Characterization Of CYP3A11 Knock-down Mice

Cytochrome P450 superfamily enzymes (CYP450) play an important role in drug metabolism, among which the 3 A subfamily(CYP3A) metabolizes about more than 60% of the clinically available drugs. Of the CYP450 subfamilies, CYP3A is particularly important because of its role in the metabolism of a wide range of pharmacologically, physiologically, and toxicologically important agents. According to relative studies, CYP3A11 is the most abundant member of mouse CYP3A subfamily,which is recognized as the counterpart of human CYP3A4 in mouse and plays key roles in metabolism of the widest range of drugs in mice, yet until now its p450s enzyme role in drug metabolism has not been fully known. In order to fully carry out its efficacy and reduce or avoid adverse events, this paper will keep on a further study about CYP3A11 expression.Knock-down is one of the most widely used technologies in drug metabolism research, particularly in protein expression. In the study, the prophase preparated CYP3A11 transgenic mice treated as laboratory animals on this technology.They mated with one another randomly in a close system, the respective offspring displaying influenrescence continued to be bred in the same way over three generations consecutively, then to detect the EGFP distribution in positive mice, explore positive rate and stability of Lentivirus transgenic animals and to research the interference efficiency Of miR-shRNA on CYP3A11 transgenic mice and the phenotype analysis of CYP3A11 transgenic mice. The completion of this project would establish a tool mouse for producing a series of more humanized CYP3A transgenic mice. The results showed that:1. An optimized and reliable fluorescence quantitive PCR (FQ-PCR) system was established for quantitive analysis of three generation transgenic EGFP expression. Results show that:the breeding rate and expression rate of the transgenic mice with single-lane inbreeding were 100%,that is to say during single line inbreeding genetically modified animal breeding groups did not appear gene separation phenomenon, so this method can be used to establish stable positive transgenic groups.2. We observed the skin, tail and pinna characteristic green fluorescent of mice under UV light in the darkroom. 3. Through the fluorescence quantitative polymerase chain reaction (PCR) analysis, the result is that the interference efficiency up to about 80% in mouse liver, it indicates virus mediated miRNA molecules also expressed and significantly reduced purpose gene expression.4. The test in vitro liver microsomal phenotype detection is that, CYP3A11 enzymes of knock-down mice liver microsomal classic substrates is 6-beta hydroxyl testosterone and its metabolic activity was reduced by 80% above than the control group. Compared with normal FVB/N mice, there is no significant difference of the enzyme activity between FVB/N mice and transgenic mice.It indicates that the transgenic process mediated by lentivirus has no significant differences to CYP3A11 enzyme activity.The following conclusions are drawn:1. The genetically modified animals could be obtained by konck-down technology. Furthermore, single line inbreeding breeding methods can be used to establish stable positive transgenic lines.2. Lentiviru mediated technology can dramatically reduce the target gene expression. So it has potential application in producing humanized CYP3A genetically modified animals.