Targeted Gene Disruption by Ectopic Induction of DNA Elimination in Tetrahymena

Abstract

Tetrahymena is a useful eukaryotic model for biochemistry and molecular cell biology studies. We previously demonstrated that targeted ectopic DNA elimination, also called co-Deletion (coDel), can be induced by the introduction of an internal eliminated sequence (IES)-target DNA chimeric construct. In this study, we demonstrate that coDel occurs at most of the loci tested and can be used for the production of somatic gene KO strains. We also showed that coDel at two loci can be simultaneously induced by a single transformation; thus, coDel can be used to disrupt multiple gene loci in a single cell. Therefore, coDel is a useful tool for functional genetics in Tetrahymena and further extends the usefulness of this model organism.KEYWORDS Tetrahymena; DNA elimination; RNAi; gene knockout T HE ciliated protozoan Tetrahymena thermophila can grow at an exceptionally high rate (its doubling time is approximately 2 hr) and can reach a high density (a few million cells per milliliter) under simple and inexpensive culture conditions (reviewed in Orias et al. 2000). In combination with robust genetic manipulation methods (reviewed in Chalker 2012), Tetrahymena is a useful model eukaryote for biochemistry and molecular cell biology studies (reviewed in Collins and Gorovsky 2005).Three strategies for loss-of-function genetic studies have been established for this organism. The first strategy is a germline knockout (KO), in which one of the two gene copies in the diploid micronucleus (MIC) is replaced with a drug-resistance gene by homologous recombination, and two heterozygous strains are then sexually crossed to obtain a homozygous germline KO strain (Cassidy-Hanley et al. 1997). The second strategy is somatic KO, in which one of the 45 copies of a gene in the polyploid macronucleus (MAC) is replaced with a drugresistance gene by homologous recombination, and "phenotypic assortment" is used to obtain cells with the drug-resistance gene at all loci (Merriam and Bruns 1988). Phenotypic assortment is possible because MAC chromosomes are randomly segregated into daughter cells in vegetative cell division and a stepwise increase of the drug in culture selects for cells that have more drug-resistance genes. The final strategy is gene knockdown (KD) by RNA interference (RNAi), in which a construct expressing long hairpin RNA that is complementary to the gene of interest is introduced into a nonessential locus in the MAC and small RNAs produced from the hairpin RNA posttranscriptionally silence the expression of the gene (HowardTill and Yao 2006).Although the first two gene KO strategies have been reliably used to study individual gene functions, they are not suitable for high-throughput genetic screening because the integration of a KO construct into the MIC germline occurs at very low efficiency and the phenotypic assortment process used in somatic KOs require the careful control of drug concentrations in each strain and culture step. Moreover, it takes 1 month to obtain KO strains using these methods. For germline KO, t...

Ciliary force-responsive striated fibers promote basal body connections and cortical interactions

“…1; Galati et al, 2014). To investigate the effect of Cro1p loss on SFs, T. thermophila CRO1 was knocked out in the T. thermophila macronucleus using CoDel (Hayashi and Mochizuki, 2015). Successful CRO1 knockout was confirmed by the loss of CRO1 genomic DNA and CRO1 transcripts (Fig.…”

Post-meiotic DNA double-strand breaks occur in Tetrahymena, and require Topoisomerase II and Spo11

“…To eliminate background autophagic DNA fragments, we created somatic knockout strains for the autophagy-related 8 isoform 2 gene ( ATG8-2 ), which encodes a central component of the Tetrahymena autophagosome (Figure 8A; Liu and Yao, 2012), using the co-Deletion technique (described in Hayashi and Mochizuki, 2015). Diagnostic PCR for the ΔATG8-2 strains of different mating types showed that ATG8-2 genomic loci were completely deleted (Figure 8B).…”

“…The post-meiotic signal in this mutant was completely absent in a triple mutant ( TOP2gi::SPO11i::ΔATG8-2 ; Figure 8E). These results show that programmed PM-DSBs are present in Tetrahymena , suggesting that PM-DSB formation requires both Top2g and Spo11.

10.7554/eLife.26176.016Figure 8. ATG8-2 gene knockout eliminates background DNA fragments resulting from autophagic degradation of unselected pronuclei.( A ) Schematic representation of ATG8-2 knockout strains by co-Deletion (as described in Hayashi and Mochizuki, 2015), confirmed by diagnostic PCR. The PCR primer set is indicated by arrows.

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“…The amplified fragment was integrated into the backbone vector pMcoDel (Hayashi and Mochizuki, 2015) using Gibson Assembly Master Mix (New England BioLabs). The resulting vector (pMcoDel-ATG8-2) was used for biolistic transformation without linearization.…”

Tetrahymenaas a Unicellular Model Eukaryote: Genetic and Genomic Tools

“…Recently, a rapid method for MAC gene knockouts has been developed that does not depend on homologous recombination (Hayashi and Mochizuki 2015;Noto et al 2015). Codeletion (CoDel) harnesses the endogenous mechanism for IES DNA elimination during MAC development to remove 1 kb of a gene of interest.…”