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The Arabidopsis genome sequencing program has provided the Plant community with the sequence of about 27,000 genes. For about two-thirds of these genes, some idea of the function of the gene can be gleaned from sequence similarity to other genes but detailed experimental data are available for only a tiny fraction of these genes. For the other 30% of the genes, we have no clues to the function of the gene product. Hence there remains a huge amount of work before we understand the function of all of the Arabidopsis genes. To accelerate this analysis, it is crucial to develop high-throughput approaches that allow the functional characterisation of hundreds of genes in parallel.

Goals of the project

The purpose of our project (funded by Genoplante and in collaboration with Invitrogen) is to use a novel cloning technique to clone thousands of Arabidopsis open reading frames (ORFs) into the expression vectors needed for functional analyses. The Gateway™ cloning system from Invitrogen based on recombinational cloning is used. We expect to clone about 5,000 genes during the project. The project has been divided into two parts:


ATOME 1 cloning strategy: from the full-length cDNA collection to ATOME 1 (clickable image map)

Cloning strategy

The cDNAs from the GSLT collection are cloned in a Gateway vector (pCMV-Sport6). These cDNAs can be transferred into Gateway destination vectors (after first transferring into a donor vector). They could be used for native protein expression in plants and probably in other eukaryotes (functional complementation or overexpression).
To make the GSLT cDNA collection "ready to use", we transfered the full-length cDNA clones from pSport to a Gateway donor vector by BP recombination.

However, these clones are not optimal for expression in bacteria of native proteins because they lack the Shine/Delgano sequence before the ATG. Furthermore, as these clones carry the 5' and 3' UTRs of genes, they cannot be used for expression of fusion proteins. Thus the cDNA clones themselves and the donor clones constructed from them are not suitable for the majority of the functional analyses that can be envisaged.
The major part of ATOME 1 was to generate "clean" ORF entry clones (i.e. minus the 5' and 3' UTRs) by amplification ready for subsequent transfer to destination vectors designed for the expression of various fusion proteins and for the expression of proteins in bacteria. Two types of ORF clones were constructed using degenerated 3' oligonucleotides: 'open clones" in which we removed the stop codon and "closed clones" in which the stop codons were cloned (see ORF structure page).

ATOME 2 cloning strategy: from the SSP ORF collection to ATOME 2 (clickable image map)

Cloning strategy

The ORF from the SSP collection are cloned in the pUNI51 vector allowing subsequent cre/lox recombination subcloning. Most of the clones from this collection are carrying the native stop codon and 10-50 bp 3'UTR. They can be used for native protein expression and/or N-terminal protein fusions using cre/lox expression vectors.

However, as these clones carry 3' UTRs of genes, they cannot be used for C-terminal fusions.
The goal of ATOME 2 is to generate "clean" ORF entry clones (i.e. minus the 5' and 3' UTRs and stop codon) by amplification ready for subsequent transfer to destination vectors designed for the expression of various fusion proteins and for the expression of proteins in bacteria. In this part of the project, only "open clones" are produced (see ORF structure page).


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The URGV is also coordinating a European-wide project to produce a genome-wide collection of hairpin RNA constructs for knock-down gene-silencing of targeted Arabidopsis genes.

 



 

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