A PCR-based strategy to generate integrative targeting alleles with large regions of homology

Abstract

Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle for which genetic and molecular techniques are well developed. The entire genome sequence of one C. neoformans strain is nearing completion. The efficient use of this sequence is dependent upon the development of methods to perform more rapid genetic analysis including gene-disruption techniques. A modified PCR overlap technique to generate targeting constructs for gene disruption that contain large regions of gene homology is described. This technique was used to disrupt or delete more than a dozen genes with efficiencies comparable to those previously reported using cloning technology to generate targeting constructs. Moreover, it is shown that disruptions can be made using this technique in a variety of strain backgrounds, including the pathogenic serotype A isolate H99 and recently characterized stable diploid strains. In combination with the availability of the complete genomic sequence, this gene-disruption technique should pave the way for higher throughput genetic analysis of this important pathogenic fungus.

Characterization and Regulation of the Trehalose Synthesis Pathway and Its Importance in the Pathogenicity of Cryptococcus neoformans

“…The nth1 mutant was produced by deletion of the gene from the ATG start site to its stop codon and replacement of the gene with the neomycin cassette. The deletion mutant was constructed using a PCR overlap technique (16). Two fragments were synthesized by PCR; 1 kb of 5Ј-untranslated region (UTR) sequence upstream of the ATG start codon, and 1 kb of 3Ј-UTR sequence downstream of the stop codon.…”

Two glutathione peroxidases in the fungal pathogen Cryptococcus neoformans are expressed in the presence of specific substrates

“…Primers used to amplify this coding region annealed to bases 5-24 and 710-730. Overlap PCR technology (Davidson et al, 2002) was used to generate the 39 GFP fusion constructs, which included the entire genomic sequence of the desired gene and~1 kb of the upstream sequence, as well as the G418 resistance marker.…”

“…An overlap PCR gene deletion technology (Davidson et al, 2002) was used to generate genespecific deletion cassettes of GPX1 (GenBank accession no. XM 570772) and GPX2 (GenBank accession no.…”

Approaches to functional genomics in filamentous fungi

“…This method has been used to create constructs for gene replacement in A. awamori [99] and for targeted in vivo promoter exchange [110]. A variation of the technique, in which overlapping segments were present on both primers at a join (termed double-jointed PCR), was used to create linear constructs for biolistic transformation of Cryptococcus neoformans [111] and for protoplast transformations of A. nidulans, A. fumigatus and Fusarium graminarium [112].…”