Gene Editing of Primary Rhesus Macaque B Cells

作者： Harald Hartweger1, Rajeev Gautam2, Yoshiaki Nishimura2, Fabian Schmidt3,5, Kai-Hui Yao1, Amelia Escolano1,6, Mila Jankovic1, Malcolm A. Martin2, Michel C. Nussenzweig1,4 1Laboratory of Molecular Immunology,The Rockefeller University, 2Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases,National Institutes of Health, 3Laboratory of Retrovirology,The Rockefeller University, 4Howard Hughes Medical Institute,The Rockefeller University, 5Laboratory of Applied Virology and Precision Medicine,King Abdullah University of Science and Technology (KAUST), 6Vaccine and Immunotherapy Center,Wistar Institute

简介：

Overview

This article presents a method for culturing and gene editing primary rhesus macaque B cells using CRISPR/Cas9 and recombinant adeno-associated virus serotype 6. The technique is designed for the study of B cell therapies and is adaptable for other cell types.

Key Study Components

Area of Science

• Neuroscience
• Gene Editing
• Cell Culture

Background

• Rhesus macaque B cells are important for studying immune responses.
• CRISPR/Cas9 technology allows precise gene editing.
• Recombinant adeno-associated virus (rAAV) is used for gene delivery.
• Preclinical research is essential for developing B cell therapies.

Purpose of Study

• To establish a reliable method for gene editing in B cells.
• To facilitate research on B cell therapies in rhesus macaques.
• To provide a protocol that can be adapted for other cell types.

Methods Used

• Preparation of B cell culture media and stimulants.
• Thawing and washing primary rhesus macaque splenocytes.
• Electroporation and transduction of B cells with CRISPR/Cas9 components.
• Genomic DNA analysis and flow cytometry for protein expression.

Main Results

• Efficient gene editing of B cells was achieved.
• The method allows for the testing of gene-edited B cells therapeutically.
• Production of rAAV6 was significantly improved compared to traditional methods.
• Editing efficiency was quantified using digital droplet PCR and Sanger sequencing.

Conclusions

• The presented method is effective for gene editing in rhesus macaque B cells.
• This approach can advance preclinical research in B cell therapies.
• Adaptability of the method for other cell types enhances its utility.

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