Any large-scale manipulation of ORFs requires cloning technologies which are free of restriction enzymes. In this respect those that utilise recombination cloning have proved to be the most suitable for the GFP-cDNA project. We have successfully used Gateway (Invitrogen) and Creator (BD Biosciences) to achieve this. Having identified novel cDNAs, their respective ORFs are PCR amplified such that specific recombination sequences are appended to the 5’ and 3’ ends of the ORF. A simple in vitro recombination reaction then allows the ORF to be shuttled into a basic recipient vector. Similar to the first reaction, a second recombination event can then be used to transfer the ORF into our CFP/YFP expression vectors, and these are then used for the localisation analysis. Generating an ‘intermediate’ clone has the advantage that the ORF can be subsequently transferred into other expression vectors, as shown in the example below.
Cloning in this way is possible in a 96-well format, and allows for both N- and C-terminal fusions to be easily and concurrently made for each source clone. Although the final product contains additional amino acid residues at the N- and C-termini of the ORF, our experience is that in the vast majority of cases these sequences have little effect on protein localisation.