Surveys for different gene expression in male and female early embryos

男性和女性早期胚胎中不同基因表达的调查

• 批准号: 11480220
• 负责人: OKABE Masaru
• 金额: $ 8.58万
• 依托单位: Osaka University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-11480220/
• 关键词: sex differentiation; sex chromosome; GFP; X chromosome; sexing of embryos; トランスジェニックマウス; 雌雄分別; ディファレンシャルディスプレイ

The purpose of this study is to separate male and female preimplantation embryos, which was not possible in the past. The strategy was to employ the transgenic animals that integrated green fluorescent gene in their sex chromosomes.Through this research project, it was proved that the sexes of preimplantation embryos are very accurately predicted by the emission of green fluorescent light. Based on this non-invasive separation, we have prepared 1500 each male and female preimplantation embryos and their mRNA was extracted to make cDNA library. For male and female mouse embryo-specific subtracted cDNA pools, purified PCR-amplified secondary PCR product was cloned into the PUC base vector. The 90% of white colonies contain plasmid with insert. Four 96-well plates containing individual clones from each library were established. Virtual northern blot analysis confirmed differential expression of three Driver (female)-specific clones.For Tester (male)-specific clones, five out of three clones are confirmed positive. The sequences of these genes are clarified and now ready for the further investigations.

这项研究的目的是分离男性和女性植入前胚胎，这在过去是不可能的。本研究的策略是利用性染色体上整合了绿色荧光基因的转基因动物，通过该研究项目证明，通过发射绿色荧光可以非常准确地预测植入前胚胎的性别。在此基础上，我们制备了1500个男性和女性的植入前胚胎，并提取其mRNA构建cDNA文库。对于雄性和雌性小鼠胚胎特异性消减cDNA池，将纯化的PCR扩增的二级PCR产物克隆到PUC基础载体中。90%的白色菌落含有带有插入物的质粒。建立含有来自每个文库的单个克隆的四个96孔板。虚拟北方印迹分析证实了三个驱动者（雌性）特异性克隆的差异表达。对于测试者（雄性）特异性克隆，三个克隆中的五个被证实为阳性。这些基因的序列已被阐明，现在为进一步的研究做好了准备。

项目成果

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Nakanishi T 等人：“通过 GFP 作为 pH 指示剂测量顶体的碱化及其与精子的关系”Dev Biol。

Keng,V.W. et al.: "Homeobox gene hex is essential for onset of mouse embryonic liver development and differentiation of the monocyte lineage"Biochem Biophys Res Commun. 276(3). 1155-61 (2000)

肯，V.W.

Nakanishi T et al.: "Alkalinization of acrosome measured by GFP as a pH indicator and its relation to sperm capacitation"Dev Biol. 237(1). 222-231 (2001)

Nakanishi T 等人：“通过 GFP 作为 pH 指示剂测量顶体的碱化及其与精子获能的关系”Dev Biol。

Fujisawa,N. et al.: "Depolarization-dependent survival of cultured mouse cerebellar granule neurons is strain-restarained"European Journal of Neuroscience. 12. 1-5 (2000)

藤泽，N.

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