The NICHD-funded Xenopus ORFeome project (R01HD069352) provides the research community with a comprehensive set of full-length, end-sequence validated, high quality open reading frame clones in the Gateway cloning system ready to use for recombinant protein expression. These ORF sequences are constructed in a format for easy transfer using recombineering (Gateway cloning) into a diverse array of expression vectors.
This set of reagents greatly decreases the time needed to characterize any protein in functional assays. Most importantly, an ORFeome set allows high-throughput in vivo functional-genomic screening of frog gnees in manner previously not feasible, This will help resesarchers take advantage of the strengths of Xenopus as a biomedical model organism.
The ORF-cloning pipeline:
- Predicted ORFs are precisely PCR-amplified between annotated initiation and termination codons, using a cDNA library as template or by RT-PCR, and specific primers are 5’-tailed with Gateway recombinational cloning sites,
- Resulting PCR products are recombined directionally into a Gateway Donor vector to create Entry vector clones,
- ORFs, validated by end-sequencing, are obtained from the Entry clones, providing experimental evidence for the existence and intron-exon structure of the corresponding coding isoform.
Xenopus laevis ORFeome (v1.0):
- ~9,000 PCR-amplified ORFs from the Xenopus Gene Collection (XGC), 7,444 valid full-length CDS clones, from Xenopus laevis from the start ATG, omitting the stop codon, cloned into the Gateway entry plasmid vectors (pDONR223).
- ~7,800 unique genes representing approximately 40% of the non-redundant X. laevis genome
- Human orthologs were identified for 7,000 of the currently validated frog clones. 2,200 of these genes are associated with human disease to date.
- Available as a complete set of 93, 96-well plates.
- DNASU Plasmid Repository at the ASU Biodesign Institute - Contact: Mitch Magee
- GE Healthcare – GE Lifesciences/Dharmacon - Contact: James Goldmeyer
- DF/HCC DNA Resource Core at Harvard Medical School - Contact: Glenn Beeman
These clones are available, without restriction, to researchers worldwide. The Xenopus community is in full support of this project and the ORFeome was voted as a top priority of needed Xenopus resources.
Xenopus ORFeome Principal Investigators:
- Michael Gilchrist, MRC National Institute for Medical Research, The Ridgeway, London, UK
- David Hill, Center for Canver Systems Biology and Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA
- Todd Stukenberg, University of Virginia, School of Medicine, Charlottesville, VA
- Aaron Zorn, Cincinnati Children's Hospital Medical Center, Cincinnati, OH
This work was supported by NIH/NICHD grant R01HD069352.
- Extend Xenopus ORFeome coverage to include clones from two Xenopus tropicalis EST collections in Gateway entry vectors.
- Clone isolation and validation of the 40% of genes not currently represented in the Xenopus ORFeome v1.0 in Gateway entry vectors.
The Gateway cloning System was developed and commercialized by Invitrogen in the late 1990s. It is a molecular biology method that enables researchers to efficiently transfer DNA-fragments between plasmids using a proprietary set of recombination sequences. These sequences were adapted from site-specific bidirectional recombination of bacteriophage gamma. Gateway Cloning techniques allow transfer of DNA fragments between different cloning vectors maintaining the proper reading frame.
Using Gateway cloning, researchers can clone or subclone DNA segments for functional analysis. The system requires the initial insertion of a DNA fragment (ORF) into a donor plasmid with the appropriate flanking recombination sequences. Destination vectors frequently enable the addition of N- or C-terminal fusions of ORFs to green fluorescent protein (GFP) or small epitope tags (e.g. HA or FLAG).
The Xenopus ORFeome sequences have been cloned from Xenopus cDNA libraries or have been chemically synthesized. This resources supports the Xenopus research community using funding from the NIH (National Institutes of Health, USA).
Gateway cloning into entry vectors for the ORFeome project requires particular initial set-up. While it is more expensive than traditional restriction endonuclease and ligase-based cloning methods, it saves research time offering simpler, highly efficient, interchangeable cloning for many down-stream applications.
- Gateway recombination cloning details
- Gateway recombination cloning manual
- pDONR223 entry vector details
- pCS2+ vector details
- pCSf107mT vector details (based on pCS2 vector)
- pCSf107mT-Gateway-3′Myc Tag
- pCSf107mT-Gateway-3′HA Tag
- pCSf107mT-Gateway-3′Flag Tag
- pCSf107mT-Gateway-3′LAP (6His-GFP) Tag - LAP: localization and purification