Abstract：Abstract: MicroRNA 505 knockout mice by the Crispr/Cas9 system were identified by fluorescence PCR technology, which can quickly detect the genotype of knockout mice, effectively speed up the process of gene function verification. Firstly, PCR primers were designed to apply to the gene loci which were edited by the Crispr/Cas9 system, and 5′end of the upstream primer was modified by 5-carboxyfluorescein. After PCR amplification, the PCR product was mixed with 6-carboxy-X-rhodamine of the known fragment size, and separated by polyacrylamide gel electrophoresis on ABI 377 Sequencer. Then, the size of PCR product was calculated according to relative molecular mass so as to achieve the purpose of identifying the genotype of mice. By application of fluorescent PCR technology, two founder mice, 16 F1 generation mice and 26 F2 generation mice were accurately identified, and among them, the microRNA 505 gene was knocked out with 17 bp or 23 bp. Therefore, the use of fluorescent PCR technology can quickly and accurately identify Crispr/Cas9 knockout mice.
引用本文:
仝 莉, 王茂春, 李 雨, 李 凯, 周宇荀, 肖君华, 金 力. 利用荧光PCR技术对Crispr/Cas9敲除microRNA 505的小鼠进行鉴定[J]. 生命科学研究, 2016, 20(4): 296-300. TONG Li, WANG Mao-Chun, LI Yu, LI Kai, ZHOU Yu-Xun, XIAO Jun-Hua, JIN Li. Genotyping of MicroRNA 505 Knockout Mice by Crispr/Cas9 Using Fluorescent PCR. Life Science Research, 2016, 20(4): 296-300.
2016,Vol. 20
