Identification and utilization of a mutated 60S ribosomal subunit coding gene as an effective and cost-efficient selection marker for Tetrahymena genetic manipulation.

Since the onset of molecular biology, the ciliate Tetrahymena thermophila has been one of the most convenient single-celled model eukaryotes for genetics, biochemistry, and cell biology. Particularly, thanks to the availability of several different selection markers, it is possible to knock out or knock in genes at multiple genetic loci simultaneously in Tetrahymena, which makes it an excellent model ciliate for tackling complex regulatory mechanisms. Despite these selection markers are efficient for genetic manipulation, the costly drugs used for selection have highlighted the urgent demand for an additional cost-efficient and effective selection marker. Here, we found that a mutated 60S ribosomal subunit component, RPL36A, confers Tetrahymena with cycloheximide resistance. On top of that, we developed a cycloheximide cassette and explored suitable transformation and selection conditions. Using the new cassette, we obtained both knock-out and knock-in strains successfully at a relatively low cost. This study also provided the first evidence that a cycloheximide resistance gene can be engineered as a selection marker to completely delete a gene from the highly-polyploid somatic nucleus in Tetrahymena.