The Neurospora Genome Project was funded by NIGMS for two successive 5 year periods, the longest period allowed by NIGMS for Program Project Grants. Because funding has expired reagents, strains and tools have been archived and are available elsewhere as described below, but descriptions of some resources are available here.
Through the efforts of the project a number of resources have been provided to the fungal genetics community including molecularly validated knockouts for all the genes in Neurospora crassa, and a large number of knockouts of Aspergillus nidulans (including a full collection of kinase knockouts) as well as primers for creating knockouts for the entire Aspergillus nidulans genome, printed microarrays, a dense SNP map for Neurospora, and a variety of other tools. These reagents and strains have all been deposited into the Fungal Genetics Stock Center where they are available for a nominal fee.
There are searchable spreadsheets available on the FGSC website that will direct users to the appropriate strain numbers.
Neurospora knockout strains are available at http://www.fgsc.net/scripts/StrainSearchForm.asp.
Because there are so many strains, these are available both individually and as arrayed sets in 96 well plates.
It should be noted that while the knockouts were being created there was ongoing annotation of the genome with the result that individual gene annotations frequently changed. As a result, while most knockouts are correct, there will be cases where a single gene is represented by two separate knockouts (if the annotation at the time the cassettes were created represented exons as separate genes) and cases where not all exons have been removed. The primers used for each knockout are available on this web site (below) and can be mapped to the genome to identify exactly the region replaced with the knockout cassette.
Most of the knockouts were molecularly validated by Southern Blotting to be certain that the targeted gene was indeed the one and only site of insertion of genetic material. However, it is well understood that transformation is mutagenic process so users should be aware of the possibility that secondary mutations could have occurred so best practice will be to send the knockout through a cross, selecting for the hygromycin resistance that marks the knockout, and then screening to be sure that phenotype(s) associated with the knockout co-segregate with the hygromycin resistance.
Multiple attempts were made to knock out every gene so if the gene knockout you seek is unavailable as a homokaryon the gene is probably essential. In most cases such knockouts are available as heterokaryons. There were however a handful of cases where knockouts were not achieved despite repeated attempts for reasons that were never clear.
As a convenience we have maintained availability here of spreadsheets describing the primers used for knockout cassette construction as well as protocols for making knockouts.
- Primers can be viewed here: PRIMERS
- Knockout Protocols are available here: KNOCKOUT PROTOCOLS
- Available Knockout Strains (updated March 7, 2016) are available here:KO STRAINS
- Aspergillus knockout cassettes and primers are available at http://www.fgsc.net/Aspergillus/KO_Cassettes.htm