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Biolistic transformation of a fluorescent tagged gene into the opportunistic fungal pathogen Cryptococcus neoformans.

Abstract
The basidiomycete Cryptococcus neoformans, an invasive opportunistic pathogen of the central nervous system, is the most frequent cause of fungal meningitis worldwide resulting in more than 625,000 deaths per year worldwide. Although electroporation has been developed for the transformation of plasmids in Cryptococcus, only biolistic delivery provides an effective means to transform linear DNA that can be integrated into the genome by homologous recombination.  Acetate has been shown to be a major fermentation product during cryptococcal infection, but the significance of this is not yet known. A bacterial pathway composed of the enzymes xylulose-5-phosphate/fructose-6-phosphate phosphoketolase (Xfp) and acetate kinase (Ack) is one of three potential pathways for acetate production in C. neoformans. Here, we demonstrate the biolistic transformation of a construct, which has the gene encoding Ack fused to the fluorescent tag mCherry, into C. neoformans. We then confirm integration of the ACK-mCherry fusion into the ACK locus.
AuthorsTonya Taylor, Indrani Bose, Taylor Luckie, Kerry Smith
JournalJournal of visualized experiments : JoVE (J Vis Exp) Issue 97 (Mar 19 2015) ISSN: 1940-087X [Electronic] United States
PMID25867491 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't, Research Support, U.S. Gov't, Non-P.H.S., Video-Audio Media)
Chemical References
  • DNA, Fungal
  • Luminescent Proteins
  • Pentosephosphates
  • red fluorescent protein
  • xylulose-5-phosphate
  • Acetate Kinase
  • Aldehyde-Lyases
  • fructose-6-phosphate phosphoketolase
Topics
  • Acetate Kinase (genetics, metabolism)
  • Aldehyde-Lyases (genetics, metabolism)
  • Artificial Gene Fusion (methods)
  • Biolistics (methods)
  • Cryptococcus neoformans (enzymology, genetics)
  • DNA, Fungal (genetics)
  • Electroporation (methods)
  • Genes, Reporter
  • Homologous Recombination
  • Luminescent Proteins (biosynthesis, chemistry, genetics)
  • Pentosephosphates (metabolism)
  • Plasmids (genetics)

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