O'Brien J A, Holt M, Whiteside G, Lummis S C, Hastings M H
Neurobiology Division, MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK.
J Neurosci Methods. 2001 Nov 15;112(1):57-64. doi: 10.1016/s0165-0270(01)00457-5.
Transfection and subsequent expression of DNA in living neuronal tissue is problematic and no technique has emerged that is completely non-damaging, efficient and reproducible. The Bio-Rad hand-held Gene Gun has overcome some of these problems by exploiting a biolistic method in which small gold particles carrying plasmid DNA are propelled into neurons whilst causing minimal detectable cell damage. In its current configuration, however, the Bio-Rad Gene Gun is optimised for transfecting cells in dispersed cultures, and therefore delivers particles superficially over a relatively wide area. Here we report modifications to the Bio-Rad Gene Gun that both enhance its accuracy by restricting its target area, and increase the depth penetration achieved by gold particles, thereby allowing smaller and deeper tissues to be transfected. These alterations make the modified Gene Gun more applicable for in vitro transfection of organotypic cultures and enhance its potential utility for in vivo gene delivery. Moreover, the modified configuration operates successfully at lower gas pressures, thereby reducing even further the degree of cell damage incurred during transfection.
在活的神经元组织中进行DNA转染及随后的表达存在问题，目前还没有出现一种完全无损伤、高效且可重复的技术。伯乐手持式基因枪通过利用一种生物弹道方法克服了其中一些问题，即携带质粒DNA的小金颗粒被推进神经元中，同时造成最小的可检测细胞损伤。然而，按照其当前配置，伯乐基因枪针对分散培养中的细胞转染进行了优化，因此在相对较宽的区域表面递送颗粒。在此，我们报告了对伯乐基因枪的改进，这些改进既通过限制其靶区域提高了其准确性，又增加了金颗粒实现的深度穿透，从而使更小且更深的组织能够被转染。这些改变使改进后的基因枪更适用于器官型培养物的体外转染，并增强了其在体内基因递送方面的潜在效用。此外，改进后的配置在较低气压下能成功运行，从而进一步降低了转染过程中产生的细胞损伤程度。
