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A streamlined method for the design and cloning of shRNAs into an optimized Dox-inducible lentiviral vectorFrank, Sander B., Schulz, Veronique V., Miranti, Cindy K. 28 February 2017 (has links)
Background: Short hairpin RNA (shRNA) is an established and effective tool for stable knock down of gene expression. Lentiviral vectors can be used to deliver shRNAs, thereby providing the ability to infect most mammalian cell types with high efficiency, regardless of proliferation state. Furthermore, the use of inducible promoters to drive shRNA expression allows for more thorough investigations into the specific timing of gene function in a variety of cellular processes. Moreover, inducible knockdown allows the investigation of genes that would be lethal or otherwise poorly tolerated if constitutively knocked down. Lentiviral inducible shRNA vectors are readily available, but unfortunately the process of cloning, screening, and testing shRNAs can be time-consuming and expensive. Therefore, we sought to refine a popular vector (Tet-pLKO-Puro) and streamline the cloning process with efficient protocols so that researchers can more efficiently utilize this powerful tool. Methods: First, we modified the Tet-pLKO-Puro vector to make it easy("EZ") for molecular cloning (EZ-Tet-pLKO-Puro). Our primary modification was to shrink the stuffer region, which allows vector purification via polyethylene glycol precipitation thereby avoiding the need to purify DNA through agarose. In addition, we generated EZ-Tet-pLKO vectors with hygromycin or blasticidin resistance to provide greater flexibility in cell line engineering. Furthermore, we provide a detailed guide for utilizing these vectors, including shRNA design strategy and simplified screening methods. Results: Notably, we emphasize the importance of loop sequence design and demonstrate that the addition of a single mismatch in the loop stem can greatly improve shRNA efficiency. Lastly, we display the robustness of the system with a doxycycline titration and recovery time course and provide a cost/benefit analysis comparing our system with purchasing pre-designed shRNA vectors. Conclusions: Our aim was twofold: first, to take a very useful shRNA vector and make it more amenable for molecular cloning and, secondly, to provide a streamlined protocol and rationale for cost-effective design, cloning, and screening of shRNAs. With this knowledge, anyone can take advantage of this powerful tool to inducibly knockdown any gene of their choosing.
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A dominant negative over expression model of mammalian MED12 functionPacker, Hans Levi 01 May 2011 (has links)
Although schizophrenia has been shown to have a substantial component, there is a paucity of known risk alleles. Furthermore, all of the known risk genes are of small effect sizes. Previously it has been shown that a 12 base pair insertional polymorphism in the in the C-terminal, opposite paired (Opa) domain of the MED12 gene known as MED1212bp represents a small but significant risk for a positive syndrome psychosis. In addition, the MED1212bp polymorphism is found in approximately 1.6 percent of X-chromosomes of northern European decent.
Studies in zebrafish show that alterations in MED12 reduces staining for monoaminergic neuronal populations, including dopaminergic and serotonergic populations. However, precise mechanisms through which these changes occur are not known. My goal for this study was to use PC6-3 cells as a mammalian cell culture for studying cellular and transcriptional effects of MED12 in a dopaminergic model system. The approach I took was based on studies that have shown that overexpression of C-terminal proline, glutamine and leucine rich (PQL) and Opa domain constructs interact in a dominant negative manner with several transcriptional regulatory proteins that interact with MED12. GFP tagged PQL and Opa domain constructs were placed into a tetracycline inducible T-REx™ regulated expression vector and introduced into a previously generated PC6-3, TR156 cell line that expresses the Tet-Repressor molecule.
In this study, I report a selection bias against stably transfected cell lines strongly expressing constructs containing the two C-terminal PQL-Opa protein domains of MED12. I also show that the described low levels of induction of that construct are associated with small, but significant alterations in nuclear morphology, possibly due to nuclear reorganization. Induction of PQL-Opa domains also increases in cell metabolism as measured by a tetrazolium salt assay, typically associated with increases in proliferation compared to the GFP controls or Opa domain alone. Interestingly, the MTS results in the stable cell lines were not reflected changes in cell numbers from direct cell counts performed by light microscopy, or changes in cell cycle distribution as measured by propidium iodide staining and fluorescence activated cell sorting (FACS).
In addition I also show microarray gene expression data for both the stable tetracycline inducible lines, as well as transiently electroporated PC6-3 cells. For both the stable and transient expression experiments, the arrays were characterized by small fold changes, which were not validated by RT-PCR. The stable arrays did not produce any robust findings. However, gene ontology (GO) data, as determined by GoMiner analysis, from the transiently electroporated cells shows that 9 of the top 31 GO categories are related to changes in proliferation and cytoskeletal reorganization. However, despite this trend, the data from the GoMiner analysis was above the level of statistical significance (á = 0.05), as is indicated by the false discovery rates (FDR > 0.3). Analysis of the directionality of expression proved intriguing and demonstrated significant evidence of skewing in the pattern of differential expression of annotated genes where there was a significant tendency for the most significantly differentially expressed probes belonging to 13568 annotated genes to be more highly expressed genes in the electroporated GFP control construct cells than those with the PQL/Opa construct. This is also consistent with a broad overlap of the expression data with ChIP-seq data suggesting that the dominant negative effect may be spread over many MED12 regulated genes, in which case the low expression levels are particularly problematic. While the data from these experiments do not present a clear mechanism for MED12 function, they are informative in developing models of MED12 alteration, and potential improvements are discussed.
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Prolactin-inducible-protein (PIP) influences host immunity by regulating intracellular signaling pathways in macrophagesIhedioha, Olivia 25 August 2015 (has links)
The human prolactin-inducible protein (PIP) or gross cystic disease fluid protein -15 (GCDFP-15) is a 15 kD protein secreted by human breast cancer cells and is abundant in fluids from gross cystic breast disease. Previous results from our laboratory showed that PIP KO mice had significantly lower numbers of CD4+ T cells in their secondary lymphoid organs, and these cells are impaired in their ability to differentiate into Th1 cells in vitro and in vivo leading to failure to control Leishmania major infection. In the present study, we further assessed the role of PIP in adaptive immunity by comparing cytokine production and intracellular signaling events in macrophages from WT and PIP KO mice following IFN-γ and lipopolysaccharide (LPS) stimulation. We show that although the expressions of IFN-γR and TLR4 on macrophages from KO and WT mice were comparable, PIP KO macrophages were significantly impaired in producing proinflammatory cytokines following IFN-γ and LPS stimulation. This was associated
with impaired phosphorylation of mitogen-activated protein kinases (MAPKs) and signal
transducers of activation of transcription (STATs) proteins in IFN-γ and LPS-stimulated
macrophages from PIP KO mice. Interestingly, the expression of suppressors of cytokine
signaling (SOCS) 1 and 3 proteins, known to suppress IFN-γ and LPS signaling, was higher in PIP KO macrophages compared to those from WT mice. Collectively, our studies clearly show that deficiency of PIP significantly affects intracellular signaling events leading to proinflammatory cytokine production in macrophages, and further confirm a role for PIP as important immunoregulatory protein involved in host defense. / October 2015
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Caveolina-1 reduce la transcripción dependiente de hif1α en un mecanismo dependiente del óxido nítrico en células tumoralesSanhueza Muñoz, Carlos Joaquín January 2013 (has links)
Doctor en Farmacología / Autorizada por el autor, pero con restricción para ser publicada a texto completo en el Portal de Tesis Electrónicas, hasta diciembre de 2018 / El cáncer es la 2° causa de muerte en Chile. El desarrollo del cáncer se ha propuesto que es consecuencia de la pérdida de función de los genes supresores de tumores beneficiando la acción de los oncogenes (genes que promueven el crecimiento de tumores). La activación del Factor inducible por hipoxia 1α (HIF1α) en un ambiente reducido en oxígeno (hipoxia) o por óxido nítrico (NO), permiten la proliferación y adaptación metabólica de las células tumorales. Por otro lado, Caveolina-1 es una proteína de andamiaje, que ha sido descrita como un supresor de tumores y promotor de metástasis. Resultados de nuestro laboratorio han demostrado que E-Cadherina y Caveolina-1 actúan cooperativamente en supresión de tumores in vivo. Sin embargo, Caveolina-1 suprime el crecimiento de tumores incluso en células carentes de E-Cadherina, de un modo menos eficiente. La isoforma endotelial de la sintasa de óxido nítrico (NOS3), es uno de los blancos inhibidos por Caveolina-1 más ampliamente descritos en la literatura, que recientemente ha sido implicado en mantenimiento tumoral. Cómo la inhibición de NOS mediada por Caveolina-1 afecta la activación de HIF1α contribuyendo a la función supresora de tumores de Caveolina-1 en ausencia de E-Cadherina, aún no ha sido evaluado. En este trabajo, evaluamos la posibilidad que la inhibición de NOS por Caveolina-1 pueda reducir la transcripción dependiente de HIF1α y contribuir así a la función supresora de tumores de Caveolina-1. Líneas celulares transfectadas con un plasmidio que codifica para Caveolina-1 [HT29(US), B16F10 y HEK293T] o células en que la expresión endógena de Caveolina-1 fue disminuida utilizando un shRNA [MDA-MB231], fueron tratadas en hipoxia (1% O2, 4-24 h). La actividad transcripcional de HIF, la expresión de genes blanco de HIF1α, la distribución subcelular de HIF1α, Caveolina-1 y NOS3, fueron evaluados mediante ensayos de gen reportero, RT-PCR/qPCR, Western Blot y microscopía confocal, respectivamente. Además, se realizaron ensayos de formación de tumores en ratones inmunosuprimidos SCID-Beige y en ratones inmunocompetentes C57BL/6. Los principales hallazgos de este trabajo fueron, que Caveolina-1 reduce la actividad transcripcional de HIF1α y la expresión de VEGF en hipoxia en las líneas celulares analizadas. Además, la reducción del crecimiento tumoral de células de melanoma murino B16F10(Cav-1) fue coincidente con la reducción de la expresión del mRNA de vegf in vivo. El tratamiento de las células con el dador de NO (DETA/NO) o el inhibidor de arginasa BEC, previnieron la inhibición de la actividad transcripcional de HIF1α mediada por Caveolina-1. Además, la inhibición de NOS con L-NAME o con el inhibidor selectivo de NOS3, L-NIO, reducen el incremento en la actividad transcripcional de HIF en hipoxia. In vivo, la sobreexpresión de HIF1α en células HT29(US)(Cav-1) revierte la supresión de tumores mediada por Caveolina-1 en ratones SCID-Beige. Finalmente, el tratamiento sistémico de ratones C57BL/6 con L-NAME, reduce el volumen tumoral a niveles comparables con los observados en los tumores formados por células B16F10(Cav-1). En resumen, nuestros resultados sugieren que la inhibición de NOS3 mediada por Caveolina-1 reduce la actividad transcripcional de HIF1α y la expresión de sus genes blanco, in vitro e in vivo, contribuyendo a la función supresora de tumores de Caveolina-1 en ausencia de E-Cadherina / Cancer, the 2nd most important cause of death in Chile, is thought to develop due to loss of tumor suppressor and gain of oncogene function. Activation of Hypoxia-inducible factor 1α (HIF1α) in the low oxygen environment (hypoxia) present in tumors or by nitric oxide (NO), favors cancer cell proliferation and metabolic adaptation. Caveolin-1 is a scaffolding protein that reportedly functions both as a tumor suppressor and promoter of metastasis. Results from this laboratory have shown that E-cadherin and Caveolin-1 cooperate in tumor suppression in vivo. However, Caveolin-1 expression suppresses tumor growth even in cancer cells lacking E-cadherin, albeit less efficiently. The endothelial isoform of nitric oxide synthase (NOS3), one of the best-established targets for inhibition by Caveolin-1, has recently been implicated in maintence of tumor growth. Whether, Caveolin-1-mediated NOS inhibition may impact on HIF1α activation and account for tumor suppression by Caveolin-1 in the absence of E-cadherin has not been yet assessed. Here, we evaluated the possibility that NOS inhibition by Caveolin-1 may reduce HIF1α - dependent transcription and thereby contribute to the tumor suppressor function of Caveolin-1. Cell lines transfected with a Caveolin-1-encoding plasmid [HT29(US), B16F10 and HEK293T] or cells where endogenous Caveolin-1 protein levels [MDA-MB231] were reduced using shRNA-technology, were exposed to hypoxia (1% O2, 4-24 h). In these cells, HIF transcriptional activity, HIF1α target-gene expression, HIF1α, Caveolin-1 and NOS3 protein levels and subcellular localization were evaluated by gene reporter assays, RT-PCR/qPCR, Western Blot and confocal microscopy, respectively. Tumor forming capacity was evaluated in immunodeficient SCID-Beige and immunocompetent C57BL/6 mouse strains. The main findings of this study are that Caveolin-1 reduced HIF1α transcriptional activity and VEGF expression in hypoxia in vitro in all cell lines. Also, reduced tumor growth of B16F10(Cav-1) melanoma cells in C57BL/6 mice correlated with reduced vegf gene expression in vivo. Treatment of cells with the NO donor (DETA/NO) o arginase inhibition with BEC, prevented Caveolin-1-mediated inhibition of HIF1α transcriptional activity. Furthermore, NOS inhibition with L-NAME or selective NOS3 inhibition with L-NIO, reduced hypoxia-enhanced HIF transcriptional activity. In vivo HIF1α overexpression in HT29(US)(Cav-1) cells reversed tumor suppression due to Caveolin-1 in SCID-Beige mice. Finally, systemic treatment of C57BL/6 mice with L-NAME, reduced tumor volumes to levels comparable to those observed for tumors formed by B16F10(Cav-1) cells. In summary, our observations suggest that Caveolin-1-mediated inhibition of NOS3 activity reduces HIF1α transcriptional activity and target gene expression, both in vitro and in vivo, and that this ability contributes to tumor suppression by Caveolin-1 in the absence of E-cadherin / CONICYT, FONDECYT, FONDAP, MECESUP
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Utilising salmonella to deliver heterologous vaccine antigenSaxena, Manvendra, s3031657@student.rmit.edu.au January 2007 (has links)
Live attenuated Salmonella vectors provide a unique alternative in terms of antigen presentation by acting as a vector for heterologous antigens. The efficiency of any live bacterial vector rests with its ability to present sufficient foreign antigen to the human or animal immune system to initiate the desirable protective immune response. Salmonella vectors encoding heterologous protective antigens can elicit the relevant immune responses, be it humoral, mucosal or cell-mediated. STM-1 is a Salmonella mutant developed by RMIT, harbours a mutation in the aroA gene that renders it attenuated, and is a well characterised vaccine strain currently in use to protect livestock against Salmonella infection. In previous work in this laboratory, STM1 was shown to be capable of eliciting immune responses in mice to plasmid-borne antigens. In this study STM-1 was analysed for its ability to vector the model antigen chicken ovalbumin and test antigen C. jejuni major outer membrane protein using in vivo inducible promoters such as pagC and nirB from the plasmid location. The determination of the architecture around the lesion in STM-1 also allowed the development of constructs expressing heterologous antigen from the chromosome. The induction of immune responses, both humoral and cell mediated, was analysed. Another issue addressed in this study was effect of pre-existing immune responses in the animal host against the vector or related strains and the effects on generation of immune responses against the subsequently vectored antigen. Humoral and cellular immune responses to vectored ovalbumin and C. jejuni Momp antigens were observed following vaccination with STM-1, when antigens were expressed from either the plasmid or chromosomal location. Up-regulation of immune responses, both humoral and cell mediated, was observed against the vectored antigens in animals which were pre-exposed to either the bacterial vector or related strains. These results indicate that STM-1 has the potential to be used as a vector to deliver heterologous vaccine antigens from a single copy gene in the field. Lastly, the results from this study indicate that pre-existing immune responses against the bacterial vector or a related strain do in fact enhance both humoral and T cell responses against the heterologous antigen.
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Role of Hypoxia-inducible Factor in Kidney CancerRoberts, Andrew Moore 14 August 2013 (has links)
Cellular adaptation to conditions of stress is critical to the survival of all organisms. In mammalian cells, reduced oxygen availability, or hypoxia, triggers a myriad of alterations within molecular pathways aimed at ensuring sustained viability and functionality of vital organs. The master regulator of the hypoxic response is the heterodimeric HIF transcription factor, composed of an oxygen-labile HIFalpha subunit and a constitutively expressed and stable HIFbeta(ARNT) subunit. While experiments in mice have demonstrated the indispensability of HIF1alpha/HIF2alpha, unchecked hyperactivation of HIF is associated with pathological complications, such as the development of clear-cell renal cell carcinoma (ccRCC), the most common form of kidney cancer. In particular, overexpression of HIF2alpha has been intimately linked to ccRCC molecular pathogenesis. Here, we report that HIF2alpha overexpression leads to Akt-mediated hyperactivation of Hdm2, resulting in decreased activity of p53 and increased resistance to apoptosis. Significantly, we show that restoration of p53 activity via inhibition of Hdm2 reverses chemoresistance of otherwise refractory ccRCC cells. We also demonstrate that the picornavirus EMCV (Encephalomyocarditis virus) efficiently destroys ccRCC tumour cells in an NF-kappaB- and HIFalpha-dependent manner both in vitro and in a mouse xenograft model. This work provides pre-clinical evidence for the potential use of EMCV in an oncolytic virus-based approach to the treatment of advanced ccRCC. In addition, using a cell biology-based approach we reveal that inhibition of the DNA methyltransferase DNMT1 leads to enhanced HIF promoter-binding affinity and transactivation activity in a manner that is independent of changes in HIFalpha protein levels. This study uncovers a novel HIF regulatory mechanism in mammalian cells. In summary, the work presented here provides insight into the molecular mechanisms governing HIF activity and the effects of HIF on the molecular pathogenesis of ccRCC. Our findings have the potential to provide new therapeutic avenues for the treatment of kidney cancer.
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Role of Hypoxia-inducible Factor in Kidney CancerRoberts, Andrew Moore 14 August 2013 (has links)
Cellular adaptation to conditions of stress is critical to the survival of all organisms. In mammalian cells, reduced oxygen availability, or hypoxia, triggers a myriad of alterations within molecular pathways aimed at ensuring sustained viability and functionality of vital organs. The master regulator of the hypoxic response is the heterodimeric HIF transcription factor, composed of an oxygen-labile HIFalpha subunit and a constitutively expressed and stable HIFbeta(ARNT) subunit. While experiments in mice have demonstrated the indispensability of HIF1alpha/HIF2alpha, unchecked hyperactivation of HIF is associated with pathological complications, such as the development of clear-cell renal cell carcinoma (ccRCC), the most common form of kidney cancer. In particular, overexpression of HIF2alpha has been intimately linked to ccRCC molecular pathogenesis. Here, we report that HIF2alpha overexpression leads to Akt-mediated hyperactivation of Hdm2, resulting in decreased activity of p53 and increased resistance to apoptosis. Significantly, we show that restoration of p53 activity via inhibition of Hdm2 reverses chemoresistance of otherwise refractory ccRCC cells. We also demonstrate that the picornavirus EMCV (Encephalomyocarditis virus) efficiently destroys ccRCC tumour cells in an NF-kappaB- and HIFalpha-dependent manner both in vitro and in a mouse xenograft model. This work provides pre-clinical evidence for the potential use of EMCV in an oncolytic virus-based approach to the treatment of advanced ccRCC. In addition, using a cell biology-based approach we reveal that inhibition of the DNA methyltransferase DNMT1 leads to enhanced HIF promoter-binding affinity and transactivation activity in a manner that is independent of changes in HIFalpha protein levels. This study uncovers a novel HIF regulatory mechanism in mammalian cells. In summary, the work presented here provides insight into the molecular mechanisms governing HIF activity and the effects of HIF on the molecular pathogenesis of ccRCC. Our findings have the potential to provide new therapeutic avenues for the treatment of kidney cancer.
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Hypoxia-inducible factors (HIFs) and biological responses in hypoxia, inflammation and embryonic vascular development /Hägg, Maria, January 2008 (has links)
Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2008. / Härtill 3 uppsatser.
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Midazolam inhibits the hypoxia-induced up-regulation of erythropoietin in the central nervous system / ミダゾラムは低酸素に誘導される脳内エリスロポイエチン発現上昇を抑制するMatsuyama, Tomonori 24 November 2015 (has links)
京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19369号 / 医博第4046号 / 新制||医||1012(附属図書館) / 32383 / 新制||医||1012 / 京都大学大学院医学研究科医学専攻 / (主査)教授 宮本 享, 教授 柳田 素子, 教授 松原 和夫 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM
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Aedes aegypti Heat Shock 70 Genes and their Inducible PromotersGross, Tiffany Lauren 21 July 2011 (has links)
Aedes aegypti is an important vector of the viruses that cause dengue fever, dengue hemorrhagic fever, and yellow fever. In depth genetic studies of vector species have been made possible due to the availability of genome sequences and techniques for producing stably transformed mosquitoes. These resources have also contributed to the establishment of new genetics-based approaches to the control of vector borne disease.
Genetic studies of Ae. aegypti have benefited from the ability to drive targeted transgene expression, however a ubiquitous inducible promoter has not been identified in this mosquito. The Drosophila melanogaster heat shock 70 promoter has been shown to drive inducible expression in heterologous systems; however, DmHsp70 possesses significant basal activity in Aedes aegypti.
This study characterized the sequence and expression of the heat shock 70 genes of Aedes aegypti. AaHsp70 genes were found to be organized in two clusters, each comprised of three divergent pairs. AaHsp70 genes exhibited robust expression upon heat shock in larvae, pupae, and adults as well as in heads, salivary glands, midguts and ovaries.
Genomic regions upstream of AaHsp70 genes were found to drive heat-inducible expression of a reporter in both cell and embryo assays. Deletion analysis of AaHsp70-derived promoters yielded two ~1.5 kb genomic fragments that maintained robust heat inducibility in these systems.
Aedes aegypti were transformed with AaHsp70-luciferase gene cassettes using the transposable element Mos1. AaHsp70-luciferase transcripts accumulated specifically after heat shock, and displayed a pattern of rapid induction and decay similar to endogenous AaHsp70 genes. Heat-induced expression of luciferase was observed in transgenic larvae, pupae and adults as well as heads, midguts and ovaries but not salivary glands, with levels varying between transgenic strains.
The effect of heat shock on the endogenous RNAi pathway as well as the effect of blood feeding on the expression of AaHsp70 genes was investigated, though reproducible results could not be obtained using the assays employed.
In conclusion, the heat shock 70 gene family of Aedes aegypti was identified and characterized. The AaHsp70 promoters described could be valuable for gene function studies as well as for the precise timing of the expression of anti-pathogen molecules. / Ph. D.
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