The particle inflow gun can be used to co-transform Paramecium using Tungsten particles.
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.
Fraga
Dean
D
Department of Biology, College of Wooster, Wooster, Ohio 44691, USA. dfraga@wooster.edu
Keenan
Erica
E
Hendel
Emily
E
Nair
Anjali
A
Schofield
Whit
W
D000818:Animals; D019470:Biolistics / Q000379:methods*; D016054:DNA, Protozoan / Q000032:analysis / Q000235:genetics; D004351:Drug Resistance; D009154:Mutation; D009355:Neomycin / Q000494:pharmacology; D010247:Paramecium / Q000235:genetics*; D010316:Particle Size; D010957:Plasmids / Q000235:genetics; D014170:Transformation, Genetic; D014414:Tungsten
D016054:DNA, Protozoan; D009355:Neomycin; D014414:Tungsten