title: Lentiviral transgenic microRNA-based shRNA suppressed mouse cytochromosome P450 3A (CYP3A) expression in a dose-dependent and inheritable manner.
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.
authors.affiliation: Department of Laboratory Animal Science, College of Basic Medical Sciences, Third Military Medical University, Chongqing, China.
authors.forename: Yong
authors.initials: Y
authors.lastname: Wang
authors.forename: Hai-Hong
authors.initials: HH
authors.lastname: Hu
authors.forename: Hao
authors.initials: H
authors.lastname: Pang
authors.forename: Xiao-Yang
authors.initials: XY
authors.lastname: Zhou
authors.forename: Lu-Shan
authors.initials: LS
authors.lastname: Yu
authors.forename: Lu-Lu
authors.initials: LL
authors.lastname: Wang
authors.forename: Cang'e
authors.initials: C
authors.lastname: Liu
authors.forename: Ke-Nan
authors.initials: KN
authors.lastname: Guo
authors.forename: Cong
authors.initials: C
authors.lastname: Zhao
authors.forename: Qin
authors.initials: Q
authors.lastname: Liu
authors.forename: Ben-Hua
authors.initials: BH
authors.lastname: Zeng
authors.forename: Huan
authors.initials: H
authors.lastname: Tang
authors.forename: Hai-Tao
authors.initials: HT
authors.lastname: Shang
authors.forename: Su
authors.initials: S
authors.lastname: Zeng
authors.forename: Hong
authors.initials: H
authors.lastname: Wei
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004305:Dose-Response Relationship, Drug; D005260:Female; D018628:Gene Dosage / Q000502:physiology; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D040582:Inheritance Patterns / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D016086:Lentivirus / Q000235:genetics; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D035683:MicroRNAs / Q000235:genetics* / Q000494:pharmacology*; D034622:RNA Interference / Q000187:drug effects / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics* / Q000494:pharmacology*; D008565:Membrane Proteins
chemical_list: D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System; D035683:MicroRNAs; D034741:RNA, Small Interfering; C496508:Cyp3a11 protein, mouse; D008565:Membrane Proteins