慢病毒介导的CYP3A11基因knock-down小鼠的建立

Establishment of CYP3A11 gene knock-down mouse by lentiviral transgenesis

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 miR RNAi lentiviral vectors of mouse cytochrome P450 3A11 and establish CYP3A11 gene knock-down mouse model. Methods The corresponding shRNA sequences of miRNA for silencing the mouse P450 3A11 gene were designed by on-line designer software on Ravi Sachidanandam＇ s Lab Website and synthesized by PCR. The PCR products were cloned into the PCI-GFP-MiR vector. Then the PCI-GFP-MiR-shRNA vector was recombined with the FUW lentiviral vector. The obtained lentiviral vectors containing P450 3All shRNA were confirmed by double enzyme digestion and sequencing. Each FUW-GFP- MiR-shRNA lentiviral vector was packaged with other vec supernatant was harvested and concentrated by high speed tested by detecting GFP expression level after transfecting tors, psPAX2 and pMD2. G, in 293FF cells. Culture centrifugation. The titre of the pseudotype virus were into 293FT cells with the solution containing the virus particles. Three recombinant vectors were transfected into the hepatic cells of FVB/N mouse. After transfection for 48 h, the expression of P450 3A11 in the hepatic cells was detected. The concentrated shRNA1 virus solution was injected into perivitelline space of mouse 12-cell eggs. The injected 22-cell egg was transferred into the oviduct of pseudo-pregnant female mice, which gave birth to F0 offspring. GFP expression was observed with a filter in mouse body under UV light. Results The DNA sequencing results showed that the sequence of 3 recombinant lentiviral vectors were all the same as the sequence of corresponding shRNA. The titre of the primary virus solution were all ≥ 10^6 TU/ml, and that after concentration were all ≥ 10^9 TU/ml. Compared with the blank control groups, transfection of 2 targeting FUW-GFP-MiR-shRNA lentiviral vectors in the hepatic cells knocked down the expression of P450 3A11 mRNA significantly. However, there was no obvious difference between the expression of negative control FUW-GFP-MiR-shRNA-NC groups and the blank control groups. The FO offspring of 3A11 gene knock-down mouse introduced by FUW-GFP-MiR-shRNA1 lentiviral vectors showed visible GFP expression under UV light. Conclusion The effective miR RNAi lentiviral vectors of murine P450 3A11 gene are constructed and selected. CYP3A11 gene knock-down mouse is established successfully.