In the framework of the AGRIKOLA programme, a genome-scale library of constructs was created for the 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 (Invitrogen). First, gene-specific sequence tags (GSTs) of 150 to 500 base pairs, designed to be as specific as possible to their cognate gene, were introduced in entry clones via a BP clonase reaction. Then, the GSTs were transferred via a double LR clonase reaction in vectors designed for the expression of hairpin RNAs in transgenic plant cells. The AGRIKOLA consortium has already generated over 25,000 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 demonstrate the high value of this resource (Hilson et al., Genome Research 14 (2004), 2176-2189).
The plasmids originally delivered by the AGRIKOLA consortium to the Arabidopsis stock centers (NASC and ABRC) were not confirmed by sequence analysis and were not carried by clonal bacterial strains (click here for more information).
Therefore, in a follow up project supported by the Belgian Science Policy, the Department of Plant Systems Biology and the BCCM/GeneCorner Plasmid Collection are systematically authenticating a subset of the AGRIKOLA plasmids. The clones validated in the context of this collaboration are distributed by the LMBP Plasmid Collection.
The available clones have been verified for purity, viability and authenticity.
The criteria to authenticate the Gateway® GST entry clones are:
- the presence of an attL1-GST-attL2 cassette into the pDONR207-derived entry clone with a size compatible with the expected GST, as demonstrated by the DNA gel analysis of PCR amplicons;
- a GST sequence obtained from the purified entry clone that shares at least 95% homology with the expected GST, over an alignment of at least 60 nucleotides.
For a detailed description of validation protocols for the entry clones, click here.
The criteria to authenticate the pAGRIKOLA hairpin RNA expression clones are:
- the presence of two attB1-GST-attB2 cassettes into the pAGRIKOLA-derived expression clone with a size compatible with the expected GST, as demonstrated by the DNA gel analysis of PCR amplicons;
- a GST sequence obtained from the purified hairpin expression clone that shares at least 95% homology with the expected GST, over an alignment of at least 60 nucleotides.
For a detailed description of validation protocols for the hairpin RNA expression clones, click here.
Entry clone: pENTR207-CATMAXyZZZZZ
Expression clone: pAGRIKOLA-CATMAXyZZZZZ
Individual clones from this library will be provided as 1 ml fresh culture of plasmid-carrying host.