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

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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.