Context
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/LMBP
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.
Validation
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.
Template plasmid information
Entry clone: pENTR207-CATMAXyZZZZZ
Expression clone: pAGRIKOLA-CATMAXyZZZZZ
Available clones
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BCCM/LMBP AGRIKOLA GST entry plasmids and hairpin RNA expression plasmids