNAi approach will have widespread applicability in
The African trypanosome, Trypanosoma brucei possesses a large and unique intraflagellar structure called the paraflagellar rod (PFR). The PFR is composed of 2 major proteins, PFRA and PFRC. We have generated an inducible mutant trypanosome cell line (snl-2) that expresses linked inverted copies of a PFRA gene, capable of forming a PFRA double-stranded (ds) RNA. When expression of this dsRNA was induced, new PFRA RNA and PFRA protein quickly disappeared and PFR construction was affected, resulting in cell paralysis. This inducible RNA interference (RNAi) effect was fast-acting, heritable and reversible. It allowed us to demonstrate that PFR proteins are able to enter both mature and growing flagella but appear to concentrate differentially in new flagella because of the construction process. The PFR is constructed by a polar assembly process at the distal end of the flagellum resulting in a stable cytoskeletal structure with low turn-over. The inducible RNAi approach will have widespread ...
Histone modifications influence gene expression in complex ways. The RNA interference (RNAi) machinery can repress transcription by recruiting histone-modifying enzymes to chromatin, although it is not clear whether this is a general mechanism for gene silencing or whether it requires repeated sequences such as long terminal repeats (LTRs). We analyzed the global effects of the Clr3 and Clr6 histone deacetylases, the Clr4 methyltransferase, the zinc finger protein Clr1, and the RNA, proteins Dicer, RdRP, and Argonaute on the transcriptome of Schizosaccharomyces pombe (fission yeast). The clr mutants derepressed similar subsets of genes, many of which also became transcriptionally activated in cells that were exposed to environmental stresses such as nitrogen starvation. Many genes that were repressed by the Clr proteins clustered in extended regions close to the telomeres. Surprisingly few genes were repressed by both the silencing and RNAi machineries, with transcripts from centromeric repeats ...
TY - JOUR. T1 - Targeting L1 cell adhesion molecule using lentivirus-mediated short hairpin RNA interference reverses aggressiveness of oral squamous cell carcinoma. AU - Hung, Shiao Chen. AU - Wu, I. Hui. AU - Hsue, Shui Sang. AU - Liao, Chia Hui. AU - Wang, Hsien Chi. AU - Chuang, Pei Hsin. AU - Sung, Shian Ying. AU - Hsieh, Chia Ling. PY - 2010/12/6. Y1 - 2010/12/6. N2 - The L1 cell adhesion molecule (L1CAM) has been implicated in tumor progression of many types of cancers, but its role in oral squamous cell carcinoma (OSCC) has not been investigated. In the present study, we demonstrated overexpression of L1CAM in OSCC cells, but not in normal keratinocytes, using both clinical specimens and cell lines. This overexpression demonstrated a strong correlation with less differentiation and a higher invasion potential of cancer cells, supporting the significance of L1CAM in human OSCC tumor progression. Targeting L1CAM gene expression in SCC4 cells overexpressing L1CAM using a lentivirus-mediated ...
AMSTERDAM, The Netherlands, December 5, 2012 /PRNewswire/ --. uniQure B.V., a leader in the field of human gene therapy, today announced a non-exclusive cross-licensing agreement with Benitec Biopharma Ltd. (ASX: BLT) giving uniQure access to Benitecs proprietary DNA-directed RNA interference (ddRNAi) technology in Huntingtons disease. In return, uniQure granted Benitec non-exclusive access to the Companys AAV5 delivery technology for the development of a ddRNAi therapy for Hepatitis B.. The cross-licensing agreement with Benitec fully capitalizes on the strength of our advanced AAV platform and our proven ability to deliver therapeutic genes to target cells with high accuracy and efficacy, says Jörn Aldag, CEO of uniQure. The agreement with Benitec opens up promising new avenues to develop therapies for high unmet medical needs such as Huntingtons disease. While our current programs focus on delivering fully functioning therapeutic genes to remedy faulty or malfunctioning genes, ...
Goodwin Procter associate Daniel Wilson looks into patenting strategies for a powerful new tool for treating disease as well as for creating models of disease.
Background Neurogenesis in the brain of adult mammals occurs throughout life in two locations: the subventricular zone of the lateral ventricle and the subgranular zone of the dentate gyrus in the hippocampus. RNA interference mechanisms have emerged as critical regulators of neuronal differentiation. However, to date, little is known about its function in adult neurogenesis. Results Here we show that the RNA interference machinery regulates Doublecortin levels and is associated with chromatin in differentiating adult neural progenitors. Deletion of Dicer causes abnormal higher levels of Doublecortin. The microRNA pathway plays an important role in Doublecortin regulation. In particular miRNA-128 overexpression can reduce Doublecortin levels in differentiating adult neural progenitors. Conclusions We conclude that the RNA interference components play an important role, even through chromatin association, in regulating neuron-specific gene expression programs. ...
Hypercapnia, elevated partial pressure of CO2 in blood and tissue, develops in many patients with chronic severe obstructive pulmonary disease and other advanced lung disorders. Patients with advanced disease frequently develop bacterial lung infections, and hypercapnia is a risk factor for mortality in such individuals. We previously demonstrated that hypercapnia suppresses induction of NF-κB-regulated innate immune response genes required for host defense in human, mouse, and Drosophila cells, and it increases mortality from bacterial infections in both mice and Drosophila. However, the molecular mediators of hypercapnic immune suppression are undefined. In this study, we report a genome-wide RNA interference screen in Drosophila S2* cells stimulated with bacterial peptidoglycan. The screen identified 16 genes with human orthologs whose knockdown reduced hypercapnic suppression of the gene encoding the antimicrobial peptide Diptericin (Dipt), but did not increase Dipt mRNA levels in air. In ...
Article A novel and quick method to avoid H|sub|2|/sub|O|sub|2|/sub| interference on COD measurement in Fenton system by Na|sub|2|/sub|SO|sub|3|/sub| reduction and O|sub|2|/sub| oxidation. Hydrogen peroxide interference on chemical oxygen demand (COD...
From the abstract: Hypercapnia, elevated partial pressure of CO2 in blood and tissue, develops in many patients with chronic severe obstructive pulmonary disease and other advanced lung disorders. Patients with advanced disease frequently develop bacterial lung infections ... We previously demonstrated that hypercapnia suppresses induction of NF-κB-regulated innate immune response genes ... However, the molecular mediators of hypercapnic immune suppression are undefined. In this study, we report a genome-wide RNA interference screen in Drosophila S2* cells stimulated with bacterial peptidoglycan. The screen identified 16 genes with human orthologs whose knockdown reduced hypercapnic suppression of the gene encoding the antimicrobial peptide Diptericin (Dipt), but did not increase Dipt mRNA levels in air. In vivo tests of one of the strongest screen hits, zinc finger homeodomain 2 (Zfh2; mammalian orthologs ZFHX3/ATBF1 and ZFHX4), demonstrate that reducing zfh2 function using a mutation or RNA ...
RNA interference (RNAi) is a post-transcriptional process triggered by the introduction of double-stranded RNA (dsRNA) which leads to gene silencing in a sequence-specific manner. The first evidence that dsRNA could achieve efficient gene silencing through RNAi came from studies on the nematode Caenorhabditis elegans. Further analyses in the fruit fly Drosophila melanogaster have contributed greatly toward understanding the biochemical nature of the RNAi pathway. Long dsRNAs are cleaved by the RNase III family member, Dicer, into 19-23 nucleotides (nt) fragments with 5 phosphorylated ends and 2-nt unpaired and unphosphorylated 3 ends.
In the present study, a genome-wide RNA interference screen was combined with an extensive biochemical analysis and quantitative proteomics to better understand the regulation of the heat-shock response (HSR) upon thermal stress. The usage of an endoribon...
TY - JOUR. T1 - A genome-wide loss-of-function screen identifies SLC26A2 as a novel mediator of TRAIL resistance. AU - Dimberg, Lina Y.. AU - Towers, Christina G.. AU - Behbakht, Kian. AU - Hotz, Taylor J.. AU - Kim, Jihye. AU - Fosmire, Susan. AU - Porter, Christopher C.. AU - Tan, Aik-Choon. AU - Thorburn, Andrew. AU - Ford, Heide L.. PY - 2017/4/1. Y1 - 2017/4/1. N2 - TRAIL is a potent death-inducing ligand that mediates apoptosis through the extrinsic pathway and serves as an important endogenous tumor suppressor mechanism. Because tumor cells are often killed by TRAIL and normal cells are not, drugs that activate the TRAIL pathway have been thought to have potential clinical value. However, to date, most TRAIL-related clinical trials have largely failed due to the tumor cells having intrinsic or acquired resistance to TRAIL-induced apoptosis. Previous studies to identify resistance mechanisms have focused on targeted analysis of the canonical apoptosis pathway and other known regulators of ...
Australia s Commonwealth Scientific and Industrial Research Organization said last week that it has signed an agreement to provide Bayer CropScience with a worldwide (except Australia) license to use its RNAi technology in developing and selling selected crop plant varieties.... Subscribers: click headline for more
Shop Systemic RNA interference defective protein ELISA Kit, Recombinant Protein and Systemic RNA interference defective protein Antibody at MyBioSource. Custom ELISA Kit, Recombinant Protein and Antibody are available.
Synthetic lethality is an attractive strategy for the design of novel therapies for cancer. Using this approach we have previously demonstrated that inhibition of the DNA repair protein, PARP1, is synthetically lethal with deficiency of either of the breast cancer susceptibility proteins, BRCA1 and …
RNA interference (RNAi) is an incredible revolution in the field of functional genomics, a breakthrough in plant molecular genetics. This technology will generate enormous potential for engineering control of gene expres-sion. The success of managing biotic stress using RNAi technology will prove to be biologically and environmentally safe. It is therapeutic in approach as the resistance induced by RNAi is triggered by ds RNA that results in silencing of specific genes before being translated in a homology dependent manner. Over the time, RNAi is significantly proving it as one of the most promiscent management strategy which eliminates certain risks associated with the development of transgenic plants. This review gives an insight into the probability of management of plant diseases caused by various biotic agents viz. fungi, bacteria and viruses using RNA interference technique and host-pathogen related targeted sites ...
ポストゲノム時代におけるRNA 干渉法の役割 [in Japanese] The role of RNA interference on post-genomic era [in Japanese] ...
Acute myeloid leukemia (AML) with an NPM1 mutation (NPMc+) has a distinct gene expression signature and displays molecular abnormalities similar to mixed lineage leukemia (MLL), including aberrant expression of the PBX3 and HOXA gene cluster. However, it is unclear if the aberrant expression of PBX3 and HOXA is essential for the survival of NPM1-mutated leukemic cells. Methods: Using the gene expression profiling of TCGA and E-MTAB-3444 datasets, we screened for high co-expression of PBX3 and HOXA9 in NPMc+ leukemia patients. We performed NPMc+ depletion and overexpression experiments to examine aberrant H3K79 methylation through epigenetic regulation. Through RNA interference technology and small-molecule inhibitor treatment, we evaluated the effect of methyl-modified H3K79 on cell survival and explored the possible underlying mechanism. Results: We showed that NPMc+ increased the expression of PBX3 and HOXA9, which are both poor prognosis indicators in AML. High PBX3 and HOXA9 expression was ...
For commonly studied genes, where there is only 1 RNAi line in the VDRC GD or KK collection at present, we aim to add a further functional RNAi line to facilitate verification of phenotypes. We chose to use the short hairpin RNAi technology as it is a simpler and more cost-effective method of creating lines than by using long double-stranded RNA. Short hairpins RNAs (shRNAs), containing a 21bp targeting sequence embedded into a micro-RNA (miR-1) backbone, have been shown to be very effective for gene knockdown in both the germline and somatic tissues (Ni et al., 2011). This technology has been used extensively by the Transgenic RNAi Project (TRiP).. To avoid direct duplication of community resources, the VDRC has collaborated with the TRiP team during shRNA design to ensure that the new VDRC lines are as distinct as possible from the TRiP resource. We have used the WALIUM20 vector (for triggering RNAi in soma and germline) in combination with the attP40 landing site, meaning that both the ...
For commonly studied genes, where there is only 1 RNAi line in the VDRC GD or KK collection at present, we aim to add a further functional RNAi line to facilitate verification of phenotypes. We chose to use the short hairpin RNAi technology as it is a simpler and more cost-effective method of creating lines than by using long double-stranded RNA. Short hairpins RNAs (shRNAs), containing a 21bp targeting sequence embedded into a micro-RNA (miR-1) backbone, have been shown to be very effective for gene knockdown in both the germline and somatic tissues (Ni et al., 2011). This technology has been used extensively by the Transgenic RNAi Project (TRiP).. To avoid direct duplication of community resources, the VDRC has collaborated with the TRiP team during shRNA design to ensure that the new VDRC lines are as distinct as possible from the TRiP resource. We have used the WALIUM20 vector (for triggering RNAi in soma and germline) in combination with the attP40 landing site, meaning that both the ...
Meacham, C. E., Lawton, L. N., Soto-Feliciano, Y. M., Pritchard, J. R., Joughin, B. A., Ehrenberger, T., Fenouille, N., Zuber, J., Williams, R. T., Young, R. A., Hemann, M. T. (March 2015) A genome-scale in vivo loss-of-function screen identifies Phf6 as a lineage-specific regulator of leukemia cell growth. Genes & Development, 29 (5). pp. 483-8. ISSN 0890-9369 Huang, C. H., Lujambio, A., Zuber, J., Tschaharganeh, D. F., Doran, M. G., Evans, M. J., Kitzing, T., Zhu, N., de Stanchina, E., Sawyers, C. L., Armstrong, S. A., Lewis, J. S., Sherr, C. J., Lowe, S. W. (August 2014) CDK9-mediated transcription elongation is required for MYC addiction in hepatocellular carcinoma. Genes and Development, 28 (16). pp. 1800-1814. ISSN 15495477 (ISSN) Zaiss, A. K., Zuber, J., Chu, C., Machado, H. B., Jiao, J., Catapang, A. B., Ishikawa, T. O., Gil, J. S., Lowe, S. W., Herschman, H. R. (July 2014) Reversible Suppression of Cyclooxygenase 2 (COX-2) Expression In Vivo by Inducible RNA Interference. PLoS One, 9 ...
Title:Therapy for Dominant Inherited Diseases by Allele-Specific RNA Interference: Successes and Pitfalls. VOLUME: 15 ISSUE: 5. Author(s):Delphine Trochet, Bernard Prudhon, Stéphane Vassilopoulos and Marc Bitoun. Affiliation:Inserm/UPMC UMR_S974, CNRS FRE3617, Institut de Myologie, Paris, France.. Keywords:Allele-specific silencing, Dominant inherited diseases, Pitfalls, RNA interference, Single nucleotide substitution, Gene-based therapy.. Abstract:RNA interference (RNAi) is a conserved mechanism for post-transcriptional gene silencing mediated by messenger RNA (mRNA) degradation. RNAi is commonly induced by synthetic siRNA or shRNA which recognizes the targeted mRNA by base pairing and leads to target-mRNA degradation. RNAi may discriminate between two sequences only differing by one nucleotide conferring a high specificity of RNAi for its target mRNA. This property was used to develop a particular therapeutic strategy called allele-specific-RNA interference devoted to silence the mutated ...
The use of small interfering RNA (siRNA) molecules in animals to achieve double-stranded RNA-mediated interference (RNAi) has recently emerged as a powerful method of sequence-specific gene knockdown. As DNA-based expression of short hairpin RNA (shRNA) for RNAi may offer some advantages over chemical and in vitro synthesised siRNA, a number of vectors for expression of shRNA have been developed. These often feature polymerase III (pol. III) promoters of either mouse or human origin. To develop a shRNA expression vector specifically for bovine RNAi applications, we identified and characterised a novel bovine U6 small nuclear RNA (snRNA) promoter from bovine sequence data. This promoter is the putative bovine homologue of the human U6-8 snRNA promoter, and features a number of functional sequence elements that are characteristic of these types of pol. III promoters. A PCR based cloning strategy was used to incorporate this promoter sequence into plasmid vectors along with shRNA sequences for RNAi. The
Article describing an optimized protocol for generating short interfering RNAs (siRNAs) or hairpin siRNAs in vitro using T7 RNA Polymerase and annealed DNA oligonucleotide templates. Two RNA interference studies in different mammalian model systems demonstrate the functionality of the synthesized siRNAs.
Examining the knockdown Once cells have been infected, it will be necessary to remove any contaminating uninfected cells. In general, there are two ways to purify and then to analyze RNAi-mediated gene knockdown in cells: one in which the whole population of infected cells are examined, and in the second approach a selected number of individual clonal cell lines are examined. Determining which strategy to perform depends on the nature of the experiment. If the entire population is to be analyzed, either flow cytometric sorting (when GFP-expressing virus is used) or drug selection (when the virus contains an antibiotic-resistance marker) may be used. If a constitutively-expressing small hairpin RNAi vector is used, it will be important to monitor the viability/growth of the cells throughout the procedure. Some gene knockdowns produce slow-growing or lethal phenotypes. This can be difficult to assess when drug selection of the infected cells is used, in which case a GFP marker may be preferred ...
We have led the way in the development of what has been hailed as a major breakthrough in molecular biology: silencing gene expression by RNA interference (RNAi). CSIROs RNAi gene silencing technology is enabling researchers around the world to protect plants and animals from diseases, and to develop new plant varieties with beneficial attributes.
TY - JOUR. T1 - Gene silencing using a heat-inducible RNAi system in Arabidopsis. AU - Masclaux, Frédéric. AU - Charpenteau, Martine. AU - Takahashi, Taku. AU - Pont-Lezica, Rafael. AU - Galaud, Jean Philippe. PY - 2004/8/20. Y1 - 2004/8/20. N2 - Controlling gene expression during plant development is an efficient tool to explore gene function. In this paper, we describe a gene expression system driven by a heat-shock gene promoter (HSP18.2), to trigger the expression of an intron-containing inverted-repeat. RNA interference became a powerful way for gene functional analysis by reverse genetic approaches. However, constitutive gene silencing cannot be used with genes involved in fundamental processes such as embryo viability. Inducible promoters provide an alternative approach for temporal and spatial gene expression control and we described here a new system, complementary to those using chemical gene inducers. To evaluate the efficiency of this system, RNA corresponding to the phytoene ...
Gene inactivation through RNA interference (RNAi) has proven to be a valuable tool for studying gene function in C. elegans. When combined with tissue-specific gene inactivation methods, RNAi has the potential to shed light on the function of a gene in distinct tissues. In this study we characterize …
Data Availability StatementThe writers declare that the info helping the results of the scholarly research can be found within this article. tumor size. The TCGA data also demonstrated how the B7-H6 mRNA manifestation level was considerably negatively correlated with the survival of HCC patients. Next, to investigate the functions of B7-H6 in HCC, we successfully constructed B7-H6 knockdown expression human HCC cell lines using the RNA interference technology. Our studies showed that reduced expression of B7-H6 in HepG2 and SMMC-7721 cells significantly attenuated cell proliferation as well as cell migration and invasion. Besides, depletion of B7-H6 greatly induced cell cycle arrest at G1 phase. And also B7-H6 knockdown in HCC cell order GSK343 lines dramatically decreased the C-myc, C-fos and Cyclin-D1 expression. Conclusions Our present findings suggested that B7-H6 played an important role in oncogenesis of HCC on cellular level, and B7-H6 could be employed to develop immunotherapeutic ...
Cholesterol levels in the blood are one of the main risk factors for cardiovascular disease. They are controlled by the amount of cholesterol that cells can take in - thus removing it from the blood - and metabolise. The researchers used RNA interference to test the function of each of the genes within 56 regions previously identified by GWAS as being linked with cardiovascular disease. They selectively decreased their action and measured what, if any, changes this induced in cholesterol metabolism. From this they could deduce which of the genes are most likely to be involved in the onset of the disease.. This is the first wide-scale RNA interference study that follows up on GWAS. It has proven its potential by narrowing down a large list of candidate genes to the few with an important function that we can now focus on in future in-depth studies, explains Rainer Pepperkok at EMBL, who co-led the study with Heiko Runz at the University of Heidelberg. In principle, our approach can be applied ...
Many invading viruses and transposons replicate and transpose through RNA intermediates. These intermediates can be detected by the host cell RNA interference machinery in plants and insects and used to generate small interfering RNAs (siRNAs), critical intermediates in silencing, which can then neutralize the invader. Lecellier et al. (see the news story by Couzin) now show that mammalian cells can also use the RNA silencing machinery to help neutralize an invading mammalian virus. Curiously, rather than siRNAs derived from the viral genome being the effector molecules that target the invader for silencing, a host microRNA tags the virus. The importance of the pathway in host defense is supported by the presence of a viral protein that can suppress the silencing effect.. C.-H. Lecellier, P. Dunoyer, K. Arar, J. Lehmann-Che, S. Eyquem, C. Himber, A. Saïb, O. Voinnet, A cellular microRNA mediates antiviral defense in human cells. Science 308, 557-560 (2005). [Abstract] [Full Text]. J. Couzin, ...
Three years ago Mark Kay MD PhD published the first results showing...Now with three human RNAi gene therapy trials under way Kays initia... Just like any other new drug it is just going to mean that we need t...In traditional gene therapy the inserted DNA produces a gene to replac...With key genes shut off viruses such as hepatitis B hepatitis C or H...,For,Stanford,scientists,,RNAi,gene,therapy,takes,two,steps,forward,,one,step,back,biological,biology news articles,biology news today,latest biology news,current biology news,biology newsletters
MK-1775 is a potent and selective inhibitor of the WEE1 kinase. As of this publication, it is the only WEE1 inhibitor that the authors are aware of currently undergoing evaluation as an anticancer agent in combination with chemotherapy in early-stage clinical trials (19, 20, 28). Previous studies using MK-1775 have shown its potentiation of DNA damage-based therapeutics by forcing unscheduled mitosis and ultimately resulting in apoptosis or mitotic catastrophe (4, 18, 29-32). However, the potential therapeutic effects of WEE1 inhibition in the absence of chemotherapies have not been widely explored. RNA interference knockdown of WEE1 is known to inhibit proliferation of cancer cell lines (13, 33), and more recently, it was shown that MK-1775 alone can induce apoptosis in sarcoma cell lines treated in vitro (34). Our results similarly highlight a requirement for WEE1 activity to maintain cellular viability and genomic stability. Furthermore, we provide the first demonstration of TGI with MK-1775 ...
MK-1775 is a potent and selective inhibitor of the WEE1 kinase. As of this publication, it is the only WEE1 inhibitor that the authors are aware of currently undergoing evaluation as an anticancer agent in combination with chemotherapy in early-stage clinical trials (19, 20, 28). Previous studies using MK-1775 have shown its potentiation of DNA damage-based therapeutics by forcing unscheduled mitosis and ultimately resulting in apoptosis or mitotic catastrophe (4, 18, 29-32). However, the potential therapeutic effects of WEE1 inhibition in the absence of chemotherapies have not been widely explored. RNA interference knockdown of WEE1 is known to inhibit proliferation of cancer cell lines (13, 33), and more recently, it was shown that MK-1775 alone can induce apoptosis in sarcoma cell lines treated in vitro (34). Our results similarly highlight a requirement for WEE1 activity to maintain cellular viability and genomic stability. Furthermore, we provide the first demonstration of TGI with MK-1775 ...
F. Leulier, P. S Ribeiro, E. Palmer, T. Tenev, K. Takahashi, D. Robertson, A. Zachariou, F. Pichaud, R. Ueda, and P. Meier (2006) Cell Death Differ, 13(10):1663-74.. ...
How to interpret the nature of biological processes, which, when perturbed, cause certain phenotypes, such as human disease, is a major challenge. The completion of sequencing of many model organisms has made reverse genetic approaches [1] efficient and comprehensive ways to identify causal genes for a given phenotype under investigation. For instance, genome-wide knockout strains are now available for Saccharomyces cerevisiae [2, 3], and diverse high throughput RNA interference knockdown experiments have been performed, or are under development, for higher organisms, including C. elegans [4], D. melanogaster [5] and mammals [6, 7].. Compared to the direct genotype-phenotype correlation observed in the above experiments, what is less obvious is how genetic perturbation leads to the change of phenotypes in the complex of biological systems. That is, we might perceive the cell or organism as a dynamic system composed of interacting functional modules that are defined as discrete entities whose ...
Who says you cant be smart, witty, or say anything of substance in 140 characters or less? Alnylam Pharmaceuticals CEO John Maraganore had a zinger ready
Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans.: RNA interference (RNAi) is a cellular defense mechanism that uses double-
Plants and fungi can use conserved RNA interference machinery to regulate each others gene expression-and scientists think they can make use of this phenomenon to create a new generation of pesticides.. 6 Comments. ...
Plants and fungi can use conserved RNA interference machinery to regulate each others gene expression-and scientists think they can make use of this phenomenon to create a new generation of pesticides.. 6 Comments. ...
Aberrant acetylation has been strongly linked to tumorigenesis, and the modulation of acetylation through targeting histone deacetylases (HDACs) is gathering increasing pace as a viable therapeutic strategy. A genome-wide loss-of-function screen identified HR23B, which shuttles ubiquitinated cargo proteins to the proteasome, as a sensitivity determinant for HDAC inhibitor-induced apoptosis. HR23B also governs tumor cell sensitivity to drugs that act directly on the proteasome. The level of HR23B influences the response of tumor cells to HDAC inhibitors, and HR23B is found at high levels in cutaneous T cell lymphoma in situ, a malignancy that responds favorably to HDAC inhibitor-based therapy. These results suggest that deregulated proteasome activity contributes to the anticancer activity of HDAC inhibitors.
Autophagy plays a critical role in cancer formation and therapeutic resistance. However, little is known about how autophagy is regulated in cancer and how it mediates therapeutic resistance. Here we elect to use chronic myeloid leukemia (CML) as a cancer model to study autophagy in that it is driven by a single onco-protein BCR-ABL, whose activity can be selectively blocked by imatinib a front-line treatment for CML. Moreover, imatinib resistance frequently occurs in CML patients. Thus, unraveling autophagy regulation in CML and its role in overcoming imatinib resistance has substantial therapeutic benefits not only for CML but also for other cancers that can be treated by imatinib. In this report, we performed a genome-wide RNA interference screen in K562 human CML cells using monodansylcadaverine (MDC) that marks autolysosomes followed by fluorescence-activated cell sorting to label and isolate autophagic cells. We have identified 336 candidate genes, knockdown of which significantly ...
To elucidate the molecular mechanisms underlying pathogen-associated molecular pattern (PAMP)-induced defense responses in potato (Solanum tuberosum), the role of the signaling compounds salicylic acid (SA) and jasmonic acid (JA) was analyzed. Pep-13, a PAMP from Phytophthora, induces the accumulation of SA, JA and hydrogen peroxide, as well as the activation of defense genes and hypersensitive-like cell death. We have previously shown that SA is required for Pep-13-induced defense responses. To assess the importance of JA, RNA interference constructs targeted at the JA biosynthetic genes, allene oxide cyclase and 12- oxophytodienoic acid reductase, were expressed in transgenic potato plants. In addition, expression of the F-box protein COI1 was reduced by RNA interference. Plants expressing the RNA interference constructs failed to accumulate the respective transcripts in response to wounding or Pep-13 treatment, neither did they contain significant amounts of JA after elicitation. In response ...
Identification of components of the intracellular transport machinery of acylated proteins by a genome-wide RNAi screen [Elektronische Ressource] / presented by Julia Ritzerfeld : IDENTIFICATIO N O F CO M PO NENTS O F TH E INTRACELLU LAR TRANSPO RT M ACH INERY O F ACYLATED PRO TEINS BY A GENO M E‐W IDE RNAI SCREEN DISSERTATIO N submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto Caro la University of Heidelberg, Germany for the degree of Doctor of Natural Sciences Julia Ritzerfeld
TY - CHAP. T1 - Short hairpin RNA-mediated gene silencing. AU - Lambeth, Luke S. AU - Smith, Craig A.. PY - 2013. Y1 - 2013. N2 - Since thefirst application of RNA interference (RNAi) in mammalian cells, the expression of short hairpin RNAs (shRNAs) for targeted gene silencing has become a benchmark technology. Using plasmid and viral vectoring systems, the transcription of shRNA precursors that are effectively processed by the RNAi pathway can lead to potent gene knockdown. The past decade has seen continual advancement and improvement to the various strategies that can be used for shRNA delivery, and the use of shRNAs for clinical applications is well underway. Driving these developments has been the many benefits afforded by shRNA technologies, including the stable integration of expression constructs for long-term expression, infection of difficultto-target cell lines and tissues using viral vectors, and the temporal control of shRNA transcription by inducible promoters. The use of different ...
RNA interference (RNAi) is a powerful tool to study the intracellular membrane transport and membrane organelle behavior in the nematode Caenorhabditis elegans. This model organism has gained popularity in the trafficking field because of its relative simplicity, yet being multicellular. C. elegans is fully sequenced and has an annotated genome, it is easy to maintain, and a growing number of transgenic strains bearing markers for different membrane compartments are available. C. elegans is particularly well suited for protein downregulation by RNAi because of the simple but efficient methods of dsRNA delivery. The phenomenon of systemic RNAi in the worm further facilitates this approach. In this chapter we describe methods and applications of RNAi in the field of membrane traffic. We summarize the fluorescent markers used as a readout for the effects of gene knockdown in different cells and tissues and give details for data acquisition and analysis ...
RNA interference (RNAi) is a gene-silencing mechanism by which a ribonucleoprotein complex, the RNA-induced silencing complex (RISC) and a double-stranded (ds) short-interfering RNA (siRNA), targets a complementary mRNA for site-specific cleavage and subsequent degradation. While longer dsRNA are endogenously processed into 21- to 24-nucleotide (nt) siRNAs or miRNAs to induce gene silencing, RNAi studies in human cells typically use synthetic 19- to 20-nt siRNA duplexes with 2-nt overhangs at the 3-end of both strands. Here, we report that systematic synthesis and analysis of siRNAs with deletions at the passenger and/or guide strand revealed a short RNAi trigger, 16-nt siRNA, which induces potent RNAi in human cells. Our results indicate that the minimal requirement for dsRNA to trigger RNAi is an approximately 42 A A-form helix with approximately 1.5 helical turns. The 16-nt siRNA more effectively knocked down mRNA and protein levels than 19-nt siRNA when targeting the endogenous CDK9 gene,
DasGupta et al. [3] developed a high-throughput assay based on the known ability of canonical Wnt signaling to activate transcription of luciferase reporter constructs in transfected cells. Improving on the widely used construct TOP-Flash [13], they generated two new reporters each containing multiple TCF-binding sites upstream of a different minimal promoter. Because only the TCF sites were common between the reporters, off-target effects unrelated to β-catenin/TCF signaling were minimized. Reporters with mutated TCF-binding sites also served as specificity controls. The authors first validated the behavior of these reporters in transfection assays of Drosophila cell lines. Then they scaled up the transfections to incorporate approximately 22,000 double-stranded RNAs (dsRNAs), so as to induce RNAi [3], and tested the individual effects on Wingless-induced signaling. The library of dsRNA sequences, previously used in other high-throughput RNAi screens, is directed at all known open reading ...
RNAi is an RNA-dependent gene silencing process that is controlled by the RNA-induced silencing complex (RISC) and is initiated by short double-stranded RNA molecules in a cells cytoplasm, where they interact with the catalytic RISC component argonaute.[5] When the dsRNA is exogenous (coming from infection by a virus with an RNA genome or laboratory manipulations), the RNA is imported directly into the cytoplasm and cleaved to short fragments by Dicer. The initiating dsRNA can also be endogenous (originating in the cell), as in pre-microRNAs expressed from RNA-coding genes in the genome. The primary transcripts from such genes are first processed to form the characteristic stem-loop structure of pre-miRNA in the nucleus, then exported to the cytoplasm. Thus, the two dsRNA pathways, exogenous and endogenous, converge at the RISC.[6] Exogenous dsRNA initiates RNAi by activating the ribonuclease protein Dicer,[7] which binds and cleaves double-stranded RNAs (dsRNAs) in plants, or short hairpin ...
RNA interference (RNAi) is a naturally occurring phenomenon that results in the suppression of a target RNA sequence utilizing a variety of possible methods and pathways. To dissect the factors that result in effective siRNA sequences a regression kernel Support Vector Machine (SVM) approach was used to quantitatively model RNA interference activities. Eight overall feature mapping methods were compared in their abilities to build SVM regression models that predict published siRNA activities. The primary factors in predictive SVM models are position specific nucleotide compositions. The secondary factors are position independent sequence motifs (N-grams) and guide strand to passenger strand sequence thermodynamics. Finally, the factors that are least contributory but are still predictive of efficacy are measures of intramolecular guide strand secondary structure and target strand secondary structure. Of these, the site of the 5 most base of the guide strand is the most informative. The capacity of
The Drosophila immune system discriminates between different classes of infectious microbes and responds with pathogen-specific defense reactions via the selective activation of the Toll and the immune deficiency (Imd) signaling pathways. The Toll pathway mediates most defenses against Gram-positive bacteria and fungi, whereas the Imd pathway is required to resist Gram-negative bacterial infection. Microbial recognition is achieved through peptidoglycan recognition proteins (PGRPs); Gram-positive bacteria activate the Toll pathway through a circulating PGRP (PGRP-SA), and Gram-negative bacteria activate the Imd pathway via PGRP-LC, a putative transmembrane receptor, and PGRP-LE. Gram-negative binding proteins (GNBPs) were originally identified in Bombyx mori for their capacity to bind various microbial compounds. Three GNBPs and two related proteins are encoded in the Drosophila genome, but their function is not known. Using inducible expression of GNBP1 double-stranded RNA, we now demonstrate ...
Researchers at the Medical University of South Carolina show in a new report that the RNA interference machinery, normally thought to reside in the nucleus or cytoplasm, predominantly localizes to these apical junctions and influences cell biology in the colon.
RNA interference has revolutionized our ability to study the effects of altering the expression of single genes in mammalian (and other) cells through targeted knockdown of gene expression. In this report we describe a web-based computational tool, siRNA Information Resource (sIR), which consists of a new open source database that contains validation information about published siRNA sequences and also provides a user-friendly interface to design and analyze siRNA sequences against a chosen target sequence. The siRNA design tool described in this paper employs empirically determined rules derived from a meta-analysis of the published data; it uses a weighted scoring system that determines the optimal sequence within a target mRNA and thus aids in the rational selection of siRNA sequences. This scoring system shows a non-linear correlation with the knockdown efficiency of siRNAs. sIR provides a fast, customized BLAST output for all selected siRNA sequences against a variety of databases so that the user
Ryan uses primary cell models (donated healthy live cells) and tissue samples from patients to investigate the cellular genetic workings of blood disorders and cancer. He tends to collect the RNA produced when genes are switched on and off, using cutting edge techniques to collect the sequences of these genes or uses microarray technologies to profile them. From this information he can identify key genes in a disease and use RNA interference technologies to switch variations of these genes off, inhibiting the production of controlling proteins for potential treatments in that disease.. As these RNA and protein molecules change in diseases we can use these changes to diagnose and help in clinical prognosis. Ryan has worked on novel sensor technologies to make such systems clinically acceptable, quicker and more sensitive.. Examples of disease work:. Manipulating gene switching in Sickle Cell Anaemia as a potential treatment; Dr D Carter, NCRNA and Chromatin Research Group, Oxford Brookes ...
Acute myeloid leukemia (AML) is an immune-susceptible malignancy, as demonstrated by its responsiveness to allogeneic stem cell transplantation (alloSCT). However, by employing inhibitory signaling pathways, including PD-1/PD-L1, leukemia cells suppress T cell-mediated immune attack. Notably, impressive clinical efficacy has been obtained with PD-1/PD-L1 blocking antibodies in cancer patients. Yet, these systemic treatments are often accompanied by severe toxicity, especially after alloSCT. Here, we investigated RNA interference technology as an alternative strategy to locally interfere with PD-1/PD-L1 signaling in AML. We demonstrated efficient siRNA-mediated PD-L1 silencing in HL-60 and patients AML cells. Importantly, WT1-antigen T cell receptor(+) PD-1(+) 2D3 cells showed increased activation toward PD-L1 silenced WT1(+) AML. Moreover, PD-L1 silenced AML cells significantly enhanced the activation, degranulation, and IFN-γ production of minor histocompatibility antigen-specific CD8(+) T ...
Previous works in the budding yeast S. cerevisiae and the fission yeast S. pombe have revealed that aslncRNAs are globally low abundant as they are extensively degraded by RNA surveillance machineries. For instance, the nuclear exosome targets a class of lncRNAs referred to as CUTs (Wyers et al, 2005; Neil et al, 2009; Xu et al, 2009), whereas the cytoplasmic 5′-3′ exoribonuclease Xrn1 degrades the so-called XUTs (Van Dijk et al, 2011), both types of transcripts being mainly antisense to protein-coding genes. However, this classification into CUTs and XUTs is not exclusive, some aslncRNAs being cooperatively targeted by the two RNA decay pathways. In fission yeast, an additional class of aslncRNAs (DUTs) was recently identified. DUTs accumulate in the absence of the ribonuclease III Dicer (Atkinson et al, 2018), highlighting the role of Dicer and RNAi in the control of aslncRNAs expression in fission yeast. This class of transcripts is absent in S. cerevisiae, which has lost the RNAi system ...
In plants, RNA- based gene silencing mediated by small RNAs functions at the transcriptional or post-transcriptional level to negatively regulate target genes, repetitive sequences, viral RNAs and/or transposon elements. Post-transcriptional gene silencing (PTGS) or the RNA interference (RNAi) approach has been achieved in a wide range of plant species for inhibiting the expression of target genes by generating double-stranded RNA (dsRNA). However, to our knowledge, successful RNAi-application to knock-down endogenous genes has not been reported in the important staple food crop banana ...
As the portfolio of RNAi methods continues to expand, options become available for even the most complex systems being studied. Until recently, synthetic siRNA was the RNAi vehicle most broadly applicable to a wide variety of systems and applications. With commercial suppliers designing and producing synthetic siRNAs, little manipulation is required for the consumer. This format is amenable to any scale of research being performed provided the system is easily transfected (e.g., standard transformed cell lines). However, obstacles for using synthetic siRNAs include being a non-renewable resource, the transient nature of silencing, and the difficulty faced in transfecting primary cells and non-dividing cell lines such as neurons, lymphocytes, and macrophages. In addition, in vivo knockdown studies are particularly cumbersome.. For those facing the above hurdles, DNA vector-based shRNA methods provide the necessary solutions. shRNA expression vectors may be propagated in Escherichia coli and, ...
Control of metabolic flux, the flow of metabolites through a complex metabolic network, is of importance to understand how an organism is sensing, and responding to, nutrient changes in its environment. Metabolic flux control can be measured for, and a control coefficient assigned to, each enzyme in a pathway. Measuring metabolic flux control in multicellular organisms is complicated by the fact that nutrient sensing and metabolic flux control may vary by tissue type. Major effects should be detectable in genomic information, as enzymes with high control coefficients will exhibit genetic patterns of adaptation when the pathway is under selection pressure. I used genetic variation within and among populations of Drosophila melanogaster, as well as divergence between D. melanogaster and the closely related D. simulans, to identify candidate genes for experimental study. I then conducted experiments with candidate genes using tissue specific RNA interference knockdown, focusing on two enzymes ...
The discovery of double-stranded RNA-mediated gene silencing has rapidly led to its use as a method of choice for blocking a gene, and has turned it into one of the most discussed topics in cell biology. Although still in its infancy, the field of RNA interference has already produced a vast array of results, mainly in Caenorhabditis elegans, but recently also in mammalian systems. Micro-RNAs are short hairpins of RNA capable of blocking translation, which are transcribed from genomic DNA and are implicated in several aspects from development to cell signaling. The present review discusses the main methods used for gene silencing in cell culture and animal models, including the selection of target sequences, delivery methods and strategies for a successful silencing. Expected developments are briefly discussed, ranging from reverse genetics to therapeutics. Thus, the development of the new paradigm of RNA-mediated gene silencing has produced two important advances: knowledge of a basic cellular ...
Bahiagrass is one of the most important warm season forage grasses. In Florida alone it is grown on more than 5 million acres. Howeve r, the high lignin content in the bahiagrass biomass significantly reduces its forage qual i ty. A key enzyme in the lignin biosynthetic pathway is th e 4coumarate CoA ligase (4CL); it catalyzes the formation of CoA thiol esters of 4 coumarate and other hydr oxycinn amates. We cloned four 4CL cDNA s from tetraploi d ba hiagrass cv. Argentine and an RNAi construct targeting a highly conserved domain was constructed using 200 bp of the coding sequences. The 4CL RNAi construct was intr o duced to bahiagrass callus by b iolistic gene transfer under transcriptional control of three alternative promoters: the constitutive e35S promoter, OsC4H promoter for xylem specific expression and the ZmdJ1 promoter for expre s sion in the green tissue. Following regeneration of plants their transgenic nature was confirmed using PCR and Southern blot analysis. Significant reduction ...
Many species, across a wide phylogenetic range, respond to aberrant/foreign RNA by degrading endogenous mRNA in a sequence-specific manner (1). This phenomenon, broadly referred to as posttranscriptional gene silencing (PTGS), can be triggered by the introduction of double-stranded RNA (dsRNA) [RNA interference (RNAi)], transformation with sense transgenes (cosuppression/quelling), or viral infection (2). RNAi acts as a cellular defense against parasitic nucleic acids and provides a fortuitous technique for biologists to reduce or eliminate a gene activity (3). RNAi-like mechanisms are also involved in the production of small noncoding RNAs that control developmental timing (4, 5). A better understanding of RNAi may then shed light on genome defense and endogenous developmental pathways.. The molecular mechanisms underlying RNAi are beginning to be elucidated. dsRNA is processed into small double-stranded fragments of 21-25 nucleotides, called small interfering RNA (siRNA; refs. 6-8), by the ...
Supplemental Figure 2 - Fig. S2. Quantification of effects on cells after CAP-D2 RNAi. (A) Growth curve showing the number of cells at different time points after CAP-D2 dsRNA treatment. Cells grew more slowly, plateaued at 72 hours, and did not change significantly after that time. (B) The percentage of mitotic cells in control and CAP-D2 RNAi cells. The percentage of mitotic cells increased two- to threefold in the CAP-D2 RNAi between 36 and 72 hours (6.7% versus 2.2% at 48 hours). (C) The percentage of abnormal mitotic cells in control and CAP-D2 RNAi cells. The majority of mitotic cells are abnormal 36 hours and later after dsRNAi treatment. (D) Histogram showing the percentage of cells in prometaphase after staining for Cyclin B/P-H3/a-tubulin in control and CAP-D2 RNAi cells. Cells delay in prometaphase in the CAP-D2-depleted cells. (E) Histogram showing the percentage of cells in anaphase after staining for Cyclin B/P-H3/a-tubulin, in control and CAP-D2 RNAi cells. The anaphase index in ...
https://doi.org/10.18632/oncotarget.4817 Aleksandra A. Pandyra, Peter J. Mullen, Carolyn A. Goard, Elke Ericson, Piyush Sharma, Manpreet Kalkat, Rosemary Yu, Janice T. Pong, Kevin R. Brown, Traver...
The success of siRNA-based therapeutics highly depends on a safe and efficient delivery of siRNA into the cytosol. In this study, we post-modified the primary amines on dendritic polyglycerolamine (dPG-NH2) with different ratios of two relevant amino acids, namely, arginine (Arg) and histidine (His). To investigate the effects from introducing Arg and His to dPG, the resulting polyplexes of amino acid functionalized dPG-NH2s (AAdPGs)/siRNA were evaluated regarding cytotoxicity, transfection efficiency, and cellular uptake. Among AAdPGs, an optimal vector with (1:3) Arg to His ratio, showed efficient siRNA transfection with minimal cytotoxicity (cell viability ≥ 90%) in NIH 3T3 cells line. We also demonstrated that the cytotoxicity of dPG-NH2 decreased as a result of amino acid functionalization. While the incorporation of both cationic (Arg) and pH-responsive residues (His) are important for safe and efficient siRNA transfection, this study indicates that AAdPGs containing higher degrees of ...
The pSUPER.retro (Oligoengine) RNA interference system was used to achieve stable expression of siRNAs. Oligonucleotides targeted to calpain 2 or PTP1B mRNA as well as a nonsilencing control were synthesized by Integrated DNA Technologies, annealed, and cloned into the pSUPER.retro.puro vector according to manufacturers instructions. Retroviral transfection was performed as described previously (Franco et al., 2004a). Wild-type MTLn3 cells were infected at 32°C for 6 h and allowed to recover in growth medium for 24 h before selection with 1 μg/ml puromycin for 4-5 d. Target sequences for calpain 2 in MTLn3 cells: control, 5′-TTCTCCGAACGTGTCACGT-3′; Capn2 si-A, 5′-AGGCCTATGCCAAGATCAA-3′; and Capn2 si-B, 5′-GAATGGCGATTTCTGCATC-3′. Target sequences for PTP1B in MTLn3 cells: PTP1B si-A, 5′-GCTGACACTGATCTCTGAA-3′; and PTP1Bsi-B, 5′-CAGGAGGAGCCTTGGTGTC-3′. Target sequences for human calpain 2 have been described previously (Su et al., 2006). Target sequences for cortactin: ...
Background: While genetic knockdown of RAS in mouse tumor models has substantiated it as a therapeutic target, there is no effective means of targeting RAS currently available in the clinic today. Numerous RNA interference-based studies targeting RAS have demonstrated therapeutic effects, however, effective delivery has been a major obstacle that has impeded this approach.. U1 Adaptors are a novel technology for oligonucleotide-mediated gene silencing that act by selectively interfering with polyadenylation of messenger RNA (mRNA) inside the cell nucleus. Polyadenosine (PolyA) tail addition is an obligatory step in mRNA maturation and function, and its failure results in rapid degradation of the nascent message by endogenous nucleases. The eukaryotic U1 small nuclear ribonucleoprotein complex (U1 snRNP) is best known for its role as a pre-mRNA splicing factor, but also acts naturally to silence some genes by suppressing polyadenylation.. U1 Adaptors are synthetic oligonucleotides that enable the ...
Ola R, Dubrac A, Han J, Zhang F, Fang JS, Larrivée B, Lee M, Urarte AA, Kraehling JR, Genet G, Hirschi KK, Sessa WC, Canals FV, Graupera M, Yan M, Young LH, Oh PS, Eichmann A: PI3 kinase inhibition improves vascular malformations in mouse models of hereditary haemorrhagic telangiectasia. Nat Commun. 2016 Nov 29; 2016 Nov 29. PMID: 27897192 Zhang F, Prahst C, Mathivet T, Pibouin-Fragner L, Zhang J, Genet G, Tong R, Dubrac A, Eichmann A: The Robo4 cytoplasmic domain is dispensable for vascular permeability and neovascularization. Nat Commun. 2016 Nov 24; 2016 Nov 24. PMID: 27882935 Kraehling JR, Chidlow JH, Rajagopal C, Sugiyama MG, Fowler JW, Lee MY, Zhang X, Ramírez CM, Park EJ, Tao B, Chen K, Kuruvilla L, Larriveé B, Folta-Stogniew E, Ola R, Rotllan N, Zhou W, Nagle MW, Herz J, Williams KJ, Eichmann A, Lee WL, Fernández-Hernando C, Sessa WC: Genome-wide RNAi screen reveals ALK1 mediates LDL uptake and transcytosis in endothelial cells. Nat Commun. 2016 Nov 21; 2016 Nov 21. PMID: 27869117 ...
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In this paper, we describe the results of a knockdown screen in mouse ES cells to identify factors required for differentiation. Grouping of the identified genes into functional pathways shows that multiple hits are involved in Ras-Mek-Erk signaling. EphB4 receptors can regulate the activity of the Ras family of GTPases, including H-Ras and R-Ras (Zou et al., 1999; Miao et al., 2001; Wang et al., 2006). When Ptpn11 (also called Shp-2), another hit from our screen, was prevented from interacting with a mutated gp130 receptor that failed to activate ERKs, this led to self-renewal (Burdon et al., 1999). These data show that our unbiased, genome-wide knockdown approach identified several factors that were previously identified to be important in self-renewal of ES cells and validate our screening strategy. An shRNA against Capn10 was found in ∼50% of the sequences and, when tested individually, this shRNA showed strong ES colony outgrowth during the first 2 wk after removal of LIF. During the 3rd ...
TY - JOUR. T1 - RNAi as a potential new therapy for HIV infection. AU - Wheeler, Lee A.. AU - Dykxhoorn, Derek M.. PY - 2008/12/1. Y1 - 2008/12/1. N2 - Controlling HIV infection continues to be a major clinical and scientific challenge. Despite the therapeutic benefits associated with HAART, the need for novel treatment approaches to combat HIV-1 remains. Effective inhibition of HIV-1 infection has been achieved by harnessing the endogenous RNAi pathway in a variety of cell types, including primary T cells and macrophages. Here we discuss the opportunities and challenges associated with translating these findings into clinically relevant therapeutic approaches.. AB - Controlling HIV infection continues to be a major clinical and scientific challenge. Despite the therapeutic benefits associated with HAART, the need for novel treatment approaches to combat HIV-1 remains. Effective inhibition of HIV-1 infection has been achieved by harnessing the endogenous RNAi pathway in a variety of cell types, ...
The field of RNA-based gene regulation has been attracting increasing interest over the past couple of years, and the regulation of gene expression by small dsRNAs is being studied intensively. Such interference can be mediated by siRNAs, which cleave a sequence-specific target mRNA, or by micro-RNAs, which inhibit translation of a target mRNA. Noncoding RNAs have also been found to play important roles in the regulation of gene expression, for example, in gene silencing by methylation of DNA or histones. Small interfering RNAs are expected to have medical application in human therapy as drugs with high specificity for their molecular targets.. A number of studies on synthetic siRNAs or DNA vector-derived small hairpin RNAs (shRNAs) in cell culture systems have been published, and there are also several animal studies (15, 16, 17, 18, 19) . McCaffrey et al. (15) cotransfected the firefly luciferase gene along with synthetic siRNAs or a shRNA expression vector into mice by hydrodynamic injection ...
RNAi is a convenient, widely used tool for screening for genes of interest. We have recently used this technology to screen roughly 750 candidate genes, in C. elegans, for potential roles in regulating muscle protein degradation in vivo. To maximize confidence and assess reproducibility, we have only used previously validated RNAi constructs and have included time courses and replicates. To maximize mechanistic understanding, we have examined multiple sub-cellular phenotypes in multiple compartments in muscle. We have also tested knockdowns of putative regulators of degradation in the context of mutations or drugs that were previously shown to inhibit protein degradation by diverse mechanisms. Here we discuss how assaying multiple phenotypes, multiplexing RNAi screens with use of mutations and drugs, and use of bioinformatics can provide more data on rates of potential false positives and negatives as well as more mechanistic insight than simple RNAi screening.
RNAi is a convenient, widely used tool for screening for genes of interest. We have recently used this technology to screen roughly 750 candidate genes, in C. elegans, for potential roles in regulating muscle protein degradation in vivo. To maximize confidence and assess reproducibility, we have only used previously validated RNAi constructs and have included time courses and replicates. To maximize mechanistic understanding, we have examined multiple sub-cellular phenotypes in multiple compartments in muscle. We have also tested knockdowns of putative regulators of degradation in the context of mutations or drugs that were previously shown to inhibit protein degradation by diverse mechanisms. Here we discuss how assaying multiple phenotypes, multiplexing RNAi screens with use of mutations and drugs, and use of bioinformatics can provide more data on rates of potential false positives and negatives as well as more mechanistic insight than simple RNAi screening.
Both ATM and ATR display a preference for phosphorylating SQ/TQ motifs in their substrates (Kim et al., 1999; Traven and Heierhorst, 2005; Shiloh, 2006). ATR is predominantly activated by UV light and stalled replication forks, whereas ATM is specifically activated by DSBs of DNA, as seen after irradiation, etoposide, or oxidative stress (Abraham, 2001; Shiloh, 2006). In contrast, treatment with the ATP-competitive kinase inhibitor, staurosporine, does not activate ATM or affect the phosphorylation status of ATM-dependent substrates (Kamer et al., 2005). We show here that DNA-damaging agents, such as IR and etoposide, trigger MEF2D phosphorylation. Moreover, MEF2D phosphorylation only increased after etoposide exposure in wt-ATM cells but not in ATM-deficient cells. These results suggest that ATM mediates MEF2D phosphorylation in response to DSBs in DNA.. Furthermore, in the present study, RNA interference-mediated knockdown experiments in cerebellar granule cells indicate that endogenous MEF2D ...
RNA interference (RNAi) is an important pathway that is used in many different organisms to regulate gene expression. This animation introduces the principles of RNAi involving small interfering RNAs (siRNAs) and microRNAs (miRNAs). We take you on an audio-visual journey through the steps of gene expression and show you an up-to-date view of how RNAi can silence specific mRNAs in the cytoplasm.. ...
Qiang Zhang is the author of this article in the Journal of Visualized Experiments: DNA Vector-based RNA Interference to Study Gene Function in Cancer
RNA interference involves the targeted knockdown of mRNA triggered by complementary dsRNA molecules applied to an experimental organism. Although this technique has been successfully used in honeybees
RNA interference (RNAi) therapeutics (siRNA, miRNA, etc.) represent an emerging medicinal remedy for a variety of ailments. However, their low serum stability and low cellular uptake signi cantly restrict their clinical applications. Exosomes are biologically derived nanodimensional vesicle ranging from a few nanometers to a hundred. In the last few years, several reports have been published demonstrating the emerging applications of these exogenous membrane vesicles, particularly in carrying different RNAi ther- apeutics to adjacent or distant targeted cells. In this report, we explored the numerous aspects of exosomes from structure to clinical implications with special emphasis on their application in delivering RNAi-based therapeutics. siRNA and miRNA have attracted great interest in recent years due to their speci c applica- tion in treating many complex diseases including cancer. We highlight strategies to obviate the challenges of their low bioavailability for gene therapy ...
The implementation of decisions affecting cell viability and proliferation is dependant on prompt detection of the problem to become addressed, formulation and transmission of the correct group of instructions and fidelity in the execution of orders. nearing mitosis might encounter, presenting the effect of post-translational adjustments (PTMs) on the right and timely working of pathways fixing errors or harm before chromosome segregation. We conclude this article having a perspective on the existing position of mitotic signaling pathway inhibitors 154235-83-3 IC50 and their potential make use of in malignancy therapy. (Mazzarello, 1999). The main occasions characterizing changeover through the cell routine are cell development, where means cells boost their size and the amount of organelles, and duplication of hereditary materials in S-phase. If not really perturbed, upon conclusion of DNA replication cells enter mitosis, a term that originally explained nuclear department (Mazzarello, 1999). ...
The most enjoyable part in following RNAi Therapeutics is to look at the rich stream of scientific data and determine the absolute maturity and competitive position of the technologies and companies involved, as well as getting a glimpse at relationship dynamics. I therefore thought to share today two examples of this that I picked up recently. One is a paper by Sirna Therapeutics/Merck shedding some light on their approach towards RNAi pharmacology and RNAi trigger design. The other is some intriguing evidence that Silence Therapeutics most important gene target, PKN3, is gaining traction in the pharmaceutical space. Studying the pharmacology of siRNA delivery. Pei and colleagues from Merck published in RNA a nice paper on better understanding the pharmacology of siRNA delivery [Pei et al. (2010). Quantitative evaluation of siRNA delivery in vivo]. Unlike small molecules or even antibodies, the pharmacology of RNAi Therapeutics is more complex as simply measuring the raw tissue abundance of an ...
Keywords: placenta, cancer of the colon, endothelium, VEGF, immunohistochemistry, angiogenesis Vascular endothelial development element A (VEFG-A), probably the most prominent person in the VEGF family, is one of the key regulators of angiogenesis in general, including the promotion of tumor progression and metastasis (Kim et al. 1993; Ferrara et al. 2003). The important role of this growth factor in different areas of biological sciences makes it therefore an interesting target in many immunohistochemical studies. At present, at least nine different primary anti-VEGF antibodies are commercially available that can be applied to formalin-fixed and paraffin-embedded tissue samples (Table 1). Considering the literature on VEGF IHC applications, there is surprisingly little discussion about the selection of the applied VEGF antibody, and no consensus on which VEGF antibody is most reliable. In an attempt to validate five VEGF antibodies, Zhang et al. (1998) reported the R and D Systems mouse ...
Caenorhabditis elegans have many benefits for genetic manipulation and research. One of the most beneficial features is that it is transparent. This is great for microscopy because it makes it easier for us to see what is different with the worms reproductive system when comparing it to the normal, not treated worm. For the experiments I perform for the microscopy element, we repeat the RNAi interference experiments with strains with fluorescent markers. GFPs are green fluorescent proteins that can stain a particular part of a cell; like a cell wall and RFP are red fluorescent protein can stain the chromosomes within the nucleus of the cell. With the strain I am working with, AJ740, I can utilize the GFP and RFP to see what is happening to the shape and overall placement of the eggs within the affect mother worm treated through RNA interference along with what is going on with the chromosomes. I have several questions. General questions like: are the eggs going to the right place and are there ...
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TOPICS-I. 1-Regulatory RNA, 2- RNA interference and micro RNA, 3-Retroviruses, 4-Transposons and Retroposons, 5-Promoters and Enhancers , 6-Activating Transcription, 7-RNA Splicing and Processing, 8-Chromosomes-Nucleosomes, 9-Controlling Chromatin Remodeling and Structure. Slideshow 6603499 by angelica-figueroa
One siRNA sequence, many cell lines - posted in siRNA, microRNA and RNAi: Hi all, Im new to process of siRNA transfection and I was wondering: Will one siRNA sequence (previously validated in the lab) be good enough to transfect multiple other cell lines from the same organism? I understand that the actual process of transfection will be different for each cell line, but I am curious as to whether I need to also worry about the sequence itself. Thanks!
Press release - Allied Market Research - RNA Interference (RNAi) Drug Delivery Market Statistics (2019-2026): Hyper Growth Recorded in the Future - published on openPR.com