Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA.

Published

Journal Article

A transformation scheme for Cryptococcus neoformans to yield high-frequency, integrative events was developed. Adenine auxotrophs from a clinical isolate of C. neoformans serotype A were complemented by the cryptococcal phosphoribosylaminoimidazole carboxylase gene (ade2) with a biolistic DNA delivery system. Comparison of two DNA delivery systems (electroporation versus a biolistic system) showed notable differences. The biolistic system did not require linear vectors and transformed each auxotrophic strain at similar frequencies. Examination of randomly selected transformants by biolistics showed that 15 to 40% were stable, depending on the recipient auxotroph, with integrative events identified in all stable transformants by DNA analysis. Although the ade2 cDNA copy transformed at a low frequency, DNA analysis found homologous recombination in each of these transformants. DNA analysis of stable transformants receiving genomic ade2 revealed ectopic integration in a majority of cases, but approximately a quarter of the transformants showed homologous recombination with vector integration or gene replacement. This system has the potential for targeted gene disruption, and its efficiency will also allow for screening of DNA libraries within C. neoformans. Further molecular strategies to study the pathobiology of this pathogenic yeast are now possible with this transformation system.

Full Text

Duke Authors

Cited Authors

  • Toffaletti, DL; Rude, TH; Johnston, SA; Durack, DT; Perfect, JR

Published Date

  • March 1, 1993

Published In

Volume / Issue

  • 175 / 5

Start / End Page

  • 1405 - 1411

PubMed ID

  • 8444802

Pubmed Central ID

  • 8444802

International Standard Serial Number (ISSN)

  • 0021-9193

Digital Object Identifier (DOI)

  • 10.1128/jb.175.5.1405-1411.1993

Language

  • eng

Conference Location

  • United States