AGRIKOLA: Systematic RNAi knockouts in Arabidopsis
Description
The AGRIKOLA project (Arabidopsis Genomic RNAi Knock-down Line Analysis) is funded by the European Union as part of the 5th Framework Programme, and started officially on the 1st November 2002. Six European labs are using high-throughput recombinational cloning to clone gene-specific tags (GSTs) into binary hairpin RNA vectors (see below for cloning strategy)
A portion of the resulting plasmids are being used to create a collection of silenced Arabidopsis lines . The GSTs used in this project were originally developed by the CATMA consortium, comprising plant genomics laboratories in eight European countries, for use in microarray studies.
To order amplicons, plates, entry-clones, delivery-clones, and seeds.
Please check your clones when you receive them - you will be reminded of this within the Compliments slip that you will receive with the stocks.
Search the line name field in the NASC catalogue for CATMA id e.g. CATMA2a16320 (this will display the entry clone, delivery clone, and seeds associated with the CATMA id). To find the plates search for the plate number in the line name field of the NASC catalogue - for example 96103. This should list the CATMA plate, AGRIKOLA entry clone plate, and the AGRIKOLA delivery clone plate. If you are not sure what the plate number is you can find it by doing a search on the CATMA id in the same line name field. It will be included in the descriptive text.
To order the amplicons please go to the CATMA pages.
Delivery clones are now available. A search using a CATMA id in the line name field of the NASC catalogue should list the delivery clone, entry clone and associated seeds for that CATMA id.
Please note that delivery plate and clone N numbers do not correspond in the same way as the entry clone numbers described below.
The pSOUP vector is now included with all delivery clone orders. The pGreen plasmid and pAGRIKOLA can replicate in Agrobacterium only if pSoup, which provides replication factors in trans for pGreen and pAGRIKOLA, is co-resident in the same strain. The plasmid pSoup carries a gene that confers resistance to tetracycline in bacteria. (10 mg/l tetracycline) . pSoup is a modified RK2 plasmid which carried the pSa replicase gene, the pAGRIKOLA vector has the pSa replication origin.
The six Agrikola consortium partners are:-
- Centro Nacional de Biotecnologia
- Imperial College at Wye - UK
- Institut fuer Biochemie und Biologie, -Genetik- c/o Max-Planck-Institut fuer molekulare Pflanzenphysiologie - Germany
- Unite de Recherche en Genomique Vegetale (URGV) - France
- VIB - Ghent University - Belgium
- Warwick HRI - UK
AGRIKOLA entry-clone numbering explanation
The 256 AGRIKOLA plate numbers correspond to the equivalent CATMA plates that they were derived (cloned) from. For example: AGRIKOLA plate 96101 (N250101*) corresponds to CATMA plate 96101(N160101*).
Although AGRIKOLA plates (clones) have the same identifying plate numbers as the CATMA plates (amplicons) they are not the same materials and therefore have different NASC numbers (see above*). For ease of ordering, the last 3 digits of the NASC number for an AGRIKOLA plate AND for the corresponding CATMA plate are the same (see above*).
Each of the individual 96 wells/DNA on each plate has also been allocated a NASC number and now available to order as separate clones. See explanation on how to order above. Each well further corresponds to a CATMA id and AGI code for the appropriate amplified locus.
AGRIKOLA pAGRIKOLA delivery clones
AGRIKOLA pEntry clones
AGRIKOLA plates delivery and entry
NOTE: Entry clones are distributed as bacterial stabs in agar. Individual entry clones and wells will consist of a mixture of both empty vector and entry vector - therefore your clones must be single colony purified and PCR/sequence confirmed before use (for example with primers designed to the appropriate CATMA amplicon sequence).
Agrikola Seed Lines and Sets
Individual seed lines are available relating to a subset of the AGRIKOLA clones. These are organised as sets containing a minimum of 1 and a maximum of 12 transformed lines derived from each clone (i.e. entry clone or delivery clone / CATMA id / AGI code). The same seeds relate to entry and delivery clones.
AGRIKOLA seed sets and individual seeds
Please note transformed plants were selected using 40 mg/l BASTA which was applied at 7, 9, 12, 14 and 16 days post germination.
Important note: All of the lines in a set are derived from the same clone but will normally have different insertion positions in the genome and may have divergent silencing phenotypes.
In addition: Seed stocks and clones are distributed 'as-is' and cannot be guaranteed to provide a given phenotype in your laboratory. These are experimental lines and individual seed stocks may not necessarily have been selected on the basis of a broad range of phenotypes in your tissue of interest under your growth conditions.
Sets of lines and each of the individual lines in a set are available to order.
Background line for Agrikola seed: Col-9 (N9573)
Seed Sets and Individual lines received
- Institut fuer Biochemie und Biologie - Thomas Altmann - 66 sets, 659 individual lines (Available).
- Institut fuer Biochemie und Biologie - Thomas Altmann - 878 sets, 8053 individual lines (Available)
- Imperial College at Wye - 720 sets, 6096 individual lines (Available).
- Imperial College at Wye - 855 sets, 7670 individual lines (Available).
Please note that we do not recommend that users wait for knock-downs in their gene of interest to be transformed into plants. We instead recommend that users order clones and make their own series of knock-downs. This will also ensure that a full range of phenotypes for your tissue of interest can be selected by you.
Agrikola cloning strategy - courtesy of Agrikola.org
This diagram shows both the steps taken to generate the available entry clone and the subsequent steps taken to integrate the entry clone into the related delivery clones.