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

鸡原始生殖细胞导入外源DNA方法的研究

• 批准号: 16380193
• 负责人: NAITO Mitsuru
• 金额: $ 9.54万
• 依托单位: National Institute of Agrobiological Sciences
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2004
• 资助国家: 日本
• 起止时间: 2004 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-16380193/
• 关键词: primordial germ cell; exogenous gene; transfection; GFP gene; chick embryo; germline chemera; breed discrimination; in vitro culture

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.

本研究旨在建立将外源DNA导入鸡原始生殖细胞并获得转基因鸡的方法。首先开发并进一步改进了从胚胎血细胞中分离原始生殖细胞的方法。然后，在体外和体内尝试通过脂质体转染和核转染来转染分离的原始生殖细胞。结果，GFP基因在体外和体内通过脂质体转染被有效地导入到原始生殖细胞中，并且所导入的GFP基因在发育胚胎的性腺中强烈表达。核转染法也是将GFP基因导入原始生殖细胞的有效方法。另一方面，从胚胎血液中分离的原始生殖细胞的命运进行了分析后，转移到阶段X胚盘受体胚胎使用线粒体D-环区域中存在的单核苷酸多态性。结果表明，移植的原始生殖细胞成功地迁移到受体胚胎的生殖嵴。该方法可用于利用精子样品分析假定的雌雄嵌合体鸡的生殖系嵌合性。此外，在体外培养的原始生殖细胞进行。部分原始生殖细胞在饲养层细胞上成功地维持和增殖。需要改善培养条件，以保持原始生殖细胞未分化以备将来使用。

项目成果

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

从胚胎血液中分离的原始生殖细胞转移至X期胚盘后迁移至性腺并通过PCR检测生殖嵌合

• 发表时间: 2004
• 期刊: British Poultry Science 45・6
• 作者: Naito;M.;Sano;A.;Harumi;T.;Matsubara;Y.;Kuwana;T.
• 通讯作者: T.

Polymerase chain reaction detection of single nucleotide polymorphisms in the chicken mitochondrial D-loop region.

聚合酶链式反应检测鸡线粒体D环区单核苷酸多态性。

• 发表时间: 2004
• 期刊: Animal Science Journal 75・6
• 作者: Harumi;T.;Sano;A.;Kagami;H.;Tagami;T.;Matsubara;Y.;Naito;M.
• 通讯作者: M.

Differentiation of female primordial germ cells in the male testes of chicken (Gallus gallus domesticus)

• DOI: 10.1002/mrd.20499
• 发表时间: 2007-01-01
• 期刊: MOLECULAR REPRODUCTION AND DEVELOPMENT
• 影响因子: 2.5
• 作者: Tagami, Takahiro;Kagami, Hiroshi;Nirasawa, Keijiro
• 通讯作者: Nirasawa, Keijiro

Differentiation of female primordial germ cells in the male tastes of chicken (Gallus gallus domesticus)

鸡（鸡内金）雄性味道中雌性原始生殖细胞的分化

• 发表时间: 2006
• 期刊: Molecular Reproduction and Development 74・1
• 作者: Tagami T;Kagami H;Matsubara Y;Harumi T;Naito M;Takeda K;Hanada H;Nirasawa K
• 通讯作者: Nirasawa K

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

新畜牧手册2.14.4家禽生物技术

• 发表时间: 2006
• 作者: Naito M;Minematsu T;Harumi T;Kuwana T;Naraolka H.;T.Unno et al.;Iwamori K;内藤 充;S.Komori et al.;Sugiura K;H.Okamoto et al.;内藤 充
• 通讯作者: 内藤 充