中国比较医学杂志2026，Vol.36Issue(9)：63-74,12.DOI:10.3969/j.issn.1671-7856.2026.09.006
一种新型神经特异遗传工具—Nestin-Cre敲入大鼠的构建与表征
Construction and characterization of a novel neural-specific Nestin-Cre knock-in rat model
摘要
Abstract
Objective To establish a Nestin-Cre site-specific knock-in rat model using CRISPR/Cas9 technology,to provide a reliable and efficient genetic tool for research on nervous system development and disease mechanisms.Methods Specific single guide RNAs(sgRNAs)targeting the downstream region of Nestin exon 5 were designed and a homologous recombination donor plasmid containing an internal ribosome-entry site-Cre expression cassette was constructed.CRISPR/Cas9 system components and the donor vector were co-microinjected into rat zygotes,followed by embryo transfer to generate F0-generation rats.Knock-in-positive individuals were identified by polymerase chain reaction and Sanger sequencing and mated with wild-type rats to obtain F1 offspring to validate germline transmission stability.To evaluate the tissue-specific activity of Cre recombinase,F1 knock-in Nestin-Cre rats were crossed with Rosa26imCherry rats and expression levels of mCherry in the brain,spinal cord,and various peripheral organs in the offspring of Nestin-Cre;Rosa26imCherry rats was examined systematically by immunofluorescence.Results High-efficiency sgRNAs and donor plasmids were prepared successfully.Microinjection and embryo transfer produced F0 founders with precise knock-in,and stable transmission of the genetic modification was confirmed.High-level expression of mCherry in the central nervous system(brain and spinal cord)in Nestin-Cre;Rosa26imCherry rats was demonstrated by immunofluorescence.Scattered fluorescence signals were observed in peripheral tissues,including the heart,liver,spleen,lung,kidney,ovary,and muscle,consistent with the endogenous developmental expression profile of Nestin.Significant co-localization of mCherry with the mature neuronal marker NeuN in brain tissues indicated that Cre recombinase activity demonstrated clear neuronal lineage specificity.Conclusions A Nestin-Cre knock-in rat model was established successfully.This model achieves efficient and specific expression of Cre recombinase within the central nervous system,with a stably inheritable modification site,providing a valuable rat strain for conducting neuron-specific gene function studies in vivo.关键词
Nestin/Cre重组酶/CRISPR/Cas9/基因敲入/转基因大鼠/大鼠模型Key words
Nestin/Cre recombinase/CRISPR/Cas9/knock-in/transgenic rat/rat model分类
医药卫生引用本文复制引用
张磊,关菲菲,刘宁,马元武,孔琪,张旭,齐晓龙,贺佳跃,宫爽,吴玥,王珏,潘烁,高珊..一种新型神经特异遗传工具—Nestin-Cre敲入大鼠的构建与表征[J].中国比较医学杂志,2026,36(9):63-74,12.基金项目
国家科技重大专项(2025ZD01900700) （2025ZD01900700）
国家重点研发计划(2024YFF0728800) （2024YFF0728800）
中国医学科学院医学与健康科技创新工程(2021-I2M-1-024,2023-I2M-2-001) （2021-I2M-1-024,2023-I2M-2-001）
全国重点实验室专项(2060204) （2060204）
中国医学科学院公益性基本科研业务费专项(2023-PT180-01) （2023-PT180-01）
细胞生态海河实验室创新基金(HH24KYZX0007). （HH24KYZX0007）
