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In JoVE (1)
Other Publications (6)
Articles by Sonali Patil in JoVE
JoVE Immunology and Infection
Optimized Protocol for Efficient Transfection of Dendritic Cells without Cell Maturation
Center for Translational Systems Biology and Department of Neurology, Mount Sinai School of Medicine
We present our optimized high-throughput nucleofection protocol as an efficient way of transfecting primary human monocyte-derived dendritic cells with either plasmid DNA or siRNA without causing cell maturation. We further provide evidence for successful siRNA silencing of targeted gene RIG-I at both the mRNA and protein levels.
Other articles by Sonali Patil on PubMed
Platelet PECAM-1 Inhibits Thrombus Formation in Vivo
Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a cell surface glycoprotein receptor expressed on a range of blood cells, including platelets, and on vascular endothelial cells. PECAM-1 possesses adhesive and signaling properties, the latter being mediated by immunoreceptor tyrosine-based inhibitory motifs present on the cytoplasmic tail of the protein. Recent studies in vitro have demonstrated that PECAM-1 signaling inhibits the aggregation of platelets. In the present study we have used PECAM-1-deficient mice and radiation chimeras to investigate the function of this receptor in the regulation of thrombus formation. Using intravital microscopy and laser-induced injury to cremaster muscle arterioles, we show that thrombi formed in PECAM-1-deficient mice were larger, formed more rapidly than in control mice, and were more stable. Larger thrombi were also formed in control mice that received transplants of PECAM-1-deficient bone marrow, in comparison to mice that received control transplants. A ferric chloride model of thrombosis was used to investigate thrombus formation in carotid arteries. In PECAM-1-deficient mice the time to 75% vessel occlusion was significantly shorter than in control mice. These data provide evidence for the involvement of platelet PECAM-1 in the negative regulation of thrombus formation.
Novel Gene Ashwin Functions in Xenopus Cell Survival and Anteroposterior Patterning
The novel gene ashwin was isolated in a differential display screen for genes activated or up-regulated early in neural specification. ashwin is expressed maternally and zygotically, and it is up-regulated in the neural ectoderm after the midgastrula stage. It is expressed in the neural plate and later in the embryonic brain, eyes, and spinal cord. Overexpression of ashwin in whole embryos leads to anterior truncations and other defects. However, a second Organizer does not form, and the secondary axial structures may result from splitting of the Organizer, rather than axis duplication. Morpholino oligonucleotide-mediated reduction in ashwin expression leads to lethality or abnormalities in gastrulation, as well as significant apoptosis in midgastrula embryos. Apoptosis is also observed in midgastrula embryos overexpressing ashwin. Coexpression of ashwin with the bone morphogenetic protein-4 antagonist noggin has a synergistic effect on neural-specific gene expression in isolated animal cap ectoderm. Ashwin has no previously characterized domains, although two nuclear localization signals can be identified. Orthologues have been identified in the human, mouse, chicken, and pufferfish genomes. Our results suggest that ashwin regulates cell survival and anteroposterior patterning.
BioPP: a Tool for Web-publication of Biological Networks
Cellular processes depend on the function of intracellular molecular networks. The curation of the literature relevant to specific biological pathways is important for many theoretical and experimental research teams and communities. No current tool supports web publication or hosting of user-developed large scale annotated pathway diagrams. Sharing via web publication is needed to allow real-time access to the current literature pathway knowledge-base, both privately within a research team or publicly among the outside research community. Web publication also facilitates team and/or community input into the curation process while allowing centralized control of the curation and validation process. We have developed new tool to address these needs. Biological Pathway Publisher (BioPP) is a software suite for converting CellDesigner Systems Biology Markup Language (CD-SBML) formatted pathways into a web viewable format. The BioPP suite is available for private use and for depositing knowledge-bases into a newly created public repository.
Validation of Efficient High-throughput Plasmid and SiRNA Transfection of Human Monocyte-derived Dendritic Cells Without Cell Maturation
Transfection of primary immune cells is difficult to achieve at high efficiency and without cell activation and maturation. Dendritic cells (DCs) represent a key link between the innate and adaptive immune systems. Delineating the signaling pathways involved in the activation of human primary DCs and reverse engineering cellular inflammatory pathways have been challenging tasks. We optimized and validated an effective high-throughput transfection protocol, allowing us to transiently express DNA in naïve primary DCs, as well as investigate the effect of gene silencing by RNA interference. Using a high-throughput nucleofection system, monocyte-derived DCs were nucleoporated with a plasmid expressing green fluorescent protein (GFP), and transfection efficiency was determined by flow cytometry, based on GFP expression. To evaluate the effect of nucleoporation on DC maturation, the expression of cell surface markers CD86 and MHCII in GFP-positive cells was analyzed by flow cytometry. We established optimal assay conditions with a cell viability reaching 70%, a transfection efficiency of over 50%, and unchanged CD86 and MHCII expression. We examined the impact of small interfering RNA (siRNA)-mediated knockdown of RIG-I, a key viral recognition receptor, on the induction of the interferon (IFN) response in DCs infected with Newcastle disease virus. RIG-I protein was undetectable by Western blot in siRNA-treated cells. RIG-I knockdown caused a 75% reduction in the induction of IFNβ mRNA compared with the negative control siRNA. This protocol should be a valuable tool for probing the immune response pathways activated in human DCs.
Signaling Network of Dendritic Cells in Response to Pathogens: a Community-input Supported Knowledgebase
Dendritic cells are antigen-presenting cells that play an essential role in linking the innate and adaptive immune systems. Much research has focused on the signaling pathways triggered upon infection of dendritic cells by various pathogens. The high level of activity in the field makes it desirable to have a pathway-based resource to access the information in the literature. Current pathway diagrams lack either comprehensiveness, or an open-access editorial interface. Hence, there is a need for a dependable, expertly curated knowledgebase that integrates this information into a map of signaling networks.
