GREAT AT SMALL THINGS

Plasmid resources for Arabidopsis reverse genetics via RNA interference

Context

In recent years, RNA interference (RNAi) has become a method of choice for reverse genetics analyses in animals and plants. Therefore, in the framework of the European Agrikola programme, we have created a genome-scale library of sequences and constructs for the RNAi post-transcriptional silencing of most genes in the model plant Arabidopsis thaliana. It is based on the manipulation of genomic DNA fragments via the Gateway recombinational cloning system. First, gene-specific sequence tags (GSTs) of 150 to 500 base pairs were introduced in entry clones via an in vitro BP clonase reaction. Then, the GSTs were transferred via an in vitro double LR clonase reaction in vectors designed for the expression of hairpin RNAs in transgenic plant cells. The Agrikola consortium has already generated over 23,000 silencing constructs that each mediates the knockdown of a different transcript. The preliminary survey of the range and frequency of phenotypes observed in Arabidopsis lines transformed with such constructs indicate that this resource is highly valuable (Hilson et al., 2004, Genome Research 14, 2176).

 

Project description

Objectives

Unfortunately, the structure and funding of the on-going Agrikola programme does not permit the comprehensive validation of the constructs it generates: their identity is not confirmed by sequence data and the bacterial strains that carry them are not clonal. This collaborative project between the Functional Genomics Division of the Department of Systems Biology (Ghent University) and the BCCM/LMBP Plasmid collection hosted in the Department of Biomedical Molecular Biology (Ghent University) specifically addresses these shortcomings. Our goals are to enhance the quality of the Agrikola resources, to ensure the durability of plasmids and bacterial strains it produced and to distribute them to the research community at large, under the strictest standards adopted by biological resource centers. In the framework of this project, we will authenticate a plasmid repertoire designed for the RNAi silencing of 2,500 Arabidopsis genes.

Methodology

The work at hand can be summarily described in four successive steps. (1) Entry clones that should carry the gene-specific sequence tags corresponding to the selected genes will be purified from the Agrikola mixed bacterial stocks. (2) Their identity will be confirmed by sequence validation and alternative clones will be recurrently isolated until correct ones are authenticated. (3) Following the transfer of the validated GSTs in a silencing hairpin RNA expression vector (a step not included in this project), the integrity of the silencing constructs will be confirmed by plasmid restriction digestion profiling. (4) All authenticated plasmids and the corresponding Escherichia coli strains will be deposited in the public LMBP collection according to its standard operating protocols, together with the related data that will be curated within the BCCM bioinformatics network. Thereby, the long-term preservation of the resources is guaranteed, as well as their distribution to third parties.

Interaction between partners

The work at hand is performed as a close and continuous collaboration between PSB and LMBP that is facilitated by the presence of the two groups involved in the same facility.

Expected deliverables

Biological materials

  • 2,500 pure Gateway entry clones (plasmid DNA and E. coli strain), each authenticated by sequence analysis of the insert
     
  • 2,500 pure hairpin RNA expression clones (plasmid DNA and E. coli strain), whose integrity is confirmed by restriction digestion profile.

All plasmids and strains will be permanently stored in duplicate at the LMBP facility, and will be publicly distributed by the LMBP staff.

Curated plasmid information

All relevant information relative to the authenticated plasmids will be introduced in the BCCM/LMBP database and published on line in a well-structured format. Furthermore, the BCCM/LMBP database web interface will include query tools to search for specific constructs according to gene names and functional annotations.

The high-quality plasmid collection delivered by this project will be a very substantial addition to the holdings of the LMBP. It will for the first time bring plant-related biological materials in BCCM. It is sure to attract international attention and to promote Arabidopsis research worldwide.

 

Partners

Partner 1: VIB, Gent University

Prof. Dr Dirk Inzé and Dr Pierre Hilson
Functional Genomics Division
Department of Plant Systems Biology
VIB - Gent University
Technologiepark 927
B-9052 Gent, Belgium
Tel: + 32 (0)9 33 13 830
Fax: + 32 (0)9 33 13 809
E-mail: pihil@psb.ugent.be
URL1: http://www.psb.ugent.be
URL2: http://www.agrikola.org

 

Partner 2: BCCM/LMBP, Gent University

Prof. Dr Roland Contreras and Lic. Martine Vanhoucke
BCCM/LMBP Plasmid collection
Department of Biomedical Molecular Biology
Ghent University
Technologiepark 927
B-9052 Gent-Zwijnaarde
Belgium
Tel1: +32 (0)9 33 13 843
Tel2: +32 (0)9 33 13 600
Fax: +32 9 33 13 504
E-mail: bccm.lmbp@irc.UGent.be
URL1: http://www.irc.UGent.be/lmbp
URL2: http://bccm.belspo.be/about-us/bccm-lmbp

 

Duration

1/11/2005 – 31/03/2008

 

User committee

Member 1

Dr Robert Ackerson, Devgen

Member 2

Prof. Geert Angenon, VUB

Member 3

Dr Henri Batoko, UCL

Member 4

Prof. Bruno Cammue, KUL

Member 5

Dr Sean May, Nottingham Arabidopsis Stock Center

Member 6

Dr Michael Metzlaff, Bayer CropScience

Member 7

Prof. Claire Périlleux, ULg

Member 8

Prof. Dr Harry Van Onckelen, UA

Member 9

Prof. Nathalie Verbruggen, ULB