一种新型神经特异遗传工具—Nestin-Cre敲入大鼠的构建与表征OA

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.