利用稳定转染细胞系设计、筛选靶向牛催乳素基因受体的sgRNA及其对胚胎发育的影响

Design, Screening, and Impact of sgRNAs Targeting Bovine Prolactin Gene Receptor on Embryonic Development Using Stably Transfected Cell Lines.

作者信息

Wang Daqing, Cao Guifang, Li Xin, Cheng Xin, Guo Zhihui, Li Lu, Su Hong, Zhang Kai, Zhang Yuanyuan, Zhang Min, Zhao Feifei, Zhao Yifan, Liang Junxi, Liu Yiyi, Zhang Yong

机构信息

College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010011, China.

Animal Embryo and Developmental Engineering Key Laboratory of Higher Education, Institutions of Inner Mongolia Autonomous Region, Hohhot 010011, China.

出版信息

Biology (Basel). 2025 Apr 15;14(4):425. doi: 10.3390/biology14040425.

DOI:10.3390/biology14040425

PMID:40282290

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC12024835/

Abstract

This study designed three sgRNAs (sgRNA139, sgRNA128, and sgRNA109) targeting the prolactin gene receptor (PRLR) in fetal cattle, utilized Cas9 to cleave endogenous DNA, and screened stable cell lines for somatic cell nuclear transfer experiments to investigate the impact of different editing sites on embryonic development. The results showed that sgRNA139 had the highest cleavage efficiency (Fcut = 0.65, Indels = 42.19%), while sgRNA109 had the lowest (Fcut = 0.45, Indels = 35.31%). No significant differences were observed in cell growth status after electroporation ( > 0.05), and the transfection efficiency exceeded 90% after five days of culture. In the evaluation of key embryonic development indicators, sgRNA109 significantly reduced the cleavage rate and blastocyst rate ( < 0.01), whereas sgRNA139 showed no significant effect on the cleavage rate ( > 0.05), but its blastocyst rate was slightly lower than that of the control group ( > 0.05). This study demonstrates that highly specific sgRNAs and stable edited cell lines used as donor cells can significantly regulate the later stages of embryonic development. This study not only provides new experimental evidence for the functional study of the PRLR but also lays an important theoretical foundation for the innovation of molecular breeding technologies in dairy cattle.

摘要

本研究设计了三种靶向胎牛催乳素基因受体（PRLR）的sgRNA（sgRNA139、sgRNA128和sgRNA109），利用Cas9切割内源性DNA，并筛选用于体细胞核移植实验的稳定细胞系，以研究不同编辑位点对胚胎发育的影响。结果表明，sgRNA139的切割效率最高（Fcut = 0.65，Indels = 42.19%），而sgRNA109的切割效率最低（Fcut = 0.45，Indels = 35.31%）。电穿孔后细胞生长状态无显著差异（> 0.05），培养五天后转染效率超过90%。在关键胚胎发育指标评估中，sgRNA109显著降低了 cleavage 率和囊胚率（< 0.01），而sgRNA139对 cleavage 率无显著影响（> 0.05），但其囊胚率略低于对照组（> 0.05）。本研究表明，高度特异性的sgRNA和用作供体细胞的稳定编辑细胞系可显著调节胚胎发育后期。本研究不仅为PRLR的功能研究提供了新的实验证据，也为奶牛分子育种技术创新奠定了重要理论基础。