Study on the methods introducing exogenous DNA into chicken primordial germ cells

• Project/Area Number: 16380193
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Applied animal science
• Research Institution: National Institute of Agrobiological Sciences
• Principal Investigator: NAITO Mitsuru National Institute of Agrobiological Sciences, Department of Developmental Biology, Laboratory Head, 遺伝子組換え家畜研究センター, 上級研究員 (70355733)
• Project Period (FY): 2004 – 2006
• Project Status: Completed (Fiscal Year 2006)
• Budget Amount: ¥14,900,000 (Direct Cost: ¥14,900,000); Fiscal Year 2006: ¥4,100,000 (Direct Cost: ¥4,100,000); Fiscal Year 2005: ¥5,200,000 (Direct Cost: ¥5,200,000); Fiscal Year 2004: ¥5,600,000 (Direct Cost: ¥5,600,000)
• Keywords: primordial germ cell / exogenous gene / transfection / GFP gene / chick embryo / germline chemera / breed discrimination / in vitro culture

Research Abstract

The present study was carried out to develop methods to introduce exogenous DNA into chicken primordial germ cells and produce transgenic chickens. A method to isolate primordial germ cells from embryonic blood cells was first developed and further improved. Then, transfection of isolated primordial germ cells was attempted by lipofection and nucleofection in vitro and in vivo. As a result, GFP gene was efficiently introduced into the primordial germ cells by lipofection in vitro and in vivo and the introduced GFP gene was expressed strongly in the gonads of developing embryos. Nucleofection method was also effective introducing GFP gene into primordial germ cells. On the other hand, the fate of primordial germ cells isolated from embryonic blood was analyzed after transfer into stage X blastoderm of recipient embryos using single nucleotide polymorphism present in the mitochondrial D-loop region. It was confirmed that the transferred primordial germ cells were successfully migrated to the germinal ridges of recipient embryos. This method can be applied to analyze the germline chimerism of putative male and female chimeric chickens using sperm samples. Furthermore, in vitro culture of primordial germ cells was carried out. A part of the primordial germ cell population was successfully maintained and proliferated on the feeder cells. It is required to improve culture conditions so as to maintain primordial germ cells undifferentiated for the future.

Research Products

• [Journal Article] Effect of soft X-ray irradiation on the migratory ability of primordial gem cells in chickens (2007)
  • Author(s): Naito M, Harumi T, Minematsu T, Tajima A, Kuwana T
  • Journal Title: British Poultry Science 48・2 Pages: 121-126
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Effect of soft X-ray irradiation on the migratory ability of primordial gem cells in chickens (2007)
  • Author(s): Naito M, Harumi T, Minematsu, Tajima A, Kuwana T
  • Journal Title: British Poultry Science 48-2 Pages: 121-126
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Conservation of a threatened indigenous fowl (Kureko Dori) using the germline chimeras transplanted from primordial germ cells. (2006)
  • Author(s): Kuwana, T., Kawashima, T., Naito, M., Yamashita, H., Matsuzaki, M., Takano, T.
  • Journal Title: Journal of Poultry Science 43・1 Pages: 60-66
  • NAID: 10017158581
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Effects of soft X-ray irradiation to stage X blastoderm on restriction of proliferation of primordial germ cells in early chicken embryos (2006)
  • Author(s): Nakamichi H, Sano A, Harumi T, Matsubara Y, Tajima A, Kosugiyama M, Naito M
  • Journal Title: Journal of Poultry Science 43・4 Pages: 394-400
  • NAID: 10018324843
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Differentiation of female primordial germ cells in the male testes of chicken (Gallus gallus domesticus) (2006)
  • Author(s): Tagami T, Kagami H, Matsubara Y, Harumi T, Naito M, Takeda K, Hanada H, Nirasawa K
  • Journal Title: Molecular Reproduction and Development 74・1 Pages: 68-75
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Evidence of in vivo transfection of circulating primordial germ cells in chickens (2006)
  • Author(s): Naito M, Harumi T, Matsubara Y, Kuwana T
  • Journal Title: Proceedings of the 12th European Poultry Conference (CD) Pages: 1-6
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Conservation of a threatened indigenous fowl (Kureko Dori) using the germline chimeras transplanted from primordial germ cells. (2006)
  • Author(s): Kuwana, T., Kawashima, T., Naito, M., Yamashita, H., Matsuzaki, M., Takano, T.
  • Journal Title: Journal of Poultry Science 43-1 Pages: 60-66
  • NAID: 10017158581
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Effects of soft X-ray irradiation to stage X blastoderm on restriction of proliferation of primordial germ cells in early chicken embryos (2006)
  • Author(s): Nakamichi H, Sano A, Harumi T, Matsubara Y, Tajima A, Kosugiyama M, Naito M
  • Journal Title: Journal of Poultry Science 43-4 Pages: 394-400
  • NAID: 10018324843
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Differentiation of female primordial germ cells in the male testes of chicken (Gallus gallus domesticus) (2006)
  • Author(s): Tragami T, Kagami H, Matsubara Y, Harumi T, Naito M, Takeda K, Hanada H, Nirasawa K
  • Journal Title: Molecular Reproduction and Development 74-1 Pages: 68-75
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Effects of soft X-ray irradiation to stage X blastoderm on restriction of proliferation of priomordial germ cells in early chicken embryos (2006)
  • Author(s): Nakamichi H, Sano A, Harumi T, Matsubara Y, Tajima A, Kosugiyama M, Naito M
  • Journal Title: Journal of Poultry Science 43・4 Pages: 394-400
• [Journal Article] Differentiation of female primordial germ cells in the male tastes of chicken (Gallus gallus domesticus) (2006)
  • Author(s): Tagami T, Kagami H, Matsubara Y, Harumi T, Naito M, Takeda K, Hanada H, Nirasawa K
  • Journal Title: Molecular Reproduction and Development 74・1 Pages: 72-80
• [Journal Article] Evidence of in vivo transfection of circulating primordial germ cells in chickens (2006)
  • Author(s): Naito M, Harumi T, Matsubara Y, Kuwana T
  • Journal Title: Proceedings of the 12th European Poultry Conference CD Pages: 1-6
• [Journal Article] Conservation of a threatened indigenous fowl(Kureko Dori) using the germline chimeras transplanted from primordial germ cells. (2006)
  • Author(s): Kuwana, T., Kawashima, T., Naito, M., Yamashita, H., Matsuzaki, M., Takano, T.
  • Journal Title: Journal of Poultry Science 43・1 Pages: 60-66
  • NAID: 10017158581
• [Journal Article] Culture of chicken embryos obtained from the anterior region of the magnum of the oviduct after removing a thin layer of dense albumen capsule from the ovum (2005)
  • Author(s): Naito, M., Sano, A., Kawashima, T., Nakamichi, H., Harumi, T., Matsubara, Y., Kuwana, T.
  • Journal Title: Journal of Poultry Science 42・2 Pages: 140-144
  • NAID: 10015494744
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] New screening test for male germline chimeric chickens by polymerase chain reaction using single nucleotide polymorphism detection primers. (2005)
  • Author(s): Sano, A., Harumi, T., Hanzawa, S., Kawashima, T., Nakamichi, H., Matsubara, Y., Naito, M.
  • Journal Title: Journal of Poultry Science 42・2 Pages: 152-157
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] An attempt at fusing primordial germ cell with blood cell in chickens using inactivated Sendai virus or electrical stimulation. (2005)
  • Author(s): Naito, M., Harumi, T., Matsubara, Y., Kuwana, T.
  • Journal Title: Journal of Poultry Science 42・4 Pages: 369-374
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Culture of chicken embryos obtained from the anterior region of the magnum of the oviduct after removing a thin layer of dense albumen capsule from the ovum (2005)
  • Author(s): Naito, M., Sano, A., Kawashima, T., Nakamichi, H., Harumi, T., Matsubara Y., Kuwana, T.
  • Journal Title: Journal of Poultry Science 42-2 Pages: 140-144
  • NAID: 10015494744
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] New screening test for male germline chimeric chickens by polymerase chain reaction using single nucleotide polymorphism detection primers. (2005)
  • Author(s): Sano, A., Harumi, T., Hanzawa, S., Kawashima, T., Nakamichi, H., Matsubara, Y., Naito, M.
  • Journal Title: Journal of Poultry Science 42-2 Pages: 152-157
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] An attempt at fusing primordial germ cell with blood cell in chickens using inactivated Sendai virus or electrical stimulation. (2005)
  • Author(s): Naito, M., Harumi, T., Matsubara, Y., Kuwana, T.
  • Journal Title: Journal of Poultry Science 42-4 Pages: 369-374
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Culture of chicken embryos obtained from the anterior region of the magnum of the oviduct after removing a thin layer of dense albumen capsule from the ovum. (2005)
  • Author(s): Naito, M., Sano, A., Kawashima, T., Nakamichi, H., Harumi, T., Matsubara, Y., Kuwana, T.
  • Journal Title: Journal of Poultry Science 42・1 Pages: 140-144
  • NAID: 10015494744
• [Journal Article] New screening test for male germline chimeric chickens by polymerase chain reaction using single nucleotide polymorphism detection primers. (2005)
  • Author(s): Sano, A., Harumi, T., Hanzawa, S., Kawashima, T., Nakamichi, H., Matsubara, Y., Naito, M.
  • Journal Title: Journal of Poultry Science 42・1 Pages: 152-157
• [Journal Article] Culture of chicken embryos obtained from the anterior region of the magnum of the oviduct after removing a thin layer of dense albumen capsule from he ovum (2005)
  • Author(s): Naito, M., Sano, A., Kawashima, T., Nakamichi, H., Harumi, T., Matsubara, Y., Kuwana, T.
  • Journal Title: Journal of Poultry Science 42・2(印刷中)
• [Journal Article] New screening test for male germline chimeric chickens by polymerase chain reaction using single nucleotide polymorphism detection primers (2005)
  • Author(s): Sano, A., Harumi, T., Hanzawa, S., Kawashima, T., Nakamichi, H., Matsubara, Y., Naito, M.
  • Journal Title: Journal of Poultry Science 42・2(印刷中)
• [Journal Article] Production of chimeric chickens (2004)
  • Author(s): Naito, M., Kuwana, T.
  • Journal Title: Methods in Molecular Biology 254・2 Pages: 245-253
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Migration of primordial germ cells isolated from embryonic blood into the gonads after transfer to stage X blastoderms and detection of germline chimaerism by PCR (2004)
  • Author(s): Naito, M., Sano, A., Harumi, T., Matsubara, Y., Kuwana, T.
  • Journal Title: British Poultry Science 45・6 Pages: 762-768
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Polymerase chain reaction detection of single nucleotide polymorphisms in the chicken mitochondrial D-loop region. (2004)
  • Author(s): Harumi, T., Sano, A., Kagami, H., Tagami, T., Matsubara, Y., Naito, M.
  • Journal Title: Animal Science Journal 75・6 Pages: 503-507
  • NAID: 10018625050
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Migration of primordial germ cells isolated from embryonic blood into the gonads after transfer to stage X blastoderms and detection of germline chimaerism by PCR (2004)
  • Author(s): Naito, M., Sano, A., Harumi, T., Matubara, Y., Kuwana, T.
  • Journal Title: British Poultry Science 45-6 Pages: 762-768
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Polymerase chain reaction detection of single nucleotide polymorphisms in the chicken mitochondrial D-loop region. (2004)
  • Author(s): Harumi, T., Sano, A., Kagami, H., Tagami, T., Matsubara, Y., Naito, M.
  • Journal Title: Animal Science Journal 75-6 Pages: 503-507
  • NAID: 10018625050
  • Description: 「研究成果報告書概要(欧文)」より
• [Journal Article] Quantitative genotyping by amplifying the polymorphic sequences of Pre-Melanosomal Protein (PMEL17) gene using real-time polymerase chain reaction in chickens
  • Author(s): Minematsu T, Harumi T, Naito M
  • Journal Title: (submitted)
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Intense expression of GFP gene in gonads of chicken embryos by transfecting circulating primordial germ cells in vitro and in vivo
  • Author(s): Naito M, Minematsu T, Harumi T, Kuwana T
  • Journal Title: (submitted)
  • NAID: 10019768378
  • Description: 「研究成果報告書概要(和文)」より
• [Journal Article] Spermatogonia or primary oocytes from 20-day incubated embryos contribute to germline lineage after transfer into bloodstream of recipient embryos
  • Author(s): Naito M, Minematsu T, Harumi T, Kuwana T
  • Journal Title: (submitted)
  • Description: 「研究成果報告書概要(和文)」より
• [Book] 新編畜産ハンドブック 2.4.4 ニワトリの育種 (2006)
  • Author(s): 内藤 充
  • Publisher: 講談社
  • Description: 「研究成果報告書概要(和文)」より
• [Book] 新編畜産ハンドブック 2.14.4 家禽のバイオテクノロジー (2006)
  • Author(s): 内藤 充
  • Publisher: 講談社
  • Description: 「研究成果報告書概要(和文)」より
• [Book] Handbook of Animal Husbandry 2.4.4 Poultry breeding (2006)
  • Author(s): Naito M
  • Publisher: Kodansha
  • Description: 「研究成果報告書概要(欧文)」より
• [Book] Handbook of animal Husbandry 2.14.4 Poultry biotechnology (2006)
  • Author(s): Naito M
  • Publisher: Kodansha
  • Description: 「研究成果報告書概要(欧文)」より