Establishment of stable MRP1 knockdown by lentivirus-delivered shRNA in the mouse testis Sertoli TM4 cell line.

Sertoli cells around germ cells are considered a barrier that protects spermatogenesis from harmful influences. The transporter multidrug-resistance-associated protein 1 (MRP1) is a xenobiotic efflux pump that can export glutathione S-conjugated metabolites and xenobiotics from cells. In this study, the Mrp1 gene was stably knocked down in a mouse Sertoli cell line (TM4) using lentivirus vector-mediated RNA interference (RNAi) technology. Four shRNA interference sequences were chosen and designed to screen for the most effective shRNA in candidate cells. The results indicate that lentivirus vectors with high titres were generated and successfully transfected into TM4 cells with high efficiency. Puromycin was added to the culture medium to maintain constant selection during the establishment of the stable cell lines. The expression levels of Mrp1 mRNA and MRP1 protein in stably transfected TM4 cells were significantly lower than those in the control group. Importantly, the transport activity of MRP1 to Calcein and 5-carboxyseminaptharhodafluor (SNARF-1) were significantly reduced because of MRP1 silencing. Moreover, the silencing of the Mrp1 gene in the transfected TM4 cell lines remained highly stable for more than 6 months. These results suggest that the lentivirus-based RNAi stably knocks down the expression of the Mrp1 gene in the established TM4 cell line. This transfected TM4 cell line will provide a new and powerful tool to study the underlying mechanism of MRP1-mediated drug resistance and detoxication in the reproductive system.

The accumulation and efflux of lead partly depend on ATP-dependent efflux pump-multidrug resistance protein 1 and glutathione in testis Sertoli cells.

Since lead accumulation is toxic to cells, its excretion is crucial for organisms to survive the toxicity. In this study, mouse testis sertoli (TM4) and Mrp1 lower-expression TM4-sh cells were used to explore the lead accumulation characteristics, and the role of ATP-dependent efflux pump-multidrug resistance protein 1 (Mrp1) in lead excretion. TM4 cells possess Mrp-like transport activity. The expression levels of mrp1 mRNA and Mrp1 increased after lead treatments at first and then decreased. The maximum difference of relative mRNA expression reached 10 times. In the presence of lead acetate, the amount of cumulative lead in TM4-sh was much higher than that in TM4. After the treatment with lead acetate at 10-40 µM for 12h or 24h, the differences were about 2-8 times. After with the switch to lead-free medium, the cellular lead content in TM4-sh remains higher than that in TM4 cells at 1,3, 6, and 9h time points (P<0.01). Energy inhibitor sodium azide, Mrp inhibitors MK571 and glutathione (GSH) biosynthesis inhibitor BSO could block lead efflux from TM4 cells significantly. These results indicate that lead excretion may be mediated by Mrp1 and GSH in TM4 cells. Mrp1 could be one of the important intervention points for lead detoxification.