Artificial chromosomes in the ciliate Oxytricha: Introduction of fluorescent reporters

Abstract

The germline genome of the ciliate Oxytricha trifallax undergoes massive deletion and rearrangement events during sexual conjugation, producing a somatic genome of over 16,000 gene-sized nanochromosomes. Non-coding RNAs produced from parental nanochromosomes program the rearrangement of germline sequences destined for the somatic nucleus. Template RNA injections have previously been used to reprogram chromosome rearrangements and produce engineered strains. A limitation of the current tools, however, is that they cannot be used to introduce novel sequences into O. trifallax that are absent from the germline genome. Two artificial chromosomes containing novel fluorescent reporters were designed and introduced into O. trifallax by microinjection. The red fluorescent protein mRuby2 was fused to the N-terminus of the conjugation-specific Piwi-homolog Otiwi1 and found to persist at high DNA copy number but did not result in significant RNA or protein expression. The fluorescent RNA aptamer Mango, on the other hand, was used to completely replace the open reading frame (ORF) of an Alba-domain protein, and this artificial chromosome was also found at high copy number, and functional RNA transcription was detected by both real-time PCR and in vitro fluorescence. The development and use of artificial nanochromosomes in O. trifallax thus presents a method for the introduction of non-native genes and markers into this model organism.