Fraga Dean, Keenan Erica, Hendel Emily, Nair Anjali, Schofield Whit
Department of Biology, College of Wooster, Wooster, Ohio 44691, USA.
J Eukaryot Microbiol. 2006 Jan-Feb;53(1):16-9. doi: 10.1111/j.1550-7408.2005.00067.x.
A particle inflow gun (PIG) was constructed and tested for its utility to transform Paramecium using tungsten or gold as the DNA carrier particle. In the first set of experiments we transformed Paramecium with a plasmid containing the neomycin-resistance gene, obtaining a transformation efficiency of 0.31+/-0.14% (mean+/-SD) for tungsten particles and 1.30+/-0.29% for gold particles. Plasmid DNA precipitated upon tungsten was shown to be stable for transformation purposes for up to 1 h prior to use and had no detectable effects on transformation efficiency. In addition, we demonstrated that at high frequency (71+/-20%) a Paramecium mutant strain could be phenotypically rescued by co-transformation with a second plasmid containing the selectable neomycin-resistance gene. The PIG coupled with tungsten particles as the carrier offers a low-cost alternative for biolistic transformation of Paramecium.
构建了一种粒子流入枪（PIG），并测试了其使用钨或金作为DNA载体粒子转化草履虫的效用。在第一组实验中，我们用含有新霉素抗性基因的质粒转化草履虫，钨粒子的转化效率为0.31±0.14%（平均值±标准差），金粒子的转化效率为1.30±0.29%。钨沉淀的质粒DNA在使用前长达1小时对转化目的而言是稳定的，并且对转化效率没有可检测到的影响。此外，我们证明，通过与含有可选择的新霉素抗性基因的第二个质粒共转化，草履虫突变菌株可以在高频（71±20%）下得到表型拯救。与作为载体的钨粒子相结合的PIG为草履虫的生物弹道转化提供了一种低成本的替代方法。
