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
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)
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
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.