A versatile and efficient gene-targeting system for Aspergillus nidulans.
Aspergillus nidulans is an important experimental organism, and it is a model organism for the genus Aspergillus that includes serious pathogens as well as commercially important organisms. Gene targeting by homologous recombination during transformation is possible in A. nidulans, but the frequency of correct gene targeting is variable and often low. We have identified the A. nidulans homolog (nkuA) of the human KU70 gene that is essential for nonhomologous end joining of DNA in double-strand break repair. Deletion of nkuA (nkuA delta) greatly reduces the frequency of nonhomologous integration of transforming DNA fragments, leading to dramatically improved gene targeting. We have also developed heterologous markers that are selectable in A. nidulans but do not direct integration at any site in the A. nidulans genome. In combination, nkuA delta and the heterologous selectable markers make up a very efficient gene-targeting system. In experiments involving scores of genes, 90% or more of the transformants carried a single insertion of the transforming DNA at the correct site. The system works with linear and circular transforming molecules and it works for tagging genes with fluorescent moieties, replacing genes, and replacing promoters. This system is efficient enough to make genomewide gene-targeting projects feasible.
Nayak
Tania
T
Department of Molecular Genetics, The Ohio State University, Columbus 43210, USA.
Szewczyk
Edyta
E
Oakley
C Elizabeth
CE
Osmani
Aysha
A
Ukil
Leena
L
Murray
Sandra L
SL
Hynes
Michael J
MJ
Osmani
Stephen A
SA
Oakley
Berl R
BR
D034961:Antigens, Nuclear / Q000235:genetics; D001232:Aspergillus fumigatus / Q000235:genetics; D001233:Aspergillus nidulans / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D018390:Gene Targeting / Q000379:methods*; D005819:Genetic Markers; D006801:Humans; D000072200:Ku Autoantigen; D009154:Mutation; D010957:Plasmids; D012689:Sequence Homology, Nucleic Acid
D034961:Antigens, Nuclear; D004268:DNA-Binding Proteins; D005819:Genetic Markers; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen