Fig 2.

Macronuclear knockdown of TtALV2. (A) Genotypic profiling PCR of DNA of the wild type (WT) and two knockdown lines displaying a phenotype (KD-C1 and KD-C2) demonstrates the correct integration of the Neo^R cassette into the Ttalv2 locus. Specific primer pairs (tKD1f and tKD1r and tKD2f and tKD2r) were used that only amplify a product upon correct 5′ and 3′ integration of the Neo^R cassette into the Ttalv2 locus. A 940-bp-long fragment of TTHERM_00127110 served as a PCR control. The primer pairs KO1f and KO1r and KO2f and KO2r were used to amplify the upstream and downstream flanking regions of the Ttalv2 gene to generate the Neo^R cassette for homologous recombination. αCentrin, anti-centrin; αTub, anti-tubulin. (B) Quantitative real-time PCR on RNA isolated from WT cells and paromomycin-resistant cells with and without a phenotype demonstrates the downregulation of TtALV2 to occur only in clones with a phenotype. (C and D) The ciliate appears to loose pellicle integrity and with it cell polarity and the ability to divide properly. No single cell axis can be defined, and multiple oral apparatuses, multinuclei, and macronuclei can be observed for each cell cluster. DIC, differential interference contrast. Scale bars, 10 μm.