RNAi-knockdown analysis of a germ-cell-specific antigen, TEX101, in mouse testis

小鼠睾丸中生殖细胞特异性抗原 TEX101 的 RNAi 敲低分析

• 批准号: 18591786
• 负责人: ISHIKAWA Tomoko
• 金额: $ 2.43万
• 依托单位: Nippon Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18591786/
• 关键词: mouse testis; TEX101; GPI; RNAi; reproductive biology; 精巣; 精子

TEX101, a unique germ-cell-specific marker protein, shows sexually dimorphic expression during mouse gonad development. To clarify the molecular basis of TEX101, we performed the RNA interference (RNAi)-knockdown study of TEX101 expressed in mouse testis.1. We examined the expression level of Tex101, by real-time PCR, in several cell lines derived from small cell carcinomas (e.g., Lu-139, and -140) and germ cell tumors (e.g., NEC-8 and -14) to find cell lines useful for in vitro analysis of TEX101. Among them, we found that Lu-140 expressed Tex101, albeit at a very weak level.2. We generated short-hairpin RNA (shRNA) expression constructs against three target sites in Tex101 and used plasmids encoding the shRNA along with Green fluorescence protein (GFP), i.e., GFP-shTex101. We validated the inhibitory efficiency of the GFP-shTex101 vectors using Tex101-transfected COS-7 cells. The vectors highly inhibited the expression of Tex101 in the culture cells.3. We next tried to transfect the GFP-shTex101 vectors into mouse testis using electroporation. Although GFP signal indicating transfection was detectable in germ cells in the testis, the transfection efficiency of the GFP-shTex101 vectors was poor. Detailed morphological changes on GFP-sh Tex101-transfeted mouse testis as well as improvement of in vivo transfection efficiency remain to be resolved.4. We also investigated the biochemical and immunohistochemical characterization of TEX101 from mice.GFP-shTex101 generated in this study would helpful in elucidating the molecular characteristics and its physiological functions of TEX101.

TEX101是一种独特的生殖细胞特异性标记蛋白，在小鼠性腺发育过程中表现出性别二态表达。为了阐明TEX101的分子基础，我们对在小鼠睾丸中表达的TEX101进行了RNA干扰(RNAi)敲除研究。我们通过实时定量聚合酶链式反应检测了来自小细胞癌(例如Lu-139和-140)和生殖细胞肿瘤(例如NEC-8和-14)的几个细胞系中Tex101的表达水平，以寻找对TEX101的体外分析有用的细胞系。其中，我们发现Lu-140表达Tex101，但表达水平很低。我们针对Tex101中的三个靶点构建了短发夹状RNA(ShRNA)表达载体，并使用编码shRNA和绿色荧光蛋白(GFP)的质粒，即GFP-shTex101。我们用转基因COS-7细胞验证了GFP-shTex101载体的抑制效率。表达载体在培养细胞中显著抑制了纹状体101基因的表达。接下来，我们尝试利用电穿孔技术将GFP-shTex101载体导入小鼠睾丸。虽然在睾丸生殖细胞中可检测到GFP信号，但GFP-shTex101载体的转染率较低。GFP-sh tex 101转基因小鼠睾丸的详细形态变化以及体内转染率的提高尚待解决。我们还对小鼠TEX101的生化和免疫组织化学特性进行了研究。本研究获得的GFP-shTex101有助于阐明TEX101的分子特征及其生理功能。

项目成果

Molecular characterization of a germ-cell-specific antigen, TEX101, from mouse testis

• DOI: 10.1017/s0967199406003753
• 发表时间: 2006-08-01
• 期刊: ZYGOTE
• 影响因子: 1.7
• 作者: Jin, Hong;Yoshitake, Hiroshi;Araki, Yoshihiko
• 通讯作者: Araki, Yoshihiko

MicroRNA (miRNA) cloning analysis reveals sex differences in miRNA expression profiles between adult mouse testis and ovary

• DOI: 10.1530/rep-08-0349
• 发表时间: 2008-12-01
• 期刊: REPRODUCTION
• 影响因子: 3.8
• 作者: Mishima, Takuya;Takizawa, Takami;Takizawa, Toshihiro
• 通讯作者: Takizawa, Toshihiro

Subcellular distribution of IgG and albumin in the first-trimester human placenta as revealed by ultrahigh-resolution immunofluorescence microscopy

超高分辨率免疫荧光显微镜揭示妊娠早期人胎盘中 IgG 和白蛋白的亚细胞分布

• 发表时间: 2007
• 作者: Toshihiro Takizawa;Takuji Kosuge;Akima Harada;Miki Mori;Osamu Ishibashi;Takami Takizawa;Yoshihiko Araki;Yoko Sato;Yoshitaka Hishikawa;Takehiko Koji;Gen Ishikawa
• 通讯作者: Gen Ishikawa

Ultrahigh-resolution immunofluorescence microscopy for histochemical analysis of germ cells

用于生殖细胞组织化学分析的超高分辨率免疫荧光显微镜

• 发表时间: 2006
• 作者: Toshihiro Takizawa;Miki Mori;Gen Ishikawa;Toshiyuki Takeshita;Shigeki Matsubara;Yoshihiko Araki
• 通讯作者: Yoshihiko Araki

Tesitcular proteins associated with the germ cell-marker, TEX101 : involvement of cellubrevin TEX101-trafficking to the cell surface during spermatogenesis

与生殖细胞标记 TEX101 相关的睾丸蛋白：在精子发生过程中参与 cellubrevin TEX101 运输至细胞表面

• 发表时间: 2006
• 期刊: Biochem Biophys Res Commun 345・1
• 作者: Hiroki Tsukamoto;Hiroshi Yoshitake;Miki Mori;Mitsuaki Yanagida;Kenji Takamori;Hideoki Ogawa;Toshihiro Takizawa;Yoshihiko Araki;Hiroki Tsukamoto
• 通讯作者: Hiroki Tsukamoto