Laboratory of Gene Therapy, Department of Biochemistry, College of Life Sciences, Shaanxi Normal University, 199 South Chang'an Road, Xi'an 710062, PR China.
J Biotechnol. 2012 Feb 10;157(3):373-8. doi: 10.1016/j.jbiotec.2011.12.022. Epub 2011 Dec 29.
The generation of hexon-modified adenovirus vector has proven difficult. In this paper, we developed a novel method for rapid generation of hexon-modified adenoviral vector via one step ligation in vitro followed by quick white/blue color screening. The new system has the following features. First, eGFP expression driven by the CMV promoter in E1 region functions as a reporter to evaluate the tropism of hexon-modified adenovirus in vitro. Second, it has two unique restriction enzyme sites with sticky ends located in the hexon HVR5 region. Third, a lacZ expression cassette under the control of plac promoter is placed between the two restriction enzyme sites, which allows recombinants to be selected using blue/white screening. To prove the principle of the method, genetically modified adenoviruses were successfully produced by insertion of NGR, RGD or Tat PTD peptide into hexon HVR5. Furthermore, the transduction efficiency of the Tat PTD modified virus was shown to be a significant enhancement in A172 and CHO-K1 cells. In conclusion, the novel system makes the production of truly retargeted vectors more promising, which would be of substantial benefit for cancer gene therapy.
六邻体修饰的腺病毒载体的产生已被证明具有一定难度。在本文中，我们开发了一种新的方法，通过体外一步连接，然后快速进行白色/蓝色筛选，从而快速产生六邻体修饰的腺病毒载体。该新系统具有以下特点。首先，E1 区 CMV 启动子驱动的 eGFP 表达可作为报告基因，用于体外评估六邻体修饰的腺病毒的亲嗜性。其次，它在六邻体 HVR5 区域具有两个独特的粘性末端的限制酶位点。第三，在两个限制酶位点之间放置了 plac 启动子控制的 lacZ 表达盒，允许使用蓝/白筛选选择重组体。为了证明该方法的原理，通过将 NGR、RGD 或 Tat PTD 肽插入六邻体 HVR5 成功生产了遗传修饰的腺病毒。此外，Tat PTD 修饰病毒的转导效率在 A172 和 CHO-K1 细胞中显著增强。总之，该新系统使真正靶向重定向载体的产生更具前景，这将对癌症基因治疗有实质性的益处。
