Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro

作者： Zejin Cui*1, Pengkun Yuan*2, Zhishan Zhao*1, Fan Zhao3, Linrong Lu1,3 1Shanghai Immune Therapy Institute,Renji Hospital, Shanghai Jiao Tong University School of Medicine, 2Zhejiang University-University of Edinburgh Institute (ZJU-UoE Institute),Zhejiang University School of Medicine, Zhejiang University, 3Institute of Immunology and Department of Rheumatology,Sir Run Run Shaw Hospital, Zhejiang University School of Medicine

简介：

Overview

This study provides a standard protocol for studying the differentiation of Th17 cells, a subset of T helper cells that play a key role in immune responses. Through retroviral transduction of guide RNA into primary T cells from Cas9 transgenic mice, scientists can study the particular function of the genes in regulating Th17 differentiation.

Key Study Components

Area of Science

• Neuroscience
• Immunology
• Gene Editing

Background

• Th17 cells are crucial for immune responses.
• Understanding gene regulation in Th17 differentiation is vital.
• CRISPR-Cas9 technology allows for precise gene editing.
• Retroviral transduction is an efficient method for gene targeting.

Purpose of Study

• To provide a protocol for gene editing in primary T cells.
• To investigate the role of specific genes in Th17 differentiation.
• To enhance understanding of immune response mechanisms.

Methods Used

• Retroviral transduction of guide RNA into primary T cells.
• Utilization of CRISPR-Cas9 technology for gene editing.
• Flow cytometry to evaluate transduction efficiency.
• In vitro Th17 polarizations to stimulate CT4+T cell differentiation.

Main Results

• Efficient transduction of guide RNA into primary T cells was achieved.
• Successful monitoring of cell differentiation using flow cytometry.
• Insights gained into the regulation of Th17 differentiation.
• Protocol established for future studies in gene editing.

Conclusions

• The protocol offers an efficient alternative for gene editing.
• It aids in understanding the genetic regulation of Th17 cells.
• Future research can build on this methodology for further insights.

Frequently Asked Questions