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The creation of transgenic pigs expressing human proteins using BAC-derived, full-length genes and intracytoplasmic sperm injection-mediated gene transfer

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Abstract

In most transgenic (Tg) animals created to date, a transgene consisting of the minimum promoter region linked to a cDNA has been used. However, transgenes on small plasmids are susceptible to the position effect, increasing the difficulty of controlling transgene expression. In this study, we attempted to create Tg pigs by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) using two large genomic transgenes derived from a bacterial artificial chromosome (BAC) containing the full genomic region encoding two human proteins, type I collagen and albumin. The production efficiencies (Tg piglets/live offspring) of type I collagen and albumin Tg pigs were 11.8% (6/51) and 18.2% (2/11), respectively. In all of the Tg pigs examined by real-time PCR analysis, tissue-specific expression of the transgene was confirmed (type I collagen: skin, tendon, vessels, genitalia; albumin: liver). The production of human proteins derived from BAC transgenes was also confirmed. Fluorescence in situ hybridization analysis indicated that the BAC transgenes transferred into porcine oocytes by ICSI-MGT were integrated into single or multiple sites on the host chromosomes. These data demonstrate that Tg pigs expressing human proteins in a tissue-specific manner can be created using a BAC transgenic construct and the ICSI-MGT method.

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Acknowledgments

This work was supported by Japan Science and Technology Agency (JST), ERATO, Nakauchi Stem Cell and Organ Regeneration Project, Tokyo, Meiji University International Cluster for Bio-Resource Research (MICBR) and the Program for Promotion of Basic Research Activities for Innovative Bioscience (PROBRAIN), Tokyo.

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Authors and Affiliations

  1. Nakauchi Stem Cell and Organ Regeneration Project, Japan Science and Technology Agency (JST), ERATO, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan

    Masahito Watanabe, Hiromitsu Nakauchi & Hiroshi Nagashima

  2. Department of Life Sciences, School of Agriculture, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa, 214-8571, Japan

    Masahito Watanabe, Hitomi Matsunari, Kazuaki Nakano, Kazuhiro Umeyema & Hiroshi Nagashima

  3. Institute of Molecular Animal Breeding and Biotechnology, Ludwig-Maximilians-University Munich, Moorversuchsgut, Hackerstr. 27, 85764, Oberschleissheim, Germany

    Mayuko Kurome

  4. International Cluster for Bio-Resource Research, Meiji University, 1-1-1 Higashimita, Tama-ku, Kawasaki, Kanagawa, 214-8571, Japan

    Kazuhiro Umeyema & Hiroshi Nagashima

  5. PhoenixBio Co., 3-4-1 Kagamiyama, Higashi-hiroshima, Hiroshima, 739-0046, Japan

    Akira Shiota

  6. Center for Stem Cell Biology and Regenerative Medicine, Institute of Medical Science, Tokyo University, 4-6-1 Shirokanedai, Minato-ku, Tokyo, 108-8639, Japan

    Hiromitsu Nakauchi

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  1. Masahito Watanabe
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  2. Mayuko Kurome
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  3. Hitomi Matsunari
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  4. Kazuaki Nakano
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  5. Kazuhiro Umeyema
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  6. Akira Shiota
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  7. Hiromitsu Nakauchi
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  8. Hiroshi Nagashima
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Correspondence to Hiroshi Nagashima.

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Watanabe, M., Kurome, M., Matsunari, H. et al. The creation of transgenic pigs expressing human proteins using BAC-derived, full-length genes and intracytoplasmic sperm injection-mediated gene transfer. Transgenic Res 21, 605–618 (2012). https://doi.org/10.1007/s11248-011-9561-3

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  • DOI: https://doi.org/10.1007/s11248-011-9561-3

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