The Study Of Function Of The Rat Epididymis-Specific Gene Ces7by RNA Interference

Recently, we isolated and cloned a novel rat epididymis-specific gene named Rat615, A BLAST search in the Gene-Bank protein database shows that Rat615protein shares50-60%similarity to the member of typical carboxylesterase (CE) family, and shares above70%similarity to the member of CES7. Our previous data has showed that Rat615was mainly expressed and secreted to the lumens of the corpus and cauda epididymis. The recombinant Rat615protein had carboxylesterase activity hydrolyzing cholesterol ester. In the present, the Rat615function was investigated by RNAi in vivo. Methods:Several siRNA duplexes were chemically synthesized to target Rat615mRNA sites and their knockdown efficiency was examined on the level of cells, and the effective siRNAs were obtained. The construction of lenti-virus vector expressing the effective siRNA was finished, and then, the recombinant virus was packed, and the titer of virus was investigated. In vivo, the packed virus was injected into cauda epdidiymis, after10days post-infection, the expression of Rat615and the capacitation of sperm were determinated.Results:1、Two effective siRNAs targeting Rat615mRNA were successfully obtained in the cells. The vector expressing these two siRNA was constructed, and then this recombinant vector and the lentivirus vectors were cotransfected to HEK293T cells to prepare the lentivus as Lenti/siRNA1-Rat615/GFP and Lenti/siRNA2-Rat615/GFP. The control lentivirus was Lenti/GFP2、After10days post-infection for in vivo cauda epididymis, the protein expression levels of Rat615in cauda epididymis and the lumen were dramatically knockdown compared with the normal. Sperm capacitation has also been inhibited.As mentioned above, Rat615plays a important role in the motility, capacitation of sperm, so the rat model of Rat615knockdown by RNAi in vivo could be a ideal tool to study the mechanism on the regulation of Rat615protein in capacitation.