Lab Anim Res.  2016 Mar;32(1):70-73. 10.5625/lar.2016.32.1.70.

Rapid and efficient identification of the mouse leptin receptor mutation (C57BL/KsJ-db/db) by tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) analysis

• Jung H
• Nam H
• Suh JG

• KMID: 2312156
• DOI: http://doi.org/10.5625/lar.2016.32.1.70

Abstract

The C57BLKS/J-Lepr(db) mouse has a point mutation in the leptin receptor gene and is one of the most useful animal model for non-insulin dependent diabetes mellitus in human. Since the homozygote of C57BLKS/J-Lepr(db) mouse is infertile, detection of point mutation in the leptin receptor gene is important for efficient maintaining strains as well as mass production of homozygotes. To develop a rapid and efficient genotyping method for C57BLKS/J-Lepr(db) mouse, the tetra-primer amplification-refractory mutation system polymerase chain reaction (ARMS-PCR) was used. The 407 and 199 bp PCR products were amplified from normal (+/+) mice; while the 407 and 268 bp PCR products were amplified from homozygotes (db/db) mice; and the 407, 268, and 199 bp PCR products were amplified from heterozygotes (db/+) mice. This result showed that the tetra-primer ARMS-PCR analysis by us is suitable to detect point mutation of the leptin receptor gene. Taken together, our method will dramatically reduce animal use for maintenance of strains as well as production of homozygote in the C57BLKS/J-Lepr(db) strains.

Keyword

Leptin receptor; genotyping; db mouse

MeSH Terms

Animals
Diabetes Mellitus
Heterozygote
Homozygote
Humans
Leptin*
Mice*
Models, Animal
Point Mutation
Polymerase Chain Reaction
Receptors, Leptin*
Leptin
Receptors, Leptin

Figure

• 

  Figure 1 Sequences of the leptin receptor mutation and normal allele (NCBI, Gene ID: 16847). The primers for ARMS-PCR were designed using the primer design web service for tetra-primer ARMS-PCR. Underline indicates transverse point mutation (G→T) in intron 18 of the leptin receptor.

• 

  Figure 2 Detection of leptin receptor mutation by the tetra-primer ARMS-PCR analysis. A. Design of PCR products. Leptin receptor mutation (268 bp, Lepr-FO and Lepr-RI), wild type (199 bp, Lepr-FI and Lepr-RO), internal control from both alleles (407 bp, Lepr-FO and Lepr-RO). B. Genotyping of leptin receptor mutant mice by the tetra-primer ARMS-PCR. C. Genotyping of double knock-out mice by the tetra-primer ARMS-PCR. M: 100 bp ladder.

Reference

1. Chen H, Charlat O, Tartaglia LA, Woolf EA, Weng X, Ellis SJ, Lakey ND, Culpepper J, Moore KJ, Breitbart RE, Duyk GM, Tepper RI, Morgenstern JP. Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor gene in db/db mice. Cell. 1996; 84(3):491–495. PMID: 8608603.

2. Coleman DL, Hummel KP. Symposium IV: Diabetic syndrome in animals. Influence of genetic background on the expression of mutations at the diabetes locus in the mouse. II. Studies on background modifiers. Isr J Med Sci. 1975; 11(7):708–713. PMID: 1099054.

3. Horvat S, Bünger L. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay for the mouse leptin receptor (Lepr(db)) mutation. Lab Anim. 1999; 33(4):380–384. PMID: 10778787.

4. Newton CR, Graham A, Heptinstall LE, Powell SJ, Summers C, Kalsheker N, Smith JC, Markham AF. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 1989; 17(7):2503–2516. PMID: 2785681.

5. Ye S, Dhillon S, Ke X, Collins AR, Day IN. An efficient procedure for genotyping single nucleotide polymorphisms. Nucleic Acids Res. 2001; 29(17):E88. PMID: 11522844.

6. Oh SH, Nam H, Suh JG. A high resolution genetic mapping of the faded (fe) gene to a region between D10mit156 and D10mit193 on mouse chromosome 10. Lab Anim Res. 2013; 29(1):33–38. PMID: 23573106.

7. Collins A, Ke X. Primer1: primer design web service for tetraprimer ARMS-PCR. Open Bioinforma J. 2012; 6:55–58.