1. Can I submit a maize gene to generate a fluorescent tagged line?
2. If I only have cDNA sequence how can I find genomic sequence for my gene?
3. Why can't you just fuse my cDNA to GFP and express using a strong promoter (e.g. 35S)?
4. Which fluorescent protein should I request?
5. What happens after I submit my request?
6. What if I don't have any microscopy experience?
7. What if I want to tag my gene right away, or don't want others to have access to the tagged lines?
8. What if my gene is expressed at a very low level?
9. What if a gene for my favorite compartment has already been tagged?
10. What are my chances of being selected?
11. Is it possible that my gene, if selected, will not be successfully tagged?
12. What if I don't hear from anyone?

Anyone can suggest a gene for tagging. However, at least 3kb of 5' and 1kb of 3' sequence, as well as the full coding region sequence (including introns) should be available, preferably from B73 (see our Protocol and Tian et al. 2004, to understand why). Priority will be given to genes with protein products that are predicted to localize to interesting sub-cellular compartments that are distinct from those that we are already generating lines for.

Use your cDNA sequence to perform a BLAST search of assembled maize genome survey sequences (e.g. the TIGR AZMs or ISU MAGIs assemblies or the sequenced maize BACs which are available through GenBank if you search the GenBank "htgs" database division). For many genes, the sequence will not yet be available, in which case you could try to isolate and sequence a genomic clone, or just wait a year or 2 until the genome is completely sequenced.

Over-expression often leads to localization artefacts, and using strong viral promoters may be more prone to gene silencing.

In our view, YFP is the easiest to image. CFP is less bright and requires a special laser (UV or similar) to image by confocal, so may be more difficult unless you are set up to do that. We do not have much experience with RFP so far, but it should work well. YFP-CFP are common "FRET" partners, if you are interested in protein-protein interactions, all three protein should work well for double or triple labeling.

For a nice summary of these proteins, see http://www.microscopyu.com/articles/livecellimaging/fpintro.html.

We will review requests periodically, and inform you when your gene is chosen for tagging. After that, it will take 1-3 months to make the construct, and 3-6 months to generate the maize plants. A further 3-6 months will be required to bulk up the seeds so we can send them out. Seeds of all lines will be freely available to all researchers through us or the Maize Genetics Stock Center. Before obtaining them, you will have to apply for an APHIS notification for shipping and growing the maize plants. Note that this process takes approximately one month.

We will do some imaging of all the lines we generate. We will also be offering imaging workshops, check back at this site for announcements.

We will happily share our protocols, advice and vectors with you. Many labs have successfully used the TTPCR tagging procedure and with care it is fairly easy to perform. The plant transformation facility at ISU can generate transgenic maize lines for a fee (http://agron-dev.agron.iastate.edu/ptf/index.aspx).

So far we have experience for one gene, abphyl1, which is expressed at a low level (no ESTs are available for this gene). We can easily detect this YFP fusion protein. Probably the expression pattern is important, if it is concentrated in a few cells during development will be easier than if it is expressed in all cells at a very low level.

We may still accept your suggestion, as our goal is to make markers that will be useful for all developmental stages. Therefore it will be useful to have more than one marker for each sub-cellular localization.

We aim to tag at least 100 genes, and there are probably about 50,000 genes in maize, so you can calculate the odds ............ but seriously, the earlier you submit your request the better, and the more information and justification you provide the better.

We have been successful with all genes attempted so far, but the answer is still yes. This is because our tagging procedure requires successful amplification of products including upstream and downstream regions. Depending on gene location, these regions may not be successfully amplified. However, we will inform you at several steps to keep you updated (see flow chart).

The project plans to consider gene requests as they arrive. If you don't hear from Dave or Anne within 2-3 weeks please feel free to email us.