The macro- and micronuclei are marked with “”a”" and “”i”", respectively. (C) Expression of HA-Cre1p suppresses growth of Tetrahymena. B2086 (wild-type) or CRE556 were diluted to 5,000 cells/mL with 1× SPP medium with or without 1 μg/mL CdCl2. At indicated time after dilution, cells were counted to monitor cell
growth. Immunofluorescence staining using an anti-HA antibody indicated that HA-Cre1p localized to the macronucleus both in the vegetative cells and conjugating cells (Fig. 2B) after its induction by CdCl2. Importantly, when the CRE556 strain was crossed with a wild-type strain, HA-Cre1p protein was detected in both cells of a pair (Fig. 2B). This result indicates that either HA-Cre1p protein or HA-Cre1p mRNA can be transferred from the CRE556 strain to the partner cell during conjugation. This is not surprising CH5183284 chemical structure Because it is known that RNA and protein is exchanged between Selleck Ro 61-8048 mating pairs . Therefore, the CRE556 strain could be used to induce homologous recombination at loxP sites introduced into the macronucleus of any cell that can mate with this strain. Expression of Cre-recombinase selleck suppresses
the growth of Tetrahymena Because Cre is a nuclease, its expression might be genotoxic to Tetrahymena cells. We tested this possibility by analyzing the growth of the CRE556 strain with and without induction of HA-Cre1p expression. Indeed, growth of the CRE556
strain was significantly suppressed when the cells were cultured in the presence of 1 μg/mL CdCl2, whereas the same amount Rolziracetam of CdCl2 had little effect on the growth of the wild-type strain (Fig. 2C). The growth defect in the CRE556 strain is not due to a reduced copy number of the MTT1 gene as expression of HA-cre1 from the BTU1 locus (Supplementary Fig. S1 in Additional file 1) caused similar growth suppression in the presence of CdCl2 (Fig. 2D). These results indicate that the expression of HA-Cre1p has a negative, possibly genotoxic effect on the growth of Tetrahymena cells. Therefore, it is necessary to minimize the exposure of cells to Cre1p when it is used for Tetrahymena transgenesis. The inducible Cre expression system aids in minimizing this toxic effect. Cre-recombinase can induce precise recombination at loxP sites To test if expression of the Cre-recombinase can induce homologous recombination at two loxP sites, we constructed a strain, loxP-neo4-loxP-EGFP-TWI1, in which the neo4 cassette was flanked by two loxP sequences in the TWI1 locus (Fig. 3A). CRE556 cells starved in 10 mM Tris (pH 7.5) were pre-treated with 50 ng/mL CdCl2 for 1.5 hr to induce the expression of HA-Cre1p and mated with a loxP-neo4-loxP-EGFP-TWI1 strain in 10 mM Tris (pH 7.5). Then, excision of the neo4 cassette was observed by PCR using the primers indicated in Fig. 3A. As shown in Fig.