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目的构建和筛选小鼠P4503A11基因miR RNAi慢病毒载体,建立CYP3A11基因knock-down小鼠。方法利用Ravi Sachidanandam’s Lab的在线软件设计沉默小鼠P4503A11基因的miRNA所对应的shRNA序列,PCR扩增后克隆到PCI-GFP-MiR质粒中,其再与慢病毒载体FUW进行重组。将psPAX2和pMD2.G两个载体和FUW-GFP-MiR-shRNA重组慢病毒载体共转染293FT细胞,包装成病毒。用包装好的病毒液感染293FT细胞后,并利用GFP蛋白表达水平进行滴度测定。将上述重组慢病毒载体分别转染FVB/N小鼠肝细胞,转染48h后,检测P4503A11基因mRNA表达水平的变化。选取干扰效果最好的FUW-GFP-MiR-shRNA1重组慢病毒载体进行制备基因knock-down小鼠模型。用显微注射法将浓缩的shRNA1病毒液注射至FVB/N小鼠12细胞期胚胎透明带下。将注射后发育至22细胞期的胚胎移植至假孕受体母鼠,得F0代小鼠。在紫外光照射下利用滤光片观察GFP在小鼠活体内的表达水平。结果DNA测序结果显示3个重组慢病毒载体均与所设计的shRNA序列一致。检测的3个浓缩前慢病毒悬液的滴度均≥106TU/ml,经过高速离心对病毒进行浓缩和纯化,其滴度达到109TU/ml以上。转染FUW-GFP-MiR-shRNA1、2的2组肝细胞的P4503A11基因mRNA表达水平相对于空白对照组均显著下降(P<0.05),而转染阴性对照FUW-GFP-MiR-shRNA-NC的肝细胞P4503A11基因mRNA表达水平相对于空白对照组无明显改变。选用FUW-GFP-MiR-shRNA1慢病毒载体制备的F0代3A11基因knock-down小鼠,在紫外光照射下,阳性小鼠可见较强的荧光。结论构建并筛选出小鼠P4503A11基因有效的靶向miR RNAi慢病毒载体,并成功建立CYP3A11基因knock-down小鼠。
Objective To construct and screen the mouse RNAi lentiviral vector of P4503A11 gene and construct CYP3A11 gene knock-down mice. Methods The shRNA sequence of miRNA of P4503A11 gene was designed by online software Ravi Sachidanandam’s Lab. After amplification by PCR, it was cloned into the plasmid PCI-GFP-MiR and recombined with lentiviral vector FUW. 293FT cells were cotransfected with psPAX2 and pMD2.G vectors and FUW-GFP-MiR-shRNA recombinant lentiviral vector and packaged into viruses. 293FT cells were infected with the packaged virus solution and the titers were measured using GFP protein expression levels. The recombinant lentiviral vector was transfected into FVB / N mouse hepatocytes respectively, and the expression of P4503A11 mRNA was detected 48 h after transfection. Select the best interference effect of FUW-GFP-MiR-shRNA1 recombinant lentiviral vector for gene knock-down mouse model. The concentrated shRNA1 virus solution was injected into the 12-cell embryo zona pellucida of FVB / N mice by microinjection. Embryos that developed to 22-cell stage after injection were transplanted into fake pregnant mice to obtain F0 mice. The expression level of GFP in vivo of mice was observed under ultraviolet light using a filter. Results DNA sequencing results showed that all three recombinant lentiviral vectors were consistent with the designed shRNA sequence. The titer of the three pre-concentration lentivirus suspensions tested was ≥106TU / ml. The virus was concentrated and purified by high-speed centrifugation and the titer reached above 109TU / ml. The mRNA expression level of P4503A11 in two groups of hepatocytes transfected with FUW-GFP-MiR-shRNA1,2 was significantly lower than that in the blank control group (P <0.05), while the transfected FUW-GFP-MiR-shRNA-NC Of hepatocyte P4503A11 gene mRNA expression relative to the blank control group had no significant change. F0 generation of 3A11 gene knock-down mice prepared with FUW-GFP-MiR-shRNA1 lentiviral vector showed strong fluorescence in positive mice under ultraviolet light irradiation. Conclusions Mouse P4503A11 gene was constructed and screened for effective lentiviral vector targeting miR RNAi and CYP3A11 gene knock-down mice were successfully established.