During 2007-2008, a 'knock in'construct was designed to introduce epitope taged coding DNA into endogenous loci by homologous recombination. To construct a knock-in of the Flag-epitope tag, a knock-in cassette was designed. The cassette contains sequences that encode a triple Flag epitope tag (3XFlag) adjacent to a URA5 gene flanked by two direct repeats of 120 nucleotides. For targeted knock-in of 3XFlag, sequences homologous to the C-terminus 5'and 3'regions flanking the C-terminus of the target locus were inserted into the two respective cloning sites flanking the knock-in cassette. A ura strain was then transformed with the targeting construct and selected for uracil prototrophs. The targeted clones were identified by genomic PCR and then the URA5 gene was excised by selecting on 5-fluorouracil containing medium. This strategy to Flag-tag the C terminus was first used for a protein that functions in cAMP pathway. This strain tagged with 3XFlag was re-used and transformed with a two HA epitope tag (2XHA) construct of another gene which was similar to the 3XFlag construct except that the 3XFlag was replaced with 2XHA. The resulting epitope-tagged proteins were readily detectable by western blot as well as by immunoprecipitation using commercially available anti-Flag and anti-HA. Moreover,the first HA-tagged protein was immunocoprecipitated with the flag-tagged second protein showing the protein-protein interaction between the two proteins. These data indicate that our targeting method is feasible for multiple loci and that 3XFlag and 2XHA can serve as universal epitopes for several antibody-based applications. The same principle can be used to tag many other proteins with different epitopes in the same strain. The knock-in approach provides a general solution for the study of proteins to which antibodies are substandard or not available. During 2009-2010, we have constructed drug marker-tagged isogenic pairs in serotype A as well as in serotype D strains. These strains are needed for the study of mixed infection in animal model in which mating type dependent difference in tissue invasion can be analyzed. In 2010, we started to construct an insertional mutant library using the Agrobacterium mediated transformation of Cryptococcus gattii, the second species that causes cryptococcosis more readily in immunocompetent individuals than C. neoformans,and isolated 30,000 mutant clones by summer of 2011. This library will serve as a tool for identification of the genes that are involved in the manifestation of pathobiological differences between the two species.
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