Abstract
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.
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Acknowledgements
This study was supported by grants from the National Institutes of Health (R01 #HL124122 and #AR067766), the American Heart Association (No 12SDG12070174) and the National Natural Science Foundation of China (No 81401155).
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Supplementary information is available on the website of Acta Pharmacologica Sinica.
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Supplementary Figure S1
The screen shot of the online software showing the design and parameters of ARMS-PCR protocol. (DOC 214 kb)
Supplementary Figure S2
Gradient PCR was used to test Tm temperature in ARMS-PCR reaction. (DOC 138 kb)
Supplementary Figure S3
To determine the 406 bp and 264 bp bands are corresponding to db/db allele and wild type allele, respectively. (DOC 105 kb)
Supplementary Figure S4
The ratio between outer primer and inner primer was adjusted from 1:4 to 4:1. (DOC 157 kb)
Supplementary Figure S5
Due to the fact that the common band (610 bp band) could potentially be weak. (DOC 123 kb)
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Peng, By., Wang, Q., Luo, Yh. et al. A novel and quick PCR-based method to genotype mice with a leptin receptor mutation (db/db mice). Acta Pharmacol Sin 39, 117–123 (2018). https://doi.org/10.1038/aps.2017.52
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DOI: https://doi.org/10.1038/aps.2017.52
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