A Rapid PCR-based Method for the Identification of ob Mutant Mice
A Rapid PCR-based Method for the Identification of ob Mutant Mice
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DOI：
10.1038/oby.2008.443
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发表时间：
2009-02-01
期刊：
影响因子：
6.9
通讯作者：
Spyropoulos, Demetri D.
中科院分区：
文献类型：
--
作者：
Ellett, Justin D.;Evans, Zachary P.;Spyropoulos, Demetri D.
随着肥胖症发病率的增加，对合适的研究和治疗模型的需求越来越大。ob/ob小鼠模型在5周龄时发生肥胖。以前，设计了一种使用DNA纯化、PCR和产物限制性消化的方法来鉴定携带ob等位基因的小鼠。在这里，我们描述了一种不需要DNA纯化的直接PCR方法。野生型和ob特异性引物在相同条件下用于两个单独且同时运行的三引物PCR。使用野生型引物混合物的标准PCR在+/+和ob/+中产生191 bp和104 bp条带，而在ob/ob动物中仅产生对照191 bp条带。ob特异性引物反应在ob/+和ob/ob中产生191 bp和123 bp条带，在+/+动物中仅产生对照191 bp条带。杂合杂交后代的表型增重用于验证基因型。这种引物特异性PCR方法允许在育种年龄之前同时鉴定+/+、ob/+和ob/ob基因型，以促进临床肥胖症的重要模型中的育种和研究。
With increasing incidence of obesity, there is greater demand for suitable research and therapeutic models. The ob/ob mouse model develops obesity by 5 weeks of age. Previously, a method using DNA purification, PCR, and restriction digestion of products was devised to identify mice bearing the ob allele. Here, we describe a direct PCR method that requires no DNA purification. Wild-type and ob-specific primers are used under the same conditions in two separate and simultaneously run three-primer PCRs. Standard PCR using the wild-type primer mix produces 191 bp and 104 bp bands in +/+ and ob/+ and only the control 191 bp band in ob/ob animals. The ob-specific primer reaction produces 191 bp and 123 bp bands in ob/+ and ob/ob and only the control 191 bp band in +/+ animals. Phenotypic weight gain in offspring of heterozygous intercrosses was used to validate genotypes. This primer-specific PCR method allows simultaneous identification of +/+, ob/+, and ob/ob genotypes prior to breeding age to facilitate breeding and research studies in an important model of clinical obesity.