Figure 3

Cre-recombinase 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 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.