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Experiment Tags: AAV (AAV), Suspension Cells, Serum-Free Media, Transfection Reagent, Lodixanol Density Gradient
Experiment Summary: In this protocol, we developed a simple scalable AAV production protocol using serum-free-media-adapted HEK293T suspension cells and VirusGEN transfection reagent.
Experiment Tags: sgRNA Design, CRISPR-Cas9 System, sgRNA Library, Gene Editing, Gene Regulation
Experiment Summary: In this protocol, we discusse how to design sgRNA sequences and genome-wide sgRNA library using CRISPR-ERA.
Experiment Tags: Retrovirus, Enveloped Virus, MLV, HIV, Capsid Core Stability Assay, Fate of Capsid Assay, Glycosylated Gag, gPr80, TRIM5α
Experiment Summary: In this protocol, we describe a method using an in vitro detergent-based approach combined with ultracentrifugation for assessing viral capsid core stability. The results show that this method is a significantly simpler and faster way to assess relative capsid core stability.
Experiment Tags: CRISPR/Cas9, AAV (AAV), Adult Mouse Brain, Gibson Assembly, Dual-sgRNA
Experiment Summary: In this protocol, we describe a detailed protocol of AAV vector construction for cell type-specific CRISPR gene editing in the adult mouse brain. The method adopts a dual-sgRNA strategy for efficient disruption of the target gene. At first, a few different sgRNAs targeting the same gene are cloned into a plasmid expressing spCas9. After evaluation of the sgRNAs by a T7 endonuclease assay, the two most efficient sgRNAs are cloned in tandem into an AAV vector using the Gibson Assembly method.
Experiment Tags: Lentiviral Transduction, CAR T cells
Experiment Summary: In this protocol, an easy and reliable approach for CAR T cell generation is described. T cells are first isolated from peripheral blood and afterwards activated for one day with anti-CD3/CD28 Dynabeads. The gene transfer is performed by lentiviral transduction and gene transfer rate can be verified by flowcytometric analysis.
Experiment Tags: Varicella Zoster Virus (VZV), Replication Assays
Experiment Summary: This protocol describes a basic VZV replication assay using a recombinant VZV-GFP reporter virus. This reporter virus-infected cell line can be used in combination with siRNA gene depletion or cDNA overexpression transfection protocols to determine the effect of individual cellular genes on virus replication.
Experiment Tags: Biological Techniques, Cryopreservation, Cooling, Freezing
Experiment Summary: In this protocol, we describe the general procedure for cryopreservation of cultured cells.
Experiment Tags: Aedes aegypti, Mosquito, Zika virus, Arbovirus, Intrathoracic Inoculation, Microinjection
Experiment Summary: This protocol describes herein a protocol for the intrathoracic inoculation of ZIKV in Ae. aegypti. This method bypasses the midgut, which leads to a more rapid and higher proportion of disseminated infections in comparison to oral challenge, and mosquitoes become infected with a consistent dose of virus.
Experiment Tags: Adeno-Associated Viruses, Acute Brain Slices, Genetically-Encoded Fluorescent Biosensors, Hippocampus, Intracranial Injection, Dentate Granule cells
Experiment Summary: In this protocol, we describe a protocol for intracranial injections of two viral vectors encoding the metabolic biosensor Peredox and the calcium biosensor RCaMP1h. When combined with 2-photon microscopy and fluorescence lifetime imaging, this protocol allows the simultaneous quantitative assessment of changes in the cytosolic NADH/NAD+ ratio and the intracellular Ca2+ levels in individual dentate granule cells from acute hippocampal slices.
Experiment Tags: Influenza A virus, Infected mouse model, H1N1 (H1N1), Virus production, Plaque assay
Experiment Summary: In this protocol, we report a protocol in infected mice with different virus doses and strains to explore how an inhibitor of lysine-specific demethylase (LSD1) impacts disease progression.
