Trasfection and reverse genetics in marine ciliates with highly amplified nanochromosomes

The study of marine ciliates of the Euplotes genus has provided significant insights into microbial ecology. These organisms have an unusual genetic organization with two genomes, that is, a micronuclear genome representing the germ line, and a macronuclear genome containing single gene nanochromosomes amplified to thousands of copies for their somatic life. Improved understanding of this unique organization has been beneficial in elucidating universal principles in the biology of telomeres. However, the application of transfection and reverse genetics techniques remains a challenge. The development of such techniques would contribute to a better understanding of the complex transposition mechanisms occurring in the new macronucleus after each sexual cycle. In this context, we worked on different methods of transfection and gene silencing by RNAi for Euplotes crassus and E. focardii. The former is mesophilic and has a cosmopolitan distribution, as it has been collected from most of the Earth's oceans, while the latter is a strictly psychrophilic Antarctic species and survives at temperatures from -2 °C to 15 °C. 1. We generated plasmids and artificial nanochromosomes ending with telomeres that contain either a GFP reporter and/or a drug selection marker. 2. We found that E. crassus and E. focardii (which has a slower growth rate) display naturally high resistance to a number of drugs/antibiotics that are typically used for selection in transfection experiments. However, they are much more sensitive to G418, paromomycin and puromycin when grown in a culture medium of 10% artificial seawater and 90% of 0.3 M glucose. 3. We demonstrated the delivery of Cy3-labelled control plasmid to Euplotes by lipofection, electroporation, and microinjection. 4. In order to set up the RNAi technology, we analysed all the steps involved in RNAi by feeding with silencing bacteria. We obtained the silencing of the telomerase gene in E.focardii, which showed a phenotype with visible changes in nuclear structure and with characteristics of senescence. (We would like to acknowledge the Moore Foundation for its financial support.)