Options for obtaining a Neurospora strain bearing
a targeted deletion in a specific gene
Knockout Team, Neurospora Genome Project
January 26, 2005
Philosophy – Our goal is to provide useful genetic strains bearing knockouts of Neurospora genes to the community in a fast, convenient, transparent, and cost effective manner. This is not a fee for service, but is a community-based effort operating at cost to serve the Fungal Genetics Community.
Background - We expect to be able to use targeted gene replacements to knock out virtually all the genes in Neurospora by the end of 2009. As outlined in the background information describing the efforts of the Neurospora Genome Project (http://www.dartmouth.edu/~neurosporagenome/), there are two major phases to this work. In the first phase knockout cassettes are being constructed to target all of the genes in Neurospora; this will be virtually complete by early summer of 2005, and the amount of cassette DNA assembled at this stage will be sufficient for just a few transformations. A list of primers used and cassettes completed is available at the Genome Project website (http://www.dartmouth.edu/~neurosporagenome/). Beginning in the spring of 2005, these cassettes will be used one-by-one at either Dartmouth or UC Riverside to knock out individual genes. A list of genes successfully deleted will be found at the Genome Project website (http://www.dartmouth.edu/~neurosporagenome/) and it will be linked to the FGSC website. All knockout strains constructed will be immediately deposited in the FGSC from which they can be obtained by standard procedures.
How do I obtain a Knockout strain?
FIRST- {available soon} Go to the listing of completed knockouts (available within Neurospora Genome Project - Proj#1). If a strain bearing the knockout has been generated as a part of this genome project, it will be listed here and will have been deposited with the FGSC. Request this strain from the FGSC. We will begin depositing strains in the spring of 2005.
SECOND - If the strain bearing the knockout of interest has not been generated you have several options.
Option 1. You may email the site (knockouts@dartmouth.edu) and request that a strain be constructed; place the term KNOCKOUT REQUEST in the subject line. Requests for up to 5 knockout strains per 12-week interval will be honored. Your request will immediately be put into the queue at either Riverside or Dartmouth and the knockout generated, typically within 8-10 weeks (depending on demand). We will let you know when the knockout cassette is completed and publicly available and will notify you when the completed knockout strain(s) are deposited in the FGSC. We expect this option to be available by mid April ’05.
Option 2. If you wish to maintain anonymity, you may make your request through the FGSC, which will forward requests to us every week to two weeks. As soon as the strain is generated, it will be deposited in the FGSC from which it may be requested.
Option 3. Although great effort has gone into streamlining the procedures for knocking out genes, this is still not a trivial undertaking. However, many labs will have the expertise to successfully carry out knockouts on their own.
Option 3a. Order the cassette and make your own knockout. You may request up to 5 knockout cassettes at a time by email (knockouts@dartmouth.edu); please place the term CASSETTE REQUEST in the subject line. You assume obligations in making this request. We will send you a few micrograms of DNA for each cassette, sufficient for 1-2 transformations. You will need to obtain the “Neurospora Knockout Strain Kit” from the FGSC. This kit includes the strain that is the recipient for all Genome Project knockout transformations and additional strains to which all primary transformants are immediately crossed. You promise in writing to make the knockout in the standard genetic background, to cross the primary knockout transformants both to the wild type (either FGSC #2489 or FGSC #4200) and his-3 strain (either FGSC #6103 or FGSC # 9716) supplied in the kit, and to deposit the knockouts in the FGSC within 3 months. The listing of currently available cassettes can be viewed within Neurospora Genome Project - Proj#1. We should be able to deliver cassettes within 2-3 weeks of receipt of a request, beginning in late February 2005.
Option 3b. Using available informatics resources, you can do it all yourself. We have developed an algorithm for designing PCR primers and applied it to the current release of the genome to generate a series of primers that can be used for construction of cassettes for making knockouts. You can go to the Genome Project web site (http://www.dartmouth.edu/~neurosporagenome/) to obtain the sequences of the recommended primers to be used for the cassette constructions, order them from your favorite supplier, and make your own cassettes. Yeast strains used for the cassette assembly and Neurospora strains used as the transformation recipients are available in the “Neurospora Knockout Strain Kit” from the FGSC, and detailed protocols for all steps in the Knockout Procedure are available at the Genome Project Website. Unfortunately, due to limited time and personnel, we can’t help to troubleshoot problems that might arise as you make your own constructs.
IMPORTANT - This effort, designed to benefit the entire community, is funded through a Program Project grant from NIGMS. Due to the nature of the work, it does not generate publications at the same rate per dollar spent as a typical grant, but publications citing the effort are essential, as for any grant. To retain funding for the completion of the effort – that is, to be able to knock out all the genes - it is essential that these resources be appropriately cited for protocols, strains, and reagents. PLEASE CITE “Colot, H.V., Park, G., Ringelberg, C., Curilla, S., Crew, C., Borkovich, KA., and Dunlap, J. C. in preparation”; also, PLEASE LET US KNOW so your paper can be listed. We will update this citation as soon as possible.
Thanks and best wishes from the Knockout team.