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性表达。为了阐明TEX101的分子基础，我们对小鼠睾丸中表达的TEX101进行了RNA干扰（RNAi）敲低研究。1．我们通过实时 PCR 检查了来自小细胞癌（例如 Lu-139 和 -140）和生殖细胞肿瘤（例如 NEC-8 和 -14）的几种细胞系中 Tex101 的表达水平，以发现可用于 TEX101 体外分析的细胞系。其中，我们发现Lu-140表达Tex101，但表达水平非常弱。2．我们针对 Tex101 中的三个靶位点生成了短发夹 RNA (shRNA) 表达构建体，并使用编码 shRNA 和绿色荧光蛋白 (GFP) 的质粒，即 GFP-shTex101。我们使用 Tex101 转染的 COS-7 细胞验证了 GFP-shTex101 载体的抑制效率。该载体高度抑制了培养细胞中Tex101的表达。 3．接下来我们尝试使用电穿孔将 GFP-shTex101 载体转染到小鼠睾丸中。尽管在睾丸生殖细胞中可检测到指示转染的 GFP 信号，但 GFP-shTex101 载体的转染效率较差。 GFP-sh Tex101转染小鼠睾丸的详细形态变化以及体内转染效率的提高仍有待解决。4．我们还研究了小鼠 TEX101 的生化和免疫组织化学表征。本研究中产生的 GFP-shTex101 将有助于阐明 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

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

生殖細胞の細胞化学的解析のための超高分解能蛍光顕微鏡法:雄性生殖細胞に特異的なGPI蛋白分子,TEX101の局在解析

用于生殖细胞细胞化学分析的超高分辨率荧光显微镜：TEX101（一种男性生殖细胞特异性 GPI 蛋白分子）的定位分析

• 发表时间: 2006
• 作者: Toshihiro Takizawa;Takuji Kosuge;Akima Harada;Miki Mori;Osamu Ishibashi;Takami Takizawa;Yoshihiko Araki;Yoko Sato;Yoshitaka Hishikawa;Takehiko Koji;Gen Ishikawa;瀧澤俊広
• 通讯作者: 瀧澤俊広

Interaction between TEX101 and cellubrevin revealed by ultrahigh-resolution immunofluorescence microscopy using ultrathin cryosections

使用超薄冷冻切片的超高分辨率免疫荧光显微镜揭示了 TEX101 和 cellubrevin 之间的相互作用

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