agrikola logoAGRIKOLA protocols

We have used high-throughput recombinational cloning to clone gene-specific tags (GSTs) into our RNAi vector pAGRIKOLA, thus generating more than 20000 plasmids, each of which can be used to reduce or eliminate expression of a single Arabidopsis gene by post-transcriptional silencing. These plasmids are available to the European (and worldwide) Arabidopsis community via NASC since mid-2005. Seeds from several thousand transformations with these constructs are also available from NASC.

Reference:

Hilson P, Allemeersch J, Altmann T, Aubourg S, Avon A, Beynon J, Bhalerao RP, Bitton F, Caboche M, Cannoot B, Chardakov V, Cognet-Holliger C, Colot V, Crowe M, Darimont C, Durinck S, Eickhoff H, Falcon de Longevialle A, Farmer EE, Grant M, Kuiper MTR, Lehrach H, Léon C, Leyva A, Lundeberg J, Lurin C, Moreau Y, Nietfeld W, Paz-Ares J, Reymond P, Rouzé P, Sandberg G, Dolores Segura M, Serizet C, Tabrett A, Taconnat L, Thareau V, Van Hummelen P, Vercruysse S, Vuylsteke M, Weingartner M, Weisbeek P J, Wirta V, Wittink FRA, Zabeau M and Small I (2004) Versatile gene-specific sequence tags for Arabidopsis functional genomics: transcript profiling and reverse genetics applications, Genome Research 14, 2176-2189.

Below are links to protocols describing how to verify AGRIKOLA resources (plasmids and/or Arabidopsis RNAi lines).

Plasmid maps:

Validation Protocols:

Disclaimer: whilst every effort has been made to assure the quality of the resources and data produced within AGRIKOLA, as in any high-throughput project, some errors are likely to have crept in. We cannot guarantee that all plasmids are correct, that all Arabidopsis lines carry correctly expressed constructions or that the target gene is efficiently silenced. Please verify the resources before use using the protocols described above and inform us of any problems you encounter.

As a follow up to the AGRIKOLA project, the Department of Plant Systems Biology (VIB-UGent) and the Belgian resource center BCCM/LMBP were funded to purify and sequence validate a subset of the clones delivered by the consortium. LMBP is already distributing a few hundred of such accessions. For more information, visit: http://bccm.belspo.be/db/lmbp_gst_clones/

Furthermore, you can request purification and sequence validation for your clones of interest. If/when these are available, you will be notified that they may be ordered via LMBP. For clone sequence validation request, visit: http://www.psb.ugent.be/reva


For those of you interested in how the AGRIKOLA resources were constructed, we started from a set of 24500 Arabidopsis gene-specific tags (GSTs) designed for use in CATMA microarray studies. These GSTs, 150-600 bp in length, are ideal for targeted gene silencing in Arabidopsis if expressed from a suitable vector capable of producing double-stranded hairpin RNA (hpRNA). The basic two-step strategy for cloning GSTs into the hpRNA vector is depicted below.


cloning scheme


Figure 1: The hairpin RNA strategy

Project protocols: