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.
Department of Toxicology, School of Public Health, Wuhan University , Wuhan , PR China and.
Zhen
Z
Li
Hong
H
Wang
Shaoxin
S
Huang
Langhuan
L
Zhou
Lu
L
Wang
Chuang
C
Du
Chunhong
C
Wang
D000818:Animals; D001578:Benzopyrans / Q000378:metabolism; D002460:Cell Line; D015536:Down-Regulation; D005452:Fluoresceins / Q000378:metabolism; D055785:Gene Knockdown Techniques; D005822:Genetic Vectors; D016086:Lentivirus / Q000235:genetics*; D008297:Male; D051379:Mice; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics / Q000378:metabolism*; D009284:Naphthols / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D012235:Rhodamines / Q000378:metabolism; D012708:Sertoli Cells / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D001578:Benzopyrans; D005452:Fluoresceins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009284:Naphthols; D034741:RNA, Small Interfering; D012235:Rhodamines; C068301:carboxy-seminaphthorhodaminefluoride; C007740:fluorexon; C421109:multidrug resistance-associated protein 1
Detoxication; MRP1; Sertoli cell; gene knockdown