Construct Saccharomyces cerevisiae Dicl1 mutants in the EM93 background using a PCR-mediated protocol with a G418-resistance cassette13.
Construct mutants in both mating types and mate them to produce homozygous ¢icl1/¢icl1 knockout strain (MLY283a/a).
Create an Dicl1 disruption construct for C. albicans by inserting a hisG±URA3±hisG cassette14 at a BglII site in the ICL1 open reading frame.
Linearize the construction and transform into CAI4 (a Ura- derivative of strain SC5314; refs 14, 15), and select by uracil prototrophy.
Identify the accurate integrants by PCR and passage on 5-FOA medium.
Use second round transformation to generate two independents homozygous ¢icl1/¢icl1 strains.
Re-introduce the wild-type ICL gene on linearized plasmid pRC2312 by transformation to produce a complemented strain.
Do the transformation of C. albicans by using the same description.
Use standard media and grow the strains at 37°C unless otherwise indicated.
Dilute overnight cultures of C. albicans strains into fresh YPD and grown for 3±4 h at 37°C.
Collect the cultures by centrifuge and wash with PBS.
Inject cells 6 x 10^5 into the tail vein of 18±20-week-old female BALB/c mice.
Monitor the mice for 3 weeks after injection and kill the moribund animals.