Transfection of the hypotrichous ciliate Stylonychia lemnae with linear DNA vectors.
We present a new protocol for the transfection of Stylonychia lemnae with linear DNA vectors containing the neomycin-resistance gene from Escherichia coli transposon Tn5. The taking up of heterologous DNA is achieved by damaging the cells' protein coat with urea prior to transfection according to the calcium phosphate co-precipitation procedure. After transfection, transformed cells can be enriched by selection with the antibiotic drug G418. Hybridization experiments show that macronuclear DNA of these G418-selected cells contains molecules homologous to the transfected vector DNA, which are altered by some recombination process. Transfected cells, which have grown for more than 100 cell cycles in antibiotic-free medium, still contain vector-homologous DNA, but recombination continued during this time. We are able to transfect Stylonychia early after conjugation, a process which is followed by complex genome rearrangements and amplification. In these experiments we observed considerable amplification of vector-homologous DNA molecules as compared to transfected vegetative cells. Again these molecules are altered by recombination in respect to the original vector DNA. As soon as suitable vectors are available, the transfection protocol presented here can be a basic tool for the study of DNA replication, transcription and macronuclear development in Stylonychia.
D000818:Animals; D016798:Ciliophora / Q000235:genetics*; D003227:Conjugation, Genetic; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D014162:Transfection
D004269:DNA, Bacterial