7 research outputs found
Lentiviral Transgenic MicroRNA-Based shRNA Suppressed Mouse Cytochromosome P450 3A (CYP3A) Expression in a Dose-Dependent and Inheritable Manner
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
Melting and standard curves of the real-time PCR systems for CYP3A, miR-shRNA1 and Rps18.
<p>The real-time PCR was performed using cDNA samples prepared from the livers as templates. The standard equation of each real-time PCR system was y = −0.3129x+8.19, y = −0.3279x+10.66 and y = −0.3008x+8.18 respectively. The amplification efficiency (E value) of each system was 1.06, 1.18 and 1.00, and correlation coefficients (r2 values) of the standard equations were 0.998, 0.997 and 0.994 respectively.</p
Correlation between CYP3A expression level and that of miR-shRNA1.
<p><b>A</b>: The correlation between the relative expression level of CYP3A and that of miR-shRNA1 detected by real-time RT-PCR. The relative CYP3A expression was calculated by Pfaffl equation, and the resulted data referred to the ratio of CYP3A expression in miR-shRNA1 transgenic mice to that of wild-type individual of the same gender. The relative miR-shRNA1 expression level was detected as the copy number of miR-shRNA1 transcript normalized to that of Rps 18 mRNA in the same sample. <b>B</b>: CYP3A protein detected by Western blot in the livers of wild-type and miR-shRNA1 transgenic mice. The GAPDH protein was simultaneously detected as internal control. WT-F: female wild-type mouse; WT-M: male wild-type mouse; 1–4: the four transgenic founder individuals displaying fluorescence of different intensities. <b>C</b>: CYP3A protein levels in the four transgenic founders and wild-type mice. CYP3A protein level in each sample was calculated by comparing the WB band density of CYP3A to that of GAPDH. <b>D</b>: The correlation between relative CYP3A protein expression level and that of miR-shRNA1. Relative CYP3A protein expression level was the CYP3A protein level normalized to that of wild-type control of the same gender, which was set as 1.</p
shRNA knock-down efficiency test <i>in vitro</i> and transgenic founder mouse production.
<p>A: CYP3A expression was detected by RT-PCR in hepatic cells expressing the two designed shRNAs (shRNA1 and shRNA2), shRNA targeting luciferase gene (negative control) and untreated hepatic cells (blank control) respectively. 1: DNA markers; 2: untreated hepatic cells (blank control); 3: hepatic cells infected with an unrelated shRNA targeting luciferase gene (negative control); 4: hepatic cells infected with shRNA1; 5: hepatic cells infected with shRNA2. B: The CYP3A expression level in hepatic cells expressing shRNA1 or shRNA2 was significantly lower than that of negative or blank control, and hepatic cells expressing shRNA1 exhibited the lowest CYP3A expression level. C: Transgenic mice were generated with the lentiviral vector expressing shRNA1, and four founder mice were detected to be transgenic by PCR using genomic DNA as templates. D. The four transgenic founder mice displayed fluorescence of different intensities. *: indicates statistic significance.</p
Lentiviral transgenic miR-shRNA1 suppressed CYP3A expression through germline transmission.
<p><b>A</b>: CYP3A protein detected by WB in the two breeding mice of each generation. WT-1: female wild-type mouse; WT-2: male wild-type mouse; F1-1, F1-2: the two breeding mice of F1 generation; F2-1, F2-2: the two breeding mice of F2 generation; F3-1, F3-2: the two breeding mice of F3 generation. <b>B</b>: Relative CYP3A expression level in the breeding mice of F1–F3 generations. The relative CYP3A expression levels were calculated as described above, and the value of each generation was the average of the two breeding mice. <b>C</b>: The inhibition rate of CYP3A expression per normalized miR-shRNA1 transcript copy over generations. The value of each generation was also the average of the two breeding mice.</p
CYP3A enzymatic activity analysis.
<p>Five adult male miR-shRNA1 transgenic mice, unrelated miR-shRNA transgenic mice and wild-type mice were subjected to CYP3A enzymatic activity analysis in parallel. The ages of used mice were 8–10 weeks. Analysis was performed using liver microsome systems prepared from each group, and testosterone was added into the systems up to a defined concentration as the probe substrate. The concentration of the oxidative metabolite of probe substrate (6β-hydroxyl-testosterone) was detected by HPLC analysis. Wild-type: wild-type mice of FVBN strain; unrelated miR-shRNA: transgenic mice expressing a miR-shRNA targeting luciferase; miR-shRNA1: transgenic mice expressing the miR-shRNA1 targeting CYP3A. * indicates statistic significance.</p
Structure of FUW-eGFP-miR-shRNA vector.
<p>The designed shRNA sequences targeting mouse CYP3A mRNAs were placed into human miR30 context downstream eGFP coding sequence (CDS). The eGFP-miR-shRNA fragments were further inserted downstream the human Ubiquitin C (UBC) promoter in the lentiviral vector FUW, as a result the eGFP-miR-shRNA fragment was under transcriptional control of human UBC promoter. Arrows P1 and P2 indicates the positions of real-time PCR primers used to detect miR-shRNA expression, and P3 and P4 the primer positions for transgenic founder mouse PCR screen.</p