The inverted terminal repeat (ITR) is the only cis element of AAV genome essential for rAAV rescue, replication and packaging. It is prone to mutation or loss when it is latent in host cell or in plasmid. Plasmids with different ITR types were cloned to compare the influence of ITR types on the AAV packaging and infectivity. The vector plasmids were transformed the competent SURE cells to get different colonies. The ITR types of plasmids were screened by digestion with SmaⅠ. AAV vector plasmid pScGFPud has two ITRs at both ends of AAV genome and plasmid pScGFPu has only one ITR at upstream end of AAV genome. When the two plasmids were co-transfected 293 cells to prepare rAAVs, 1.08?1013 viral particles (AAV1-GFPud) were produced from 20-dishes of 293 cells cotransfected with plasmid pScGFPud, 4.28?1012 viral particles (AAV1-GFPu) were produced from 20-dishes of 293 cells cotransfected with plasmid pScGFPu. Virus AAV1-GFPud infected 293, HeLa and NCI H446 cells more efficiently than did virus AAV1-GFPu. This suggests that defect ITRs in AAV genome is deleterious to AAV packaging and AAV infectivity and vector with complete ITRs is favorable to the yield and activity of rAAV.

Recombinant AAV serotype 8 (rAAV8) vector is new and promising for gene delivery,which transduces muscular and hepatic cells highly efficiently. The transduction varies with the administration routes. It was observed that the rAAV8 virus injected intraperitoneally had been delivered to many different tissues through the blood circulation and resulted in extensive and prolonged gene expression. The interest gene (human coagulation factor Ⅸ) has been highly expressed via the rAAV8 vector injected intraperitoneally into mice at the dose of 5?1010 gc/ mouse. Considerable expression was detected in mouse plasma at two weeks after the administration. Expression peak up to 1 000% level compared to the UCRP occurred between 1~2 months after administration, and then the expression declined gradually but obvious expression persisted at 4 months post administration. APTT test proved the clotting activity of the recombinant hFⅨ in mouse plasma. Immunohistochemical assay showed that the human coagulation factor has been expressed significantly in several organs including liver, kidney, heart and muscles (abdominal and hindleg). These suggest that the rAAV8 virus injected intraperitoneally has been delivered via blood circulation and transduced different cells of multi-organs, and the product of the gene mediated by the rAAV8 vector is biologically active.

OBJECTIVETo analyze the effect of small interfering RNA (siRNA) targeting mouse epididymis-specific colipase-like (meClps) gene on mouse sperm mobility.

METHODSThe eukaryotic expression vector pDsRed2.0-C1-meClps was constructed and transfected into NIH-3T3 cells, and the protein expression was detected with anti-meClps serum. Three interfering sequences targeting meClps (RNAi-251, 224 and 286) were inserted into lentiviral vectors pRNAT-U6.2/lenti, which were co-transfected with pDsRed2.0-C1-meClps into NIH-3T3 cells. The RNA interfering efficiency was confirmed by semi-quantitative PCR and Western blotting. The lentivirus, packed with the lentiviral vector with the highest interfering efficiency, was injected into the caput tissues of mouse epididymis, and its effect on sperm mobility of the cauda epididymis was evaluated.

RESULTSAll the 3 lentiviral RNAi vectors targeting meClps could inhibit the mRNA and protein expressions of meClps, among which pRNAT-U6.2/lenti-RNAi-251 had the highest interfering efficiency. The lentivirus packed with pRNAT-U6.2/lenti-RNAi-251 significantly reduced the path velocity of cauda sperm after injection into the caput epididymis of the mice (P<0.05).

CONCLUSIONKnock-down meClps expression by lentiviral-mediated RNA interference can lower sperm mobility of mice.

Animals ; Epididymis ; Gene Targeting ; Genetic Vectors ; Lentivirus ; Male ; Mice ; NIH 3T3 Cells ; RNA Interference ; RNA, Messenger ; RNA, Small Interfering ; Sperm Motility ; Spermatozoa ; Transfection