Protocol for evaluating the activity of R2 retrotransposons in mammalian cells

Yangcan Chen; Shengqiu Luo; Yanping Hu; Qi Zhou; Wei Li

doi:10.1016/j.xpro.2024.103538

Protocol for evaluating the activity of R2 retrotransposons in mammalian cells

STAR Protoc. 2025 Jan 6;6(1):103538. doi: 10.1016/j.xpro.2024.103538. Online ahead of print.

Authors

Yangcan Chen¹, Shengqiu Luo², Yanping Hu³, Qi Zhou⁴, Wei Li⁵

Affiliations

¹ Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China; Bejing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China. Electronic address: chenyangcan@ioz.ac.cn.
² Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
³ Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China; Bejing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China.
⁴ Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Bejing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China. Electronic address: zhouqi@ioz.ac.cn.
⁵ Key Laboratory of Organ Regeneration and Reconstruction, State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China; Institute for Stem Cell and Regeneration, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China; Bejing Institute for Stem Cell and Regenerative Medicine, Beijing 100101, China. Electronic address: liwei@ioz.ac.cn.

PMID: 39764849
DOI: 10.1016/j.xpro.2024.103538

Abstract

R2 retrotransposons can be harnessed to insert genes at targeted sites by all-RNA delivery, presenting a new technology for next-generation biotherapeutics. Here, we report a protocol for evaluating the gene integration activity of R2 retrotransposons in mammalian cells. We describe the construction of vectors separately expressing R2 protein and donor, the process of liposome transfection, and flow cytometry. This protocol provides a useful reporter system, which can be applied to evaluate the activity of other new retrotransposon systems. For complete details on the use and execution of this protocol, please refer to Chen et al.¹.

Keywords: CRISPR; biotechnology and bioengineering; molecular biology.