Establishment Of CYP3A11 Gene Knock-down Mouse By Lentiviral Transgenesis

1. BackgroundCytochromes P450 (CYPs) are enzymes known for their role in metabolism of compounds of a rather nonpolar character. Cytochrome P450 enzymes constitute an important detoxification system that contributes to primary metabolism of more than 60% of all prescribed medications. Of the CYP subfamilies, CYP3A is particularly important because of its role in the metabolism of a wide range of pharmacologically, physiologically, and toxicologically important agents.Some animal models of drug metabolism have been established, however, no one can reflect metabolism of human beings thoroughly. Species differences with respect to cytochrome P450 enzyme activities were investigated.In animal species, the levels of related enzymes/subfamilyes may be very different from those of human body.The advent of RNA interference has led to the ability to interfere with gene expression and greatly expanded our ability to perform genetic screens in mammalian cells. If a transgenic animal model can express human CYP3A and suppress the expression of its own CYP3A by RNAi technology, it will be an ideal model for evaluation of new drugs under development, as well as a perfect tool for better understanding of metabolism pathways of various toxicants and compounds.In this work, three recombinant Lentiviral vectors expressing miRNA-based shRNA molecules targeting CYP3A11 gene were constructed and packaged to generate respective recombinant pseudotyped lentiviruses. The gene knock-down efficiency of the three recombinant lentiviral vectors were tested by transducing the primary hepatic cells derived from FVB/N mouse strain. At 48 h post-transduction, expression of P450 3A11 gene in hepatic cells was quantitatively analyzed. To produce transgenic mice, the recombinant lentiviral vector with higher knock-down efficiency was used to transduce mouse 1-cell egg by injecting the virus solution into perivitelline space. 2. ResultsIn the first part, recombinant lentiviral vectors expressing shRNAs were constructed. The lentiviral vectors were packaged and concentrated to transfect the hepatic cell of FVB/N mouse to test the knock-down efficiency against the target gene CYP3A11. The results were the following:1. The recombinant lentiviral vectors FUW-GFP-MiR-shRNA were constructed and identificated by double digestion and DNA sequencing.2. Through the three-plasmid packgeing system, the titres of the concentrated lentivial suspension of was tested by means of detecting GFP expression level after infecting 293FT cells with the solution containing the virus particles. By RT-PCR method, the mRNA expression of P450 3A11 gene in hepatic cells was detected after transfection of concentrated lentiviral vectors into the hepatic cell of FVB/N mouse. According to the normal control group and the negative control group, the FUW-GFP-MiR-shRNA1, 2 respectively down-regulate (74.9±0.92)%, (55.3±1.06) % of mRNA expression of the CYP3A11 gene in the hepatic cell of FVB/N mouse.In the second part, based on RNA interference (RNAi) strategy, we produced transgenic knock-down mice targeting CYP3A11 gene by lentiviral transgenesis. The vector for expressing shRNA molecules based on the FUW vector was constructed in the first part. To trace the shRNA expression visibly, the eGFP coding sequence was placed downstream the promoter in the lentiviral vectors. The results are the following:1. The shRNA expression cassette was detected in the genomic DNAs of the founder mice by PCR.2. The eGFP expression was observed in the skin of ear concha under fluorescent microscope. These results indicated that transgenic mice harboring the miRNA expression cassette was produced.3. By Fluorenscent Quantitative PCR (FQ-PCR) analysis, the CYP3A11 expression in the liver of FVB/N mouse was shown to be reduced by 72.7%, indicating that the miRNA molecule delivered by lentiviral vector was expressed and knocked down the target gene expression significantly.3. ConclusionsA recombinant lentiviral vector FUW-GFP-miR-shRNA expressing miRNA-based shRNA molecules targeting mosue CYP3A11 gene was constructed.Using"three plasmid package system", the recombinant FUW-GFP-miR-shRNA vectors were packaged and the concentrated recombinant lentivirus solution with titres more than 1×109 TU/mL were prepared. By transfecting primary hepatic cells derived from FVB/N inbred mouse strain, it was shown that the recombinant vectors can specifically knock-down the expressing of mosue endogenous CYP3A11 gene effectively.Transgenic mice expressing miR30-based shRNA molecules were efficiently generated using the concentrated lentivirus. Primary data indicated that the mouse endogenous CYP3A11 expression was specifically inhibited with an efficiency of 72.7%.