Dear Ciliate Community, At the last Ciliate Molecular Biology Conference, I describe the use of recombination-based (Gateway) cloning as the means for rapid analysis of Tetrahymena genes. I am writing a proposal to NIH to further develop a comprehensive set of these vectors for reverse genetic studies. In the proposal, I hope to secure funds to have such DNA resources distributed by the Stock Center. I have already sent these types of vectors to several labs. If you support this initiative, I need your help by providing a letter of support to me. The proposal is due Sept. 25th, 2008. Sorry for the short notice, but if you can spare a few minutes to right a brief letter of support and sent it to me by Sept 22nd. I'd greatly appreciate it. The more evidence for community support, the more likely this proposal will be reviewed favorably. Even a short letter saying you would benefit from this resource will go along way. Thank you in advanced Doug Chalker PS. a draft the aims of the proposal are below. Title: A uniform toolbox for Tetrahymena functional genomics. (Submitted in Response to Funding Opportunity Announcement PA-07-457) Specific Aims: Aim 1) Generate a comprehensive set of expression vectors for reverse genetic and genomic approaches in Tetrahymena. Cloned DNA can be efficiently introduced into Tetrahymena using either autonomously replicating vectors or constructs targeted for integration into specific loci via homologous recombination. We proposed to create a uniform set of vectors for epitope tagging and other reverse genetic approaches that employ Gateway Cloning Technology. Gateway recombination-based cloning greatly facilitates DNA manipulations and enables high-throughput approaches. It has proven to be an effective strategy for genetic and genomic resources in widely-used model organisms such as C. elegans and Arabidopsis. The resources that we plan to create include plasmid DNAs (called the “destination” vectors) that contain tags for protein localization and interaction studies (e.g. Green Fluorescent Protein – GFP -- and split GFP), protein expression and immunoprecipitation studies (e.g. Hemoagglutanin –HA and c-myc epitopes), and proteomic approaches (tandem affinity purification tags). One of the major advantages of recombination-based cloning for reverse genetics approaches is that a Gateway compatible “entry” vector containing one’s ‘gene of interest’ can be rapidly recombined into any desired “destination” vector without the need for labor intensive re-cloning of the coding sequence. Employing this strategy for studies in Tetrahymena will promote the generation of a Tetrahymena “ORFeome” collection (a uniform set of entry containing cloned open reading frames-- ORFs) from the research community utilizing these resources, distributed throught the National Tetrahymena Stock Center. 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. Aim 2) Create Gateway recombination compatible cDNA libraries for high-throughput screens. Currently few, if any, Tetrahymena cDNA libraries are widely available to even identify specific clones for individual genes, let alone for use in phenotypic screens. We will generate libraries from RNA isolated from vegetatively growing cells and cells at various stages of development (e.g. meiosis, nuclear differentiation). These libraries will be cloned into vectors that contain the Gateway compatible attachment sites for easy recombination into destination vectors generated in Aim 1. The availability of cDNA libraries in a Gateway compatible format will allow these to be easily incorporated in genetic and cytological screens (see Aim 3). Aim 3) Develop strategies for high-throughput over-expression and RNA interference (RNAi) screens. The generation of cDNA libraries in Gateway compatible entry vectors (Aim 2) will provide a tremendous resource for the development of reverse genetic screens. When these are combined with the destination vectors designed for Aim 1, the research community will have necessary reagents to identify genes important for any investigator’s pathway or process of interest. In this aim, we will develop the methodologies necessary to routinely achieve high rates of transformation with these libraries that are necessary for maximal utility of this type of approach. The autonomously replicating vectors commonly used for protein expression in Tetrahymena contain the rRNA gene replication origin, which leads to high copy amplification upon transformation, thus making over-expression phenotypic screens feasible. Combining overexpression with GFP tagging allows additional cytological screening approaches to be employed. Furthermore, gene suppression by RNA interference (RNAi) has been achieved in Tetrahymena, but it has yet to be exploited in genetic screens. RNAi is ideal for genomic-level screens applied for gene discovery when combined with the cDNA libraries generated in Aim 2. We plan to generate Gateway compatible RNAi destination vectors containing convergent promoters flanking the recombination cassette gateway to enable such approaches. Dissemination of developed resources. All plasmid DNAs and libraries will be deposited at the National Tetrahymena Stock Center for easy access. This proposal therefore seeks to enhance the utility of the Stock Center by equipping it to distribute DNA resources as well as strains. To further promote the use of these approaches, yearly workshops will be offered to train researchers in the use of these tools and to train investigators new to Tetrahymena in the basic experimental techniques available for this important, but underused model organism.