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Experiment Tags: CRISPR, Genome Engineering, Cardiac Gene Editing, rAAV Delivery, Dystrophin Restoration
Experiment Summary: This protocol describes how to use the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system to perform postnatal cardiac gene editing in mdx/utrophin+/- mice. Specifically, the protocol uses the Staphylococcus aureus CRISPR-associated protein 9 (SaCas9) and guide RNAs (gRNAs) delivered via recombinant adeno-associated virus (rAAV) serotype rh.74 (rAAVrh.74) to carry out the gene editing. Restoring dystrophin expression in these mice's cardiac muscles is the main goal in hopes of developing a treatment for Duchenne muscular dystrophy (DMD).
Experiment Tags: Breast Cancer, Mouse Model, Intraductal Injection, Adenoviral Cre, Temporal Control
Experiment Summary: This protocol outlines a technique for injecting adenoviral Cre (Ad-Cre) intraductally into mice mammary glands to create animal models of breast cancer. By employing cell-type-specific promoters to target mammary epithelial cells (MECs), this strategy enables fine temporal control of cancer induction and cell-type specificity. The process is described in the protocol, which includes creating and caring for floxed mice, monitoring the growth of breast tumors, and providing postoperative care.
Experiment Tags: Mitochondria Transfer, Mesenchymal Stem Cells, Glioblastoma Stem Cells, Cell Interaction, Cell Biology.
Experiment Summary: This protocol facilitates the transfer of mitochondria from mesenchymal stem cells (MSCs) to glioblastoma stem cells (GSCs). This protocol allows for the investigation of the effects of mitochondria on GSC metabolism, proliferation, and response to therapy by isolating mitochondria from MSCs and transferring them to GSCs.
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.
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