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The full-ORF clone resource of the German cDNA Consortium.

BMC genomics (2007-11-02)
Stephanie Bechtel, Heiko Rosenfelder, Anny Duda, Christian Peter Schmidt, Ute Ernst, Ruth Wellenreuther, Alexander Mehrle, Claudia Schuster, Andre Bahr, Helmut Blöcker, Dagmar Heubner, Andreas Hoerlein, Guenter Michel, Holger Wedler, Karl Köhrer, Birgit Ottenwälder, Annemarie Poustka, Stefan Wiemann, Ingo Schupp
ABSTRACT

With the completion of the human genome sequence the functional analysis and characterization of the encoded proteins has become the next urging challenge in the post-genome era. The lack of comprehensive ORFeome resources has thus far hampered systematic applications by protein gain-of-function analysis. Gene and ORF coverage with full-length ORF clones thus needs to be extended. In combination with a unique and versatile cloning system, these will provide the tools for genome-wide systematic functional analyses, to achieve a deeper insight into complex biological processes. Here we describe the generation of a full-ORF clone resource of human genes applying the Gateway cloning technology (Invitrogen). A pipeline for efficient cloning and sequencing was developed and a sample tracking database was implemented to streamline the clone production process targeting more than 2,200 different ORFs. In addition, a robust cloning strategy was established, permitting the simultaneous generation of two clone variants that contain a particular ORF with as well as without a stop codon by the implementation of only one additional working step into the cloning procedure. Up to 92 % of the targeted ORFs were successfully amplified by PCR and more than 93 % of the amplicons successfully cloned. The German cDNA Consortium ORFeome resource currently consists of more than 3,800 sequence-verified entry clones representing ORFs, cloned with and without stop codon, for about 1,700 different gene loci. 177 splice variants were cloned representing 121 of these genes. The entry clones have been used to generate over 5,000 different expression constructs, providing the basis for functional profiling applications. As a member of the recently formed international ORFeome collaboration we substantially contribute to generating and providing a whole genome human ORFeome collection in a unique cloning system that is made freely available in the community.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
CMP-Sialic Acid Synthetase from Neisseria meningitidis group B, recombinant, expressed in E. coli BL21, ≥10 units/mg protein
Sigma-Aldrich
Hexokinase from Saccharomyces cerevisiae, Type F-300, lyophilized powder, ≥130 units/mg protein (biuret)
Sigma-Aldrich
L-Glutamine Synthetase from Escherichia coli, lyophilized powder, 400-2,000 units/mg protein
Sigma-Aldrich
Adenosine 5′-Triphosphatase from porcine cerebral cortex, lyophilized powder, ≥0.3 units/mg protein, pH 7.8
Sigma-Aldrich
Enolase from baker′s yeast (S. cerevisiae), lyophilized powder, ≥50 units/mg protein
Sigma-Aldrich
Hexokinase from Saccharomyces cerevisiae, lyophilized powder, ≥350 units/mg protein, Protein ≥10 % by biuret
Sigma-Aldrich
Phospholipase A2 from honey bee venom (Apis mellifera), salt-free, lyophilized powder, 600-2400 units/mg protein
Sigma-Aldrich
Dihydrofolate Reductase human, ≥80% (SDS-PAGE), recombinant, expressed in E. coli, ≥1 units/mg protein
Sigma-Aldrich
Neuron-specific enolase from human brain, ≥95% (SDS-PAGE), buffered aqueous solution
Sigma-Aldrich
Galactosyltransferase from bovine milk, lyophilized powder
Sigma-Aldrich
Phospholipase A2 from porcine pancreas, ammonium sulfate suspension, ≥600 units/mg protein
Sigma-Aldrich
α-Ketoglutarate Dehydrogenase from porcine heart, buffered aqueous glycerol solution, 0.1-1.0 units/mg protein (Lowry)
Sigma-Aldrich
Phospholipase A2 from bovine pancreas, lyophilized powder, ≥20 units/mg protein