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Experiment Tags: RMCE, Mouse Embryonic Stem Cells, Structure-Function Studies, Recombinase-Mediated Cassette Exchange, Gene Targeting, ROSALUC Mice, R26-iPSC Mice, pRMCE-DV1, Cre-Excised pRMCE-DV1, pCAG-FlpE-IRES-Puro-pA, Gateway pDONR221 Vector.
Experiment Summary: This protocol describes how to employ recombinase-mediated cassette exchange (RMCE) to perform structure-function research in mouse embryonic stem cells (mESCs). The process entails separating KO mESCs that are compatible with RMCE, creating a targeting vector that is compatible with RMCE, and using RMCE to target rescue constructs to the ROSA26 locus using mESCs.
Experiment Tags: Oncolytic viruses, tissue processing, viral infection, fluorescence microscopy, luciferase expression, plaque assay
Experiment Summary: As cancer treatments, oncolytic viruses (OVs) have potential, and determining whether living tissue specimens are infectious ex vivo prior to treatment offers a distinct benefit. This protocol describes the steps involved in preparing tissues for oncolytic virus ex vivo infection and the subsequent measurement of viral activity.
Experiment Tags: CRISPR-Cas9, Gene Editing, Lentivirus, AAV, Brown Adipose Tissue, Mouse Model
Experiment Summary: This Protocol optimizes CRISPR-Cas9 gene editing in brown adipose tissue (BAT) using lentivirus and AAV systems. Ucp1-Cre/Cas9 mice are used in in vivo research after screening effective sgRNAs in cultured cells and cloning them into AAV vectors. Western blot analysis is used to validate the effectiveness of gene deletion. By using this approach, the biology of BAT and its potential for treatment in metabolic diseases are better understood.
Experiment Tags: CRISPR, Gene Editing, Jurkat T Cells, Lentivirus, dCas9, gRNA, IFNG-AS1
Experiment Summary: This protocol outlines a comprehensive procedure for CRISPR-based gene editing in Jurkat T cells using lentiviral vectors encoding dCas9 and guide RNAs (gRNAs). The experiment includes vector design and generation, virus generation and particle count, dCas9-VP64 transduction, selection, clone creation, and screening.
Experiment Tags: HBV Infection Model, cell culture, HepG2, Co-culture
Experiment Summary: Current HBV research primarily focuses on hepatocytes, limiting insights into HBV-related pathology in non-hepatic tissues. This study introduces a protocol using non-hepatic 293T and hepatoma cells to investigate HBV infection. Two HBV infection methods were developed based on 293T-NE-3NRs.
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.
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