Workplan
Introduction - general description and milestones
The main objective of our proposed research programme is the generation of a comprehensive collection of C. elegans strains carrying transposon-tagged genes. The use of heterologous transposable elements instead of resident Tc transposons allows for rapid and controllable genetic and molecular maneuvers, such as gene identification and cloning, and when necessary, efficient generation of knock out alleles. This collection will be an extremely valuable resource for the European and international scientific community, and will greatly facilitate functional genomics approaches to disease mechanisms, and gene function, in an organism with exceptional experimental advantages. To achieve the main objective we plan to:
· Optimize and automate technology based on the Mos1 transposable element.
· Develop alternative tools for mutagenesis and transgenesis based on the Minos transposon.
· Develop and deploy platform technologies and infrastructure required to achieve our main objective and to manage and maintain a large resource.
· Test/evaluate our final product and tools in case studies involving forward genetic screens.
The Workplan is broken down to activities that partially or totally depend on each other, and is further dissected into workpackages, which correspond to major subdivisions of the programme. The structure of the project reflects the complementarity of the approaches and of the expertise of the individual participants. Hence, each participant contributes to several workpackages. The tight integration of individual contributions and activities is of fundamental importance to the successful completion of the project (see the NemaGENETAG Project Organization Diagram). Our activities are categorized into 2 major types:
· Research activities, which are distributed into 4 workpackages as follows:
o Optimization/automation of Mos1 transposon-based technologies
o Development of alternative systems for mutagenesis and transgenesis in C. elegans based on the Minos transposon
o Generation of a comprehensive, ordered library of tagged nematode genes
o Case-studies/evaluation of the resource
· Technological development, innovation and demonstration related activities, which shape workpackage 5:
o Platform technology development/deployment
Below we outline these activities, explain the structure of the plan and present the overall methodology used to achieve the objectives. We also identify significant risks, propose contingency measures.
A. Research activities
1. Optimization/automation of Mos1 transposon-based technologies (WP1)
The goal of WP1 is to validate the utility of the Mos1 system at the whole genome scale and to generate new tools to engineer the C. elegans genome. It breaks into three major points:
· Evaluation of a pilot collection of Mos1 insertions that is being generated by the Segalat group. Localization and statistical analysis of insertion distribution will be performed by the Segalat group. A subset of insertions will be selected and analyzed for phenotypic alterations between the Segalat, Bessereau and Ewbank groups.
· Development of a strategy for efficient recovery of gene deletions by Mos1 excision.
· Development of a strategy for engineering mutations by transgene-instructed double stranded DNA break repair following Mos1 excision.
The last two points will be developed in the Bessereau group.
Workplan
1. Analysis of the distribution of 1,000 Mos1 insertions. (Months 0-2)
2. Selection of a subset of strains containing inserts in predicted exons and phenotypic analysis. Comparison with RNAi data and known mutant phenotypes (Months 2-6)
3. Generation of extrachromosomal transgenes co-expressing the Mos1 transposase and the Red Fluorescent Protein. Generation of low copy number integrated transgenes by bombardment. Comparative tests of transposition efficiency with the different transgenes. (Months 0-8)
4. Identify a phenotypically silent Mos1 insertion in a gene, which is known to cause an easy phenotype to score after mutation. Reintroduce heat-shock and glh-2 Mos1 transposase expression constructs and score the frequency of imprecise excision events. Put the insertion over a balancer chromosome and score excision frequency. RNAi components of DNA repair machinery and score excision frequencies (Months 2-18).
5. Generate imprecise excision of 5 additional Mos1 insertions using optimised excision conditions (Months 18-24)
6. Generate transgenic arrays to be used as templates for repair of DSB in unc‑5(ox171::Mos1) and unc-22(kr5::Mos1) strains. (Month 4-12).
7. Use optimized Mos1 re-excision conditions to demonstrate repair from a transgene. Determine the minimum homology sequence for recombination. Evaluate the amount of exogenous sequence that can be introduced at and proximal to the Mos1 excision site (Month 12-36)
2. Development of alternative systems for mutagenesis and transgenesis in C. elegans based on the Minos transposon (WP2)
The objective of this workpackage is to develop alternative systems to the Mos1 system, which is presently the only fully-controllable transposable element which can be used in C. elegans to construct libraries of tagged genes. Minos has been chosen because 1) Minos has a large spectrum of hosts and 2) Minos can support extensive modification of their internal sequences without hampering the ability to transpose. Efficient transposition based on these systems would not only facilitate the construction of the insertion library (WP3), but it would also benefit to the C. elegans community as new tools and pave the way for a much needed enhancer trap system.
Methodology:
We will follow the strategy successfully taken by JL Bessereau (participant #2) to adapt Mos1 transposition to C. elegans (Nature, Vol. 413, p. 70). This consists in designing a binary system, in which the two functional elements of the transposon (namely the transposase enzyme and the template) are separated and transformed independently in transgenic strains. The strains are later crossed together to bring the two elements together and allow transposition.
Role of the participants:
Participant #1 (N. Tavernarakis) will carry out the experiments in parallel and have weekly exchanges on data obtained. Inputs from participant #2 (JL Bessereau), who has extensive experience on the subject and will serve as internal reviewer for this workpackage will be incorporated regularly. In addition, N. Tavernarakis will benefit from adjacent presence of Prof. Charalambos Savakis who pioneered the Minos work and used this element to achieve transformation of the medfly Ceratitis capitata for the first time.
Description of operations:
Construction of vectors. Vectors in which the open reading frame of each transposase will be placed downstream of a heat-inducible promoter will be constructed by standard molecular biology techniques. In addition, we will double these constructs with non-inducible germ-line specific promoters to maximize the chances of success. In addition, wild-type transposon sequences, to serve as templates, will be introduced in vectors suitable to transformation.
Generation of transgenic strains. Transgenic strains will be obtained by the standard transformation technique of micro-injection. Alternatively, bombardment with gold particles can be used to favor germ-line expression.
Detection of transposition events. After appropriate crosses are made between the strains, transposition will be detected by PCR. The progeny of positive animals will be retested to eliminate possible amplification artifacts. Moreover, transposition will be confirmed by Southern analysis, and the location of the insertion will be determined by inverse PCR, a technique both laboratories master.
Upgrades of the systems. When a reliable transposition system is proven effective, we will add features in the template to 1) insert a GFP marker to make the transposon easier to follow and 2) pieces of genomic DNA increasing the mutagenic properties of the transposons. Corresponding transgenic strains will be generated and tested similarly to the first instance.
Timing:
We will construct the vectors for transposition and generate the first transgenic strains during year 1. Within year 2, we will test transposition and--if necessary--generate additional transgenic strains. We will also make effort to improve the systems depending on the results obtained during the development period.
Potential difficulties and contingency plans:
Difficulties to express transposase in the germ-line of the transgenic animals. Our strategy necessitates expression of transposase in the germ-line of transgenic animals. Expression of transgenes in the C. elegans germ-line has long been a problem with this organism because transgenes are repressed in this tissue by mechanisms, which are not completely understood. If no satisfactory expression can be obtained by conventional transgenesis methods (micro-injection), we will use biolistic techniques to generate transgenic animals. The Segalat laboratory has experience with such protocols.
Absence of transposition. Even if transposase is successfully expressed in the germ-line, transposition might not be obtained initially. We will then multiply the number of transgenic strains because strains variations are often encountered in such experiments. We will also try to generate strains carrying more template copies to facilitate transposition. However, it must be noted here that transposition of Minos in C. elegans is not guaranteed beforehand. Although analysis of the literature shows that the probability that Minos will transpose in C. elegans is high, there is no experimental proof of this at this time.
Low level of transposition. Transposition may be detected experimentally but at levels insufficient to use it as tools for large-scale use. In such a case, we will generate additional strains carrying more copies of the transgenes in a first attempt to boost transformation. We will also cross the transgenes in genetic backgrounds known to favor transposition.
3. Generation of a comprehensive, ordered library of tagged nematode genes (WP3)
The objective of this workpackage is to generate an ordered collection of C. elegans strains carrying transposon-tagged genes. The aim is to achieve genome-wide coverage through the delivery of at least 17,000 tagged genes (85% of the nematode gene complement).
Tools: During the first phase of the workpackage (estimated between 6 and 18 months), we will take advantage of the Mos1 system which has been developed by one of the participants and is currently the best available tool to conduct this project. During the second phase of the workpackage, we will progressively implement upgraded and alternative tools resulting from the work conducted in workpackages 1 and 2. Depending on the efficiency of these new tools, we will then totally and partially replace the current Mos1 system by these new tools.
General scheme of insertion production. For each cycle of mutagenesis, one transgenic strain carrying a transposon template will be crossed with a second transgenic strain carrying controllable source of transposase (Bessereau et al., Nature, Vol. 413, p. 70). Upon the induction of transposase expression animals carrying both transgenes will undergo a burst of transposition, which will result in transposon insertions through the genome in a non-directed manner. The subsequent loss of the transposase-containing transgene stabilises the transposon insertion. Strains in which a transposition event has been detected are amplified and the site of transposon insertion is determined by a series of molecular Biology steps. Insertions are stabilised by keeping descendants not carrying the source of transposase. The mutated strains we be frozen in triplicate for long –term storage. We aim to automate the procedure as much as possible and plan to institute barcode-based identification of strains to facilitate strain tracking and to minimise the risk of handling errors.
Role of individual participants. Participant #3 (J. Ewbank) will carry out the upstream steps (1-4) and participant #6 (L. Segalat) will carry out the downstream steps (5-9; see below).
Detailed description of operations. (Descriptions are made on the basis of existing tools (Mos1) that will serve during the initial phase of the project. Some details of the protocol may vary when other tools are implemented).
1. Generation of strains carrying both the template and the source of transposase. Crosses will be made between transgenic strains carrying the template with strains carrying the source of the transposase. Both types of strains are marked with fluorescent markers. The sorting of animals of the desired genotype will be done with the worm sorter. Such crosses must be redone every 2 months because strains become non-competent for transposition with time.
2. Induction of transposition. Double transgenic animals will be exposed to a heat shock to induce Mos1 transposase expression. This step is not time-consuming and will be done manually by putting animals in an incubator for two hours.
3. Sorting of fluorescent animals. Animals carrying neither the source of transposase nor the template are singled for subsequent tests. This will be done by the worm sorter.
4. Initial PCR test to detect transposition. Until we develop transposons carrying internal GFP markers (which is part of Workpackages 1 and 2), effective transposition in animals submitted to the Heat-Shock treatment can only be verified by the PCR detection of Mos1 sequences in their genome. This is done by a single PCR reaction performed on isolated whole worms. This will be done on adult worms after their progeny is secured. This will be done on 96-well plates by a robot. PCR-products will be loaded on agarose gels. Positive samples will be amplified for determination of the insertion sites (steps 5-8) and freezing (step 9). This step will be removed when transposons tagged with a readily distinguishable fluoresecent marker are available, or if optimization of the protocols lead to 100% of animals positive for transposition.
5. Inverse PCR to amplify transposon flanking sequences. Inverse PCR is a molecular biology technique aimed at amplifying a DNA fragment by PCR when only one side of the fragment is known. It is based on random digestion of the DNA, intramolecular ligation, and amplification. These steps will be carried out by a robot available at the CGMC.
6. Sequencing information. PCR products are then sequenced. Although the CGMC has extensive sequencing capacities, sequencing of the PCR fragment may be subcontracted to a service company. No final decision has been made yet on this point.
7. Determination of the insertion site by sequence comparison. Determination of the insertion point of insertions is done by comparing the sequence generated in the previous step with that of the C. elegans genome by a Blast program. Software development is currently being done to perform this task automatically.
8. Database update. Data are compiled in a database that primarily links each strain number to an insertion site. As for step 7, the automatic update of the database is already being worked out, so as this step is fully operational when the project begins.
9. Freezing and testing. Freezing of each insertion positive strain will be done in triplicate. C. elegans strains are typically maintained frozen in -80°C freezers or in liquid nitrogen. 1 copy will be frozen at -80°C and the remaining two will be frozen in liquid nitrogen. The third copy will be stored at a location different from CGMC as a backup. The Caenorhabditis Genetic Center of Minneapolis (USA), which is already the main repository for C. elegans strains, is being considered for storage of the third copy of the collection. Alternatively, other locations in Europe might also been considered. No final decision has been made regarding this point.
Timetable. We expect that 4 people working full-time on production of the insertions (1 in Marseille, 1 in Cambridge, and 2 in Lyon) will be able to produce at least 6000 insertions during year 1, 12000 during year 2, and 24000 during year 3. These are conservative numbers based 1) on our experience with pilot experiments currently being run by participant #6, 2) on estimated gains of productivity realized as protocols are optimized and streamlined as the result of workpackage 1.
Significant risks and contingency plans.
· Strong bias of insertions. A difficulty with approaches such as this one may reside in a bias in the distribution of insertions, due to the preferential insertion of transposons in some genome regions. This point is one of the questions addressed in the pilot experiment run by participant #6. Preliminary data obtained on 800 insertions do not reveal any obvious bias at the level of chromosome distribution nor between chromosome regions. Further analysis carried out as part of WP1 will address this question more thoroughly. In any case, the use of alternative systems, to be developed in WP2 should level potential biases that may exist with each transposon.
· Failure to generate a high level of transposition. The generation of insertions described in this workpackage requires a high frequency of insertions, which is conditioned by the ability of transgenes to trigger efficient transposition. Extinction of transposition in the transgenic strains after several generations has been observed by us and others. However, this can be circumvented by keeping strains at 25°C or by thawing new isolates regularly. The participants of this project have now gained sufficient experience to manage this problem efficiently.
· Difficulties with implementing automation. The proposed work includes a progressive scale-up of operations that will double the number of insertions produced every year. This scale-up will be made possible by increased experience and also by optimization and automation of the operations. Although such gains have been estimated on a conservative basis, they cannot be taken for granted. However, we are confident in our ability to manage these developments.
· Temporary bottleneck in the sequence of operations. We cannot exclude that insertion production may be temporarily slowed by some unexpected event, such as breakdown of a robot, or a similar event. In such a case, efforts will be made to reabsorb this bottleneck, possibly by reallocating resources temporarily.
4. Case-studies/evaluation of the resource (WP4)
The objective of this workpackage is to evaluate, and to provide added value to, the bank of C. elegans transposon-tagged genes generated in workpackage 3 using:
Forward genetic screens.
Two main forward genetic screens will be undertaken:
Description
We anticipate that our activities and their final product, an ordered, comprehensive resource of transposon-tagged genes will be extremely valuable for both forward and reverse genetic approaches aiming at deciphering gene structure and function, and understanding the molecular mechanisms of disease. To obtain proof-of-principle and to evaluate the usefulness of our resource and tools we will perform representative tasks, typically expected to be undertaken by end-users. Specifically, we will conduct two types of genetic screenings described briefly below.
Nicotinic neurotransmission. Participant 2 will conduct two types of pharmacological screens that will be used for phenotypic characterization of the mutant collections. First, the nicotinic agonist DMPP causes larval development arrest. In a preliminary screen, six genes have been identified that confer resistance to the drug after being mutated. This screen is a selection screen that can be performed in a 96-well plaque format and is well suited to the characterization of large numbers of strains. Second, a screen will be conducted to identify mutants that fail to adapt to the nicotinic drug levamisole. These two screens will identify genes involved in function and plasticity of nicotinic synapses. Such genes represent candidate drug targets, especially for the treatment of nicotine addiction.
Necrotic cell death and neurodegeneration. Participant 1 has developed and extensively characterized genetic models of neurodegeneration in the nematode C. elegans. The establishment of such models provides the unique opportunity to utilize a genetically tractable organism such as C. elegans to genetically and molecularly dissect the process of necrotic cell death. Transposon insertion libraries will be screened for mutant nematode strains with either increased or decreased resistance to neurodegeneration. Suppressor strains will be phenotypically analyzed and microscopically examined for effects on neuronal cell death. Standard genetic and molecular methodologies will be used to characterize and map the suppressor mutations. The corresponding genes will be cloned, their temporal and spatial expression profile will be analyzed and their involvement in the process of neurodegeneration will be determined by genetic epistasis and overexpression studies. This approach should yield a wealth of information on the molecular events that ensue during necrosis. Upon completion of this work, we should be able to describe in molecular detail how degeneration is initiated and what proteins are needed to enact the death. Given that apoptotic cell death mechanisms are conserved between nematodes and humans (Hengartner M. O., Nature, Vol. 407, pp. 770-6.), our analysis of C. elegans degenerative, necrotic cell death mechanisms should lead to insights into causes of, and potential therapeutic intervention with, human neurodegenerative diseases.
B. Technological development, innovation and demonstration related activities
5. Platform technology development/deployment (WP5)
Workpackage 5 incorporates the development of platform technologies required for the timely and successful completion of the project. Our participant, MAIA Scientific will decisively contribute to technology development and support of research activities. Today, MAIA Scientific COPAS Biosort technology is well embedded in the C. elegans research community. Two contributing teams are currently using it and will apply it within context of this project. MAIA Scientific will continue actively to the smooth introduction of new developments on COPAS. The aim is to introduce a new ultra sensitive automated microscopic imaging device (MIAS-2) into the consortium and to apply it to the advantage of its core objective: genome-wide application of current and novel transposon technologies. The MIAS-2 reader will be ready by the fourth quarter of 2003 and available for acquisition of images starting day 1 of the project. In addition to providing automated image acquisition and analysis, it is expected that the very high sensitivity in low light conditions of the MIAS-2 microscopic reader will increase the success rate of the project in several ways e.g.: (1) investigate very low expression levels of reporter proteins in the nematode body (way below the sensitivity of the eye) and (2) the reagent cost of micro array experiments is likely to be decreased through low-light image capture.
The first objective will be to develop a panel of dedicated bright field and (low-light) fluorescent image-analysis applications for relevant mutant phenotypes that will help substitute for hands-on visual inspection of transposon-induced mutant collections. Starting from image collections from MIAS-2 or from other microscopes (in standard image file formats), proof of principles (POP) will be generated that 15 relevant phenotypic features can be quantified in images using scripts of the eaZYX imaging software platform. Once a POP software script is approved for full development, the script will be refined to differentiate ‘baseline’ from ‘increased or decreased’, to identify outliers and to derive alphanumerical descriptors that are exportable to spreadsheet, biostatistics or genomic databases. For the investigator, visual auditing and proofing will be built in the application that facilitate to browse through the results sing on image projection of the relevant quantifier. This part of the work is considered RTD. All developed scripts will then be embedded into platform software (eaZYX-C. elegans) on a computer, dedicated to nematode analysis. Successful completion of a number of image analysis applications that will further the consortium’s main objective is a major milestone for WP5. Subsequently, the platform software will be tested on its robustness and performance with the following case studies generated by the other participants. This part of the work is considered demonstration activity.
A second objective is robot integration of COPAS flow sorting and MIAS-2 image capture and analysis. To achieve this goal, MAIA Scientific will design a new automation concept by which the two instruments (slaves) are being integrated onto a single robot station (master) implement its new technology as specified in work package 5. This work is considered RTD. Once a concept has been developed, components of the concept will be demonstrated in a prototype [COPAS-Biosort + robot + MIAS-2] test assembly. The building and the testing of the concept are considered as demonstration activity. The overall allocation of resources is 80% RTD and 20% demonstration activities.
Contingency plans for WP5: The MIAS-2 reader development will be completed before the start of the project. Our experience with image analysis application developments is that >90% of the desired features can be extracted from noise-intensive image collections acquired in low light conditions. By starting with 12 proofs of principle and assuming 25% attrition, we expect to develop 9 applications for the consortium. The challenge for MAIA Scientific will be to build a new concept for integration. The fallback strategy is that COPAS Biosort and MIAS-2 readers are today equipped with functional robotics for 80 and 300 plates respectively. This will allow the user to achieve est. 50% of the automation possibilities.
