Objective  To construct miR RNAi lentiviral vectors of mouse cytochrome P450 3A11 and establish CYP3A11 gene knock-down mouse model.   Methods  The corresponding shRNA sequences of miRNA for silencing the

mouse P450 3A11 gene were designed by on-line designer software on Ravi Sachidanandam’s Lab Website and synthesized by PCR. The PCR products were cloned into the PCI-GFP-MiR vector. Then the PCI-GFP-MiR-shRNA vector was recombined with the FUW lentiviral vector. The obtained lentiviral vectors containing P450 3A11 shRNA were confirmed by double enzyme digestion and sequencing. Each FUW-GFP-MiR-shRNA lentiviral vector was packaged with other vectors

psPAX2 and pMD2.G

in 293FT cells. Culture supernatant was harvested and concentrated by high speed centrifugation. The titre of the pseudotype virus were tested by detecting GFP expression level after transfecting into 293FT cells with the solution containing the virus particles. Three recombinant vectors were transfected into the hepatic cells of FVB/N mouse. After transfection for 48 h

the expression of P450 3A11 in the hepatic cells was detected. The concentrated shRNA1 virus solution was injected into perivitelline space of mouse 12-cell eggs. The injected 22-cell egg was transferred into the oviduct of pseudo-pregnant female mice

which gave birth to F0 offspring. GFP expression was observed with a filter in mouse body under UV light.   Results  The DNA sequencing results showed that the sequence of 3 recombinant lentiviral vectors were all the same as the sequence of corresponding shRNA. The titre of the primary virus solution were all ≥10

6

TU/ml

and that after concentration were all ≥10

9

TU/ml. Compared with the blank control groups

transfection of 2 targeting FUW-GFP-MiR-shRNA lentiviral vectors in the hepatic cells knocked down the expression of P450 3A11mRNA significantly. However

there was no obvious difference between the expression of negative control FUW-GFP-MiR-shRNA-NC groups and the blank control groups. The F0 offspring of 3A11 gene knock-down mouse introduced by FUW-GFP-MiR-shRNA1 lentiviral vectors showed visible GFP expression under UV light.   Conclusion  The effective miR RNAi lentiviral vectors of murine P450 3A11 gen

e are constructed and selected.  CYP3A11 gene knock-down mouse is established successfully．