Possible Mechanisms for the Testis-Mediated Gene Transfer as a New Method for Producing Transgenic Animals.

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  • Chang Kyu-Tae
    Department of Veterinary Physiology, Veterinary Medical Science, The University of Tokyo, Tokyo 113-8657, Japan
  • Ikeda Akihiro
    Department of Veterinary Physiology, Veterinary Medical Science, The University of Tokyo, Tokyo 113-8657, Japan
  • Hayashi Katsuhiko
    Department of Animal Production, Ikuta Campus, Meiji University, Kanagawa 214-0033, Japan
  • Furuhata Yasufumi
    Department of Veterinary Physiology, Veterinary Medical Science, The University of Tokyo, Tokyo 113-8657, Japan
  • Bannai Makoto
    Department of Veterinary Physiology, Veterinary Medical Science, The University of Tokyo, Tokyo 113-8657, Japan
  • Nishihara Masugi
    Department of Veterinary Physiology, Veterinary Medical Science, The University of Tokyo, Tokyo 113-8657, Japan
  • Ohta Akihiko
    Department of Animal Production, Ikuta Campus, Meiji University, Kanagawa 214-0033, Japan
  • Ogawai Shyoso
    Department of Animal Production, Ikuta Campus, Meiji University, Kanagawa 214-0033, Japan
  • Takahashi Michio
    Department of Veterinary Physiology, Veterinary Medical Science, The University of Tokyo, Tokyo 113-8657, Japan

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Abstract

We have recently shown that DNAs injected into the testis of either rats or mice can be transferred into eggs via sperm at fertilization and expressed and transmitted in the descendants (testis-mediated gene transfer). In the present study, we investigated the association of epididymal and ejaculated sperm with DNA injected into the testis of rats. In the first experiment, a chimeric gene of mouse metallothionein-I promoter (MT)/human growth hormone receptor (hGHR) labeled with FITC was prepared. This gene construct was mixed with cationic liposome and injected into rat testis, and 4 days later the cauda epididymis was dissected out and prepared for histological examination. A confocal microscopic observation showed that not only the head, but also the tail, of almost all spermatozoa in the epididymis was labeled with FITC. In the next experiment, 4 days after the injection of MT/hGHR gene encapsulated by liposome into the testis, the male was allowed to mate with females and spermatozoa were recovered from the uterus in the next morning. Spermatozoa were incubated in the presence or absence of deoxyribonuclease (DNase) I and then genomic DNA was extracted. The MT/hGHR gene was detected only in the spermatozoa incubated without DNase by means of PCR. These results support the notion that, in the case of our testis-mediated gene transfer, exogenous DNA injected with liposomes is not integrated into genome of the sperm and the integration occurs after fertilization.

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