Muliawan Hary Sakti, Nakayama Kazuhiko, Yagi Keiko, Ikeda Koji, Yagita Kazuhiro, Hirata Ken-ichi, Emoto Noriaki
Division of Cardiovascular Medicine, Department of Internal Medicine, Kobe University Graduate School of Medicine, Kobe, Japan.
Department of Clinical Pharmacy, Kobe Pharmaceutical University, Kobe, Japan.
Kobe J Med Sci. 2015 Oct 7;61(2):E47-53.
Gene delivery to the lung has rapidly progressed as an important method for studying various chronic lung diseases. Viral vectors, albeit highly efficient, are limited by the host immune response. Electroporation, a well-known non-viral method, can efficiently deliver genes to the lung, but is unable to induce stable gene expression. The Tol2 transposon is another non-viral method that can induce stable gene expression by reinserting its genes into the host genome. In this study, we combined electroporation and Tol2 transposons to obtain stable, high-level gene expression in the mouse lung. Tol2 transposon plasmids (pT2A-EGFP; Tol2, pCAGGS-TP; transposase) were optimized in vitro, and the electroporation procedure (pCAG-EGFP) was optimized in mouse lungs. After optimization, a combination of electroporation plus the Tol2 transposon was used in a comparative analysis with electroporation plus pCAG-EGFP. GFP expression levels were quantified and visualized on days 4 and 7 post-electroporation. We successfully reproduced the Tol2 transposon system in vitro and the electroporation procedure in vivo. We observed sustainable GFP expression using electroporation plus the Tol2 transposon on days 4 and 7, while electroporation plus pCAG-EGFP resulted in decreased GFP expression on day 7. We were able to induce high-level, stable gene expression in mouse lungs using a combination of electroporation and the Tol2 transposon. This represents a safer method for lung gene delivery that can be used as an alternative to viral vectors.
作为研究各种慢性肺病的一种重要方法，向肺部进行基因递送已取得迅速进展。病毒载体尽管效率很高，但受到宿主免疫反应的限制。电穿孔是一种众所周知的非病毒方法，它能有效地将基因递送至肺部，但无法诱导稳定的基因表达。Tol2转座子是另一种非病毒方法，它可以通过将其基因重新插入宿主基因组来诱导稳定的基因表达。在本研究中，我们将电穿孔与Tol2转座子相结合，以在小鼠肺部获得稳定的高水平基因表达。Tol2转座子质粒（pT2A-EGFP；Tol2，pCAGGS-TP；转座酶）在体外进行了优化，电穿孔程序（pCAG-EGFP）在小鼠肺部进行了优化。优化后，将电穿孔加Tol2转座子的组合用于与电穿孔加pCAG-EGFP的比较分析。在电穿孔后第4天和第7天对绿色荧光蛋白（GFP）表达水平进行定量和可视化。我们在体外成功重现了Tol2转座子系统，在体内成功重现了电穿孔程序。我们观察到，在第4天和第7天，使用电穿孔加Tol2转座子可实现可持续的GFP表达，而电穿孔加pCAG-EGFP在第7天导致GFP表达下降。我们能够通过电穿孔与Tol2转座子的组合在小鼠肺部诱导高水平的稳定基因表达。这代表了一种更安全的肺部基因递送方法，可作为病毒载体的替代方法。
