This neo4-excised locus will be referred to as loxP-EGFP-TWI1. DNA sequencing of the shorter

PCR product confirmed that this product resulted from the precise excision of neo4 by homologous recombination of two loxP sites (Fig. 3C). Figure 3 Cre-recombinase BTK inhibitor induces precise recombination at loxP sites. (A) Diagrams of the wild-type TWI1, loxP-neo4-loxP-EGFP-TWI1 and loxP-EGFP-TWI1 loci. The loxP-neo4-loxP-EGFP-TWI1 construct was introduced to the TWI1 locus by homologous recombination. The neo4 cassette was removed from the loxP-neo4-loxP-EGFP-TWI1 locus by Cre-mediated recombination to produce the loxP-EGFP-TWI1 locus. The arrowheads represent the primers used for the DNA excision analysis shown in Fig. 3B and Fig. 4B. (B) Cre-induced recombination at loxP-neo4-loxP-EGFP-TWI1 locus. Total genomic DNA was extracted from starved CRE556 or loxP-neo4-loxP-EGFP-TWI1 cells, or click here mating CRE556 and loxP-neo4-loxP-EGFP-TWI1 PCR cells at 2, 4, 6 and 8 hr post-mixing (hpm) and PCR-amplified using the primers shown in Fig. 3A. The products corresponding to the non-excised loxP-neo4-loxP-EGFP-TWI1 locus (+neo4) and the excised loxP-EGFP-TWI1 locus (-neo4) are marked by arrows. (C) Sequence analysis of the loxP-EGFP-TWI1

locus. DNA sequence of the 1.1 kb PCR product from mating CRE556 and loxP-neo4-loxP-EGFP-TWI1 PCR cells at 8 hpm was analyzed. The Cre/loxP system can be used for N-terminal epitope tagging In the loxP-neo4-loxP-EGFP-TWI1 locus, the loxP-neo4-loxP sequence is inserted directly before the first methionine-coding codon of the EGFP-TWI1 fusion gene. Therefore, EGFP-TWI1 can be expressed only after the excision of the neo4 cassette by HA-Cre1p. This system allows us to express N-terminal EGFP-tagged Twi1p from the endogenous TWI1 promoter. Because the parental PJ34 HCl macronucleus is eventually destroyed at the end of conjugation, the loxP-neo4-loxP-EGFP-TWI1 locus or the neo4-excised loxP-EGFP-TWI1 locus is lost in the sexual progeny. Therefore, to use the loxP-EGFP-TWI1 locus for analyses

of EGFP-Twi1p, parental cells must be recovered after the induction of conjugation between the CRE556 and the loxP-neo4-loxP-EGFP-TWI1 strains. Around a quarter of mating wild-type Tetrahymena cells aborts conjugation before producing zygotic nuclei and haploid meiotic micronuclear products are endoreplicated to regenerate a diploid micronucleus. Parental macronuclei are preserved in this process [15]. We established a method to efficiently recover cells after aborting conjugation and to distinguish the loxP-neo4-loxP-EGFP-TWI1 (or neo4-excised loxP-EGFP-TWI1) strain from CRE556. The method is schematically shown in Fig. 4A. First, individual mating pairs were isolated into drops of 1× SPP at 2 hpm and cells aborting conjugation in these drops by 6 hpm were isolated into drops of fresh 1× SPP.