中文摘要：

利用荧光PCR技术对Crispr/Cas9系统敲除的micro RNA 505小鼠进行鉴定,能快速检测出敲除小鼠的基因型,有效加快敲除小鼠基因功能验证的进程。首先,在Crispr/Cas9系统敲除的基因位点区域设计PCR引物,并在上游引物的5’端使用羟基荧光素（5-carboxyfluorescein,FAM）荧光修饰。模板经过PCR的扩增后,将PCR产物与已知片段大小的6-羧基-X-罗丹明琥珀酰亚胺酯（6-carboxy-X-rhodamine,ROX）混合,在377测序仪上通过聚丙烯酰胺凝胶电泳分离。随后,根据相对分子质量内标法计算出PCR产物大小,从而达到鉴定小鼠基因型的目的。将上述荧光PCR技术应用于实践发现,两只奠基鼠、16只F1代小鼠以及26只F2代小鼠都能得到准确的鉴定,其中包括micro RNA 505基因区段被敲除了17 bp和23 bp。因此,利用荧光PCR技术可以快速、准确地鉴定Crispr/Cas9敲除小鼠。

英文摘要：

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.