Functional analysis of germ line cells using gene manipulated mouse

使用基因操作小鼠进行生殖系细胞的功能分析

• 批准号: 11234203
• 负责人: OKABE Masaru
• 金额: $ 54.4万
• 依托单位: OSAKA UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research on Priority Areas
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11234203/
• 关键词: gene manipulate animals; transgenic mouse; GFP; sex differentiation; sex chimera; ACE; GPI-anchor; 生殖細胞; 複数分裂; 雌雄キメラ; Cre; 1oxP; loxP; 雌雄選別; キメラ

We have developed an easy and very efficient system to separate male and female embryos before implantation stage with non invasive manner. When male and female eggs prepared by this system were aggregated to produce sex chimera, the advantage of this system is the easiness of pointing out the sex of individual cells that form organs by observing the transgenically incorporated green fluorescent protein gene in X chromosome. By analyzing the genital organs in thus prepared sex chimeras, we have found many spermatogonia like cells of female origin in phenotypically male testes. The pattern of the genomic imprinting of these genes was found out to be male type in spite of their original sex was female.With the collaboration to Prof. Nakatsuji's group, it was also found out that these "female spermatogonia" cells entered meiosis in early stages of development However, on the other hand, some cells originated from female became huge cells like eggs in the testis of sex chimera animals, whi … More le the majority of the female cells developed into male type spermatogonia. The complexity of the sex differentiation of germ cells was elucidated by producing these sex chimera mice and simultaneously provided a tool to analyze the mechanism of male-female differentiation in germ line cells.Pig-a, an X-linked gene, is a key component of glycosylphosphatidylinositol (GPI) anchor biosynthesis based on the fact that lack of this gene causes deficiencies of hundreds of GPI-anchored proteins. Dr. Kondoh has produced a transgenic mouse line that expressed enhanced green fluorescent protein in GPI-anchored form and monitored GPI-anchor-positive cells in situ. It was found out that ACE has phospholipase activity other than the dipeptidyl-peptidase. It was suggested that testicular ACE is indispensable because it may release GPI anchored proteins from sperm surface to make fertile sperm. The results indicated that the necessity of re-evaluation of the involvement of ACE in reproductive biology. Less

我们开发了一种简单且非常有效的系统，可以在植入阶段之前以非侵入性方式分离男性和女性胚胎。当通过该系统制备的雄性和雌性卵子聚集在一起产生性别嵌合体时，该系统的优点是通过观察X染色体中转基因掺入的绿色荧光蛋白基因，可以轻松地指出形成器官的单个细胞的性别。通过分析由此制备的性嵌合体中的生殖器官，我们在表型为男性的睾丸中发现了许多女性起源的精原细胞样细胞。这些基因的基因组印记模式被发现是雄性型，尽管它们的原始性别是雌性。通过与中辻教授课题组的合作，还发现这些“雌性精原细胞”在发育早期就进入了减数分裂。但另一方面，一些源自雌性的细胞在性嵌合动物睾丸中变成了像卵子一样的巨大细胞，而大多数雌性细胞却保留了下来。 发育成雄性型精原细胞。通过生产这些性嵌合体小鼠，阐明了生殖细胞性别分化的复杂性，同时为分析生殖系细胞中雌雄分化的机制提供了工具。Pig-a是一种X连锁基因，它是糖基磷脂酰肌醇（GPI）锚定生物合成的关键组成部分，因为缺乏该基因会导致数百种细胞的缺陷。 GPI 锚定蛋白。 Kondoh 博士培育了一种转基因小鼠系，该小鼠系以 GPI 锚定形式表达增强型绿色荧光蛋白，并可原位监测 GPI 锚定阳性细胞。发现ACE具有二肽基肽酶以外的磷脂酶活性。有人认为，睾丸ACE是不可或缺的，因为它可以从精子表面释放GPI锚定蛋白，使精子具有受精能力。结果表明重新评价ACE参与生殖生物学的必要性。较少的

项目成果

Kimura SH. et al.: "Cyclin G1 is involved in G2/M arrest in response to DNA damage and in growth control after damage recovery"Oncogene. 20. 3290-3300 (2001)

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Koresawa,Y. et al.: "Synthesis of a New Cre Recombinase Gene Based on Optimal Codon Usage for Mammalian Systems"J Biochem (Tokyo). 127(3). 367-72 (2000)

是泽，Y.

Gao, X.H.et al.: "Rapid compensation for glycosylphosphatidylinositol anchor deficient keratinocytes after birth: visualization of glycosylphosphatidylinositol-anchored proteins in situ"J Invest Dermatol. 118. 998-1002 (2002)

高，X.H.等人：“出生后糖基磷脂酰肌醇锚定缺陷角质形成细胞的快速补偿：原位糖基磷脂酰肌醇锚定蛋白的可视化”J Invest Dermatol。

Baba, D. et al.: "Mouse Sperm Lacking Cell Surface Hyaluronidase PH-20 Can Pass through the Layer of Cumulus Cells and Fertilize the Egg"J Biol Chem. 277(33). 30310-30314 (2002)

Baba, D. 等人：“缺乏细胞表面透明质酸酶 PH-20 的小鼠精子可以穿过卵丘细胞层并使卵子受精”J Biol Chem。

Nomura M et al.: "Mechanism of host cell prptection from complement in murine cytomegalovirus (CMV) infection : identification of a CMV-responsive element in the CD46 promoter region"Eur J Immunol. 32(10). 2954-2964 (2002)

Nomura M等人：“鼠巨细胞病毒（CMV）感染中补体对宿主细胞的保护机制：CD46启动子区域CMV反应元件的鉴定”Eur J Nutrition。