Abstract
Cytochomosome P450 enzymes (CYP) are heme-containing monooxygenases responsible for oxidative metabolism of many exogenous and endogenous compounds including drugs. The species difference of CYP limits the extent to which data obtained from animals can be translated to humans in pharmacodynamics or pharmacokinetics studies. Transgenic expression of human CYP in animals lacking or with largely reduced endogenous CYP counterparts is recognized as an ideal strategy to correct CYP species difference. CYP3A is the most abundant CYP subfamily both in human and mammals. In this study, we designed a microRNA-based shRNA (miR-shRNA) simultaneously targeting four members of mouse CYP3A subfamily (CYP3A11, CYP3A16, CYP3A41 and CYP3A44), and transgenic mice expressing the designed miR-shRNA were generated by lentiviral transgenesis. Results showed that the CYP3A expression level in transgenic mice was markedly reduced compared to that in wild type or unrelated miR-shRNA transgenic mice, and was inversely correlated to the miR-shRNA expression level. The CYP3A expression levels in transgenic offspring of different generations were also remarkably lower compared to those of controls, and moreover the inhibition rate of CYP3A expression remained comparable over generations. The ratio of the targeted CYP3A transcriptional levels was comparable between knockdown and control mice of the same gender as detected by RT-PCR DGGE analysis. These data suggested that transgenic miR-shRNA suppressed CYP3A expression in a dose-dependent and inheritable manner, and transcriptional levels of the targeted CYP3As were suppressed to a similar extent. The observed knockdown efficacy was further confirmed by enzymatic activity analysis, and data showed that CYP3A activities in transgenic mice were markedly reduced compared to those in wild-type or unrelated miR-shRNA transgenic controls (1.11±0.71 vs 5.85±1.74, 5.9±2.4; P<0.01). This work laid down a foundation to further knock down the remaining murine CYP3As or CYPs of other subfamilies, and a basis to generate CYP knockdown animals of other species.
Highlights
RNA interference (RNAi) is a post transcriptional gene silencing mechanism, which is conserved among a broad variety of eukaryotic organisms including mammalian species [1,2,3,4]
We found that the transgenic miRshRNA targeting murine Cytochromosome P450 3A (CYP3A), which was delivered by lentiviral vector, suppressed CYP3A expression in a dosedependant and inheritable manner in mice
The two shRNAs both exhibited no homology to human CYP3A mRNA sequences, suggesting they would not silence human CYP3A expression if human CYP3A were transgenically expressed in the resulted knock-down mice
Summary
RNA interference (RNAi) is a post transcriptional gene silencing mechanism, which is conserved among a broad variety of eukaryotic organisms including mammalian species [1,2,3,4]. Traditional method for generating gene-disrupted animals is through ES cell-based homologous recombination. This method is effective, it is limited by the low efficiency of DNA homologous recombination, time-consuming and laborintensive cross strategies for obtaining homozygous mutant individuals, and more importantly, the lack of ES cells derived from other mammalian species limits its application to other important mammalian model animals such as rats, pigs and monkeys. Since RNAi is a highly conserved gene silence mechanism, RNAi provides an alternative method for disrupting mammalian gene expression on both cell and individual level. Compared to conventional shRNA molecules, gene silence mediated by miRNA-shRNA has advantages. It has higher efficacy in knock-down of target gene expression, for it works through the existing natural mechanisms or pathways in cells which are used by endogenous miRNA molecules [10]. Its expression can be driven by polymerase II promoter, as that for endogenous miRNA molecules, which rendered the gene silence mediated by RNAi to be more controllable
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