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

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两性二态表达。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。我们通过实时PCR检测了Tex101在几种来自小细胞癌（例如，Lu-139和-140）和生殖细胞肿瘤（例如，NEC-8和-14）的细胞系中的表达水平，以寻找可用于体外分析Tex101的细胞系。其中，我们发现Lu-140表达Tex101，但表达水平非常弱。我们在Tex101的三个靶点上生成了短发夹RNA （shRNA）表达构建体，并使用编码该shRNA和绿色荧光蛋白（GFP）的质粒，即GFP- shtex101。我们用转染tex101的COS-7细胞验证了GFP-shTex101载体的抑制效率。载体对Tex101在培养细胞中的表达有较强的抑制作用。接下来我们尝试用电穿孔法将GFP-shTex101载体转染到小鼠睾丸中。虽然在睾丸生殖细胞中检测到GFP信号，但GFP- shtex101载体的转染效率较差。GFP-sh tex101转染后小鼠睾丸的详细形态学变化以及体内转染效率的提高仍有待解决。我们还研究了小鼠TEX101的生化和免疫组织化学特性。本研究生成的GFP-shTex101有助于阐明TEX101的分子特性及其生理功能。

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.