鼠原始生殖细胞报告基因系统的建立及验证

目的:构建小鼠原始生殖细胞（PGCs）报告基因系统。方法:根据CRISPR基因编辑技术结合同源重组原理，针对鼠Prdm1和Dppa3基因的翻译起始位点ATG附近位置分别设计一对sgRNA，并根据Cas9切割位点以及拟插入目的片段构建相应的用于同源重组的供体质粒，将sgRNA与Donor质粒同时转染入小鼠胚胎干细胞（ESCs）中，并利用质粒携带的抗性基因筛选出目标单克隆细胞，并进行基因型鉴定。结果:通过提取单克隆细胞的基因组DNA进行基因型鉴定，PCR和Sanger测序结果显示，单克隆细胞的基因组中，Prdm1和Dppa3基因翻译起始位点后分别成功插入eGFP和mCherry荧光蛋白编码序列。利用细胞因子诱导eGFP-Prdm1/mCherry-Dppa3mESCs（mouse ESCs）定向分化为mPGCs（mouse PGCs），流式细胞仪检测显示诱导分化的mPGCs特异表达eGFP和mCherry荧光信号。结论:成功构建mPGCs报告基因系统，可用于后续研究mPGCs命运决定的分子机制，对于研究小鼠早期胚胎发育过程具有重要意义。

The establishment and validation of genetic reporter systems in mouse primordial germ cells

Objective: To establish a genetic reporter system in mouse primordial germ cells（PGCs）. Methods:Using CRISPR（Clustered Regularly Interspaced Short Palindromic Repeats） gene editing technology and homologous recombination，a pair of sgRNAs were designed near the ATG，the translation initiation sites of the mouse Prdm1 and Dppa3 genes，respectively. At the same time，the corresponding Donor plasmids for homologous recombination were constructed according to the sgRNA cleavage site and the inserted target fragment. The sgRNA and Donor fragment were simultaneously transferred into the mouse embryonic stem cells（ESCs），and the target monoclonal cells are screened for genotype identification by corresponding resistance. Results:Genotype identification was performed by extracting the genomic DNA of the monoclonal cells. The results of PCR and Sanger sequencing showed that eGFP and mCherry fluorescent tags were successfully inserted into the genome of the monoclonal cells after the translation initiation sites of mouse Prdm1 and Dppa3 genes，respectively. Cytokines were used to induce directional differentiation of eGFP-Prdm1/m Cherry-Dppa3 mESCs（mouse ESCs） into mPGCs（mouse PGCs）. Flow cytometry showed that the differentiated mPGCs specifically expressed eGFP and mCherry fluorescent signals. Conclusion:mPGCs reporter gene system is successfully constructed，which can be used to study the molecular mechanism of the mPGCs fate determination，and also has positive significance for the study of early embryonic development in mice.