Development of an Efficient Genotyping Method to Detect Obese Mutation in the Mouse Leptin Gene for Use in SPF Barrier Facilities

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  • AYABE Hiroaki
    Production Department, Plasma Team, CHARLES RIVER LABORATORIES JAPAN, Inc., 795 Shimofurusawa, Atsugi-shi, Kanagawa 243–0214, Japan
  • IKEDA Shogo
    Department of Biochemistry, Faculty of Science, Okayama University of Science, 1–1 Ridaicho, Kita-ku, Okayama-shi, Okayama 700–0005, Japan
  • MARUYAMA Shigeri
    Monitoring Center, CHARLES RIVER LABORATORIES JAPAN, Inc., 4049–3 Nakatsu, Aikawa-machi, Aikou-gun, Kanagawa 243–0303, Japan
  • SHIOYAMA Seiji
    Tsukuba Breeding Center, CHARLES RIVER LABORATORIES JAPAN, Inc., 955 Kamibayashi, Ishioka-shi, Ibaraki 315–0138, Japan
  • KIKUCHI Mayumi
    Monitoring Center, CHARLES RIVER LABORATORIES JAPAN, Inc., 4049–3 Nakatsu, Aikawa-machi, Aikou-gun, Kanagawa 243–0303, Japan
  • KAWAGUCHI Ayako
    Monitoring Center, CHARLES RIVER LABORATORIES JAPAN, Inc., 4049–3 Nakatsu, Aikawa-machi, Aikou-gun, Kanagawa 243–0303, Japan
  • YAMADA Tetsuo
    Hino Breeding Center, CHARLES RIVER LABORATORIES JAPAN, Inc., 735 Shimokomaduki, Hino-cho, Gamou-gun, Shiga 529–1633, Japan
  • IKEDA Takuya
    Production Department, CHARLES RIVER LABORATORIES JAPAN, Inc., 3–17–6 Shin-Yokohama, Kohoku-ku, Yokohama-shi, Kanagawa 222–0033, Japan

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We have developed a rapid and efficient genotyping method for detection of the mouse leptin obese mutation (Lepob) using tetra-primer amplification refractory mutation system-polymerase chain reaction (tetra-primer ARMS-PCR). In this method, whole blood collected onto gamma-ray sterilized Flinders Technology Associates (FTA) filter paper is used as PCR template without a DNA purification step. Three genotypes (Lepob/Lepob, Lepob/+ and +/+) differentiated by single-tube PCR and electrophoresis were perfectly consistent with those determined by PCR-restriction fragment length polymorphism (PCR-RFLP). This method can save material costs and operation time, because it does not require restriction enzyme digestion and could be set up in most specific pathogen-free (SPF) barrier facilities.

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