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CILIATEMOLBIO-L  August 2010

CILIATEMOLBIO-L August 2010

Subject:

Re: Development of Tetrahymena Genetics/Genomics Resources

From:

"B. Franz Lang" <[log in to unmask]>

Date:

Mon, 23 Aug 2010 15:22:57 -0400

Content-Type:

text/plain

Parts/Attachments:

Parts/Attachments

text/plain (297 lines)

Hi Doug

I note that all listed tools are experimental, none
bioinformatics. A reviewer might easily point out
that for instance genomics without a substantial bioinformatics
component is unlikely to be effective

Cheers Franz


Doug Chalker wrote:
> *Tetrahymena Molecular Genetics Resource Survey (From Douglas Chalker)*
> Dear colleagues,
> In an effort to further develop research tools to facilitate molecular
> genetics and genomics approaches in Tetrahymena, I previously
> submitted a resource development proposal to NIH. While the proposal
> received overall good reviews, one major criticism was that the need
> for these tools was not well documented and that the many letters of
> support supplied did not specifically mention how these tools would
> aid individual research programs. I am planning to resubmit this
> proposal, and would like your help to prioritize the effort to develop
> the resources that would be most useful to the Tetrahymena research
> community. In the short survey (word file attached and pasted below),
> please indicate your likeliness of using each of the proposed tools or
> technologies in your research. If you would like more information or
> would like to supply a letter of support, please let me know.
>
>
> ------------------------------------------------------------------------
>
>
> Please return by Sept 2, 2010 if possible.
> Thanks for your time.
> Doug
>
> Doug Chalker, Ph. D.
> Associate Professor
> Dept. of Biology -- campus box 1137 Monsanto Lab room 304 (office)
> Psychology bdlg, room 452 (lab)
> Washington University
> One Brookings Drive
> St. Louis, MO 63130
> office 314-935-8838; fax 314-935-4432
> lab 314-935-7143
> [log in to unmask] <mailto:[log in to unmask]>
> http://www.biology.wustl.edu/faculty/chalker/main.php
>
> Survey:
> For each proposed tool, indicate whether you are already developing or
> using such a tool (Curr.), would use it immediately if available
> (Immed.), would use it within five years if available (long term), or
> would never use it (never). Mark your selections by inserting “X” in
> the appropriate boxes. Additionally, please indicate the THREE tools
> or technologies that are of the highest priority to your research.
> Finally, if you wish to do so, please provide any comments about how
> you might use such a tool or technology in the space below according
> to the number in the table. This information will be extremely helpful
> in the grant application.
> If possible, please return by September 2, 2010.
> Thank you very much for your assistance in this endeavor! Doug
>
> Tool or Technology
>
>
>
> Aim
>
>
>
> Curr.
>
>
>
> Immed.
>
>
>
> Long Term
>
>
>
> Never
>
>
>
> Top 3
>
> 1. Vectors for adding fluorescent tags (GFP, CFP, YFP, mCherry) to
> genes of interest.
>
>
> 1
>
>
>
>
>
>
> 2. Vectors for adding eptiope tags (Myc, HA) to genes of interest.
>
>
> 1
>
>
>
>
>
>
> 3. Vectors adding protein complex purification tags (tandem tags) to
> genes of interest.
>
>
> 1
>
>
>
>
>
>
> 4. In vivo protein/protein interaction detection by split-GFP.
>
>
> 1
>
>
>
>
>
>
> 5. Inducible promoters (Heat shock, Tet-on)
>
>
> 2
>
>
>
>
>
>
> 6. Repressible promoters
>
>
> N/A
>
>
>
>
>
>
> 7. cDNA libraries from developmental timepoints
>
>
> 3
>
>
>
>
>
>
> 8. Techniques for high-throughput gene overexpression screens
>
>
> 4
>
>
>
>
>
>
> 9. Techniques for high-throughput gene silencing screens
>
>
> 4
>
>
>
>
>
>
> 10. Techniques for high-throughput cytological screens (GFP tagging of
> cDNA libraries)
>
>
> 4
>
>
>
>
>
>
>
>
>
>
>
>
>
>
>
> Comments (numbered as above):
> 1.
> 2.
> 3.
> 4.
> 5.
> 6.
> 7.
> 8.
> 9.
> 10.
>
> *Research Plan: A uniform toolbox for Tetrahymena functional genomics.*
>
> *Specific Aims: *The ciliate /Tetrahymena/ /thermophila/ has been the
> vehicle for serveral important discoveries (e.g ribozymes, telomerase,
> chromatin regulators, etc). Given its many experimental attributes, it
> has great potential to become a leading model organism. Currently, no
> organisms from the protozoan lineage of eukaryotes can be considered
> major model systems, which is striking as they generally have
> extensive genetic and biochemical diversity. This proposal aims to
> generate key resources for functional genomics studies in Tetrahymena,
> thus enabling the science community to take full advantage of its
> ~27,000 genes
>
> *Aim 1) Generate a comprehensive set of expression vectors for reverse
> genetics and genomics.* We propose to create a uniform set of vectors
> for epitope tagging that employ Gateway cloning technology. Gateway
> recombination-based cloning greatly facilitates DNA manipulations and
> enables high-throughput approaches. We will generate both high- and
> low-copy vectors (called “destination” vectors) designed to tag
> proteins on either their amino(N)- or carboxy(C)- termini. The tags
> will include fluorescent proteins to facilitate in vivo localization
> studies, short epitope tags useful for protein expression and
> immunoprecipitation assays, and tandem affinity purification tags that
> enable proteomic approaches. By using a uniform recombination cloning
> platform, one’s genes of interest, once cloned in a Gateway compatible
> “entry’ vector can be rapidly assembled into any or all of the
> “destination” vectors with minimal effort. In addition, the wide
> implementation of this strategy will promote the creation of the
> Tetrahymena “ORFeome” collection (a uniform set of entry vectors
> containing cloned _o_pen _r_eading _f_rames-- ORFs) by the research
> community.
>
> *Aim 2) Develop promoters for regulated expression of transgenes.* The
> /Metallothionine/ /1/ (/MTT1/) promoter provides for inducible
> transgene gene expression. To enable greater experimental flexibility,
> we will develop additional promoters for our vectors. We will test the
> utility of the Tetrahymena /HSP70/ heat shock protein promoters as
> effective alternatives. In addition, we will adapt the
> doxycycline-inducible Tetracycline repressor (Tet-On) for regulated
> expression in Tetrahymena. **
> *Aim 3) Create Gateway compatible cDNA libraries for high-throughput
> screens*. Currently few Tetrahymena cDNA libraries are widely
> available. We will generate libraries from RNA isolated from cells at
> various life-cycle stages (e.g. growth, meiosis, nuclear
> differentiation, etc). These libraries will be cloned into vectors
> that contain the Gateway compatible attachment (att) sites that direct
> recombination into the destination vectors generated in Aim 1. The
> availability of cDNA libraries in a Gateway compatible format will
> allow these resources to be easily incorporated in genetic and
> cytological screens (see Aim 4).
> *Aim 4) Develop strategies for high-throughput over-expression and RNA
> interference (RNAi) screens.* By recombining the Gateway compatible
> cDNA libraries with destination vectors similar to those designed for
> Aim 1, the research community will have valuable reagents with which
> to devise screens capable of identifying principal genes acting in a
> pathway or process of interest. We will develop the methodologies
> necessary to routinely achieve high rates of transformation with such
> libraries, which is necessary for maximal utility of these approaches.
> These strategies will require the creation of additional destination
> vectors. These vectors will permit over-expression, phenotypic (e.g.
> suppression) screens. We will augment such over-expression strategies
> with methodologies to perform cytological screens using fluorescent
> protein tags. In addition, we plan to develop gene knock-down screens
> that will further diversify the repertoire of possible screens. Gene
> suppression by RNA interference (RNAi) has been achieved in
> Tetrahymena, but it has not been exploited for genetic screens. We
> will generate a Gateway compatible RNAi vector that can incorporate
> library cDNA clones between convergent promoters.
> *Resource Sharing Plan.* All plasmid DNAs and libraries will be
> deposited at the National Tetrahymena Stock Center for easy access.
> This proposal therefore seeks to enhance the capability of the Stock
> Center by equipping it to distribute DNA resources in addition to its
> current mission of housing strains. After the duration of the
> requested funding, the center should be self-sustaining requiring only
> a nominal fee for shipping/handling to order plasmids, etc. To further
> promote the use of these approaches, yearly workshops will be offered
> to train researchers in the use of the ‘toolbox’ and to introduce
> investigators unfamiliar with Tetrahymena to the basic experimental
> techniques available for this important, but underused model organism.
> We believe these resources will promote important studies in this
> emerging model eukaryote and catalyze the generation of additional
> genomic resources by the research community.
>
>
>
>

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