Department of Surgery, Davis Heart and Lung Research Institute, the Ohio State University Wexner Medical Center, Columbus, OH 43210, USA.
Children's Hospital of Chongqing Medical University, Chongqing 400000, China.
Acta Pharmacol Sin. 2018 Jan;39(1):117-123. doi: 10.1038/aps.2017.52. Epub 2017 Jul 27.
db/db mice is one of most widely used animal models in studying the cellular and molecular mechanisms of metabolic disorders, such as diabetes, hyperlipidemia, and obesity. The mice carry spontaneous point mutations in the gene encoding the leptin receptor, leading to leptin receptor inactivation. Since homozygous db/db mice are sterile, the maintenance of db/db mice requires breeding between heterozygous pairs, which makes genotyping essential for the identification of offspring. The aim of this study was to develop a quick and highly repeatable method for genotyping db/db mice, which comprised only three simple steps: genomic DNA is extracted from either tail tips or ear notches via alkaline lysis (∼20 min); samples are then subjected to tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) using specially designed and validated primer sets (∼1.5 h); finally, genotypes are be determined by resolving PCR products on regular DNA electrophoresis (∼10 min). The entire db/db mice genotyping procedure can be performed using regular Taq polymerase and PCR amplification within 2 h. Other advantages of this method include high sensitivity and reproducibility. Minimal amounts of tissue from mice are required, and genomic DNA samples can be stably stored at room temperature for up to one month. In conclusion, the method is simple, cost effective, sensitive and reliable, which will greatly facilitate studies using db/db mice.
db/db 小鼠是研究代谢紊乱（如糖尿病、高血脂和肥胖）的细胞和分子机制的最广泛使用的动物模型之一。这些小鼠携带编码瘦素受体的基因的自发点突变，导致瘦素受体失活。由于纯合子 db/db 小鼠是不育的，因此需要在杂合子对之间进行繁殖来维持 db/db 小鼠，这使得基因分型对于鉴定后代至关重要。本研究旨在开发一种快速且高度可重复的 db/db 小鼠基因分型方法，该方法仅包含三个简单步骤：通过碱性裂解从尾尖或耳缺提取基因组 DNA（约 20 分钟）；然后使用专门设计和验证的引物组对样品进行四引物扩增受阻突变系统-聚合酶链反应（ARMS-PCR）（约 1.5 小时）；最后，通过在常规 DNA 电泳上解析 PCR 产物来确定基因型（约 10 分钟）。整个 db/db 小鼠基因分型过程可以在 2 小时内使用常规 Taq 聚合酶和 PCR 扩增来完成。该方法的其他优点包括高灵敏度和可重复性。只需要从小鼠身上取少量组织，基因组 DNA 样品可在室温下稳定保存长达一个月。总之，该方法简单、经济有效、灵敏可靠，将极大地促进 db/db 小鼠的研究。
