Researchers are exploring whether these ubiquitous fluorinated molecules might worsen infections or hamper vaccine effectiveness.
Image credit: Shutterstock/Dmitry Naumov.
Edited by David D. Perkins, Stanford University, Stanford, CA, and approved March 29, 2006
↵†H.V.C. and G.P. contributed equally to this work. (received for review February 21, 2006)
Abstract
The low rate of homologous recombination exhibited by wild-type strains of filamentous fungi has hindered development of high-throughput gene knockout procedures for this group of organisms. In this study, we describe a method for rapidly creating knockout mutants in which we make use of yeast recombinational cloning, Neurospora mutant strains deficient in nonhomologous end-joining DNA repair, custom-written software tools, and robotics. To illustrate our approach, we have created strains bearing deletions of 103 Neurospora genes encoding transcription factors. Characterization of strains during growth and both asexual and sexual development revealed phenotypes for 43% of the deletion mutants, with more than half of these strains possessing multiple defects. Overall, the methodology, which achieves high-throughput gene disruption at an efficiency >90% in this filamentous fungus, promises to be applicable to other eukaryotic organisms that have a low frequency of homologous recombination.
Footnotes
- ↵‖To whom correspondence should be addressed. E-mail: jay.c.dunlap{at}dartmouth.edu
Author contributions: H.V.C., G.P., G.E.T., K.A.B., and J.C.D. designed research; H.V.C., G.P., G.E.T., C.R., C.M.C., and L.L. performed research; H.V.C., G.P., G.E.T., C.R., C.M.C., R.L.W., K.A.B., and J.C.D. contributed new reagents/analytic tools; H.V.C., G.P., G.E.T., and K.A.B. analyzed data; and H.V.C., G.P., G.E.T., K.A.B., and J.C.D. wrote the paper.
Conflict of interest statement: No conflicts declared.
This paper was submitted directly (Track II) to the PNAS office.
- Abbreviation:
- VM,
- Vogel’s minimal medium.
- Received February 21, 2006.
- © 2006 by The National Academy of Sciences of the USA
Freely available online through the PNAS open access option.
A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors
Sign up for Article Alerts
Jump to section
You May Also be Interested in
Recent experiments and simulations are starting to answer some fundamental questions about how life came to be.
Image credit: Shutterstock/Radoslaw Lecyk.
Archaeologists have long tried to define the transition between the two time periods.
Image credit: Ceri Shipton.
Jonathon Yuly, David Beratan, and Peng Zhang investigate how electron bifurcation reactions work.
A study finds that giant pandas roll in horse manure to increase their cold tolerance.
Image credit: Fuwen Wei.