利用hrm技术区分ob糖尿病小鼠不同基因型-中国细胞生物学学报.pdfVIP

中国细胞生物学学报 Chinese Journal of Cell Biology 2014, 36(10): 1403– 1408 DOI: 10.11844/cj cb.2014.10.0149 技术与方法 利用HRM技术区分ob糖尿病小鼠不同基因型 刘 苏 周舒雅 左 琴 王辰飞 贺争鸣 李保文 刘佐民 范昌发* ( 中国食品药品检定研究院, 北京100059) 摘要 ob/ob 小鼠是糖尿病相关研究中使用最广泛的动物模型之一, 近年来, 市场需求呈上 升趋势。与野生型小鼠相比, 其瘦素蛋白基因105号密码子发生CT点突变, 导致不能产生正常 的瘦素蛋白。该模型4周前外观表型与野生型无异, 而ob/ob 纯合子不育, 因而在繁殖建系过程中, 需要通过基因型鉴定纯合、杂合和野生三种基因型。该文建立了基于高分辨率溶解曲线分析(high resolution melting analysis, HRM) 的基因型鉴定方法, 基因型分型结果与常规PCR加酶切方法一 致, 也与DNA测序结果吻合; 通过该方法分型后获得纯合子小鼠外观表型、血糖浓度参数符合 预期。该方法较常规方法省时、高效、节省实验成本, 可用于大规模ob 小鼠繁殖中的基因型鉴定。 关键词 ob小鼠; 基因型鉴定; HRM Efficient Genotyping ob Mice Using High Resolution Melting Analysis Liu Susu, Zhou Shuya, Zuo Qin, Wang Chenfei, He Zhengming, Li Baowen, Liu Zuomin, Fan Changfa* (National Insitute for Food and Drug Control, Beijing 100059, China) Abstract The ob/ob mouse is a well-characterized model of obesity in diabetes research, and market demands are increasing in recent years. For the CT transition occurred in ob mouse, normal leptin protein cannot be produced. The ob homozygous mouse has similar exophenotype with wild type before 4 weeks old, and ob/ob mouse issterile and breeding of complete knockouts is impractical. Thus, fertile heterozygotes must be identified by genotyping methods to maintain efficient breeding colonies for the production of wild type, heterozygous and homozygous test animals. Here we described a new genotyping method based on high resolution melting analysis (HRM). The identification results by HRM are consistent with that of conventional PCR products gel-based method and DNA sequencing. The ob mice have expected exophenotype and blood glucose parameter when observed at 8 weeks old. This method is rapid, efficient and economical compared with other genotyping techniques. Key words ob mice; g