Identification of positive GATEWAY expression clones when both the pENTRY and pDEST vectors contain the same marker for bacterial selection.

GATEWAY cloning technology (Invitrogen) takes advantage of bacteriophage λ site-specific recombination. The life cycle of λ alternates between the lytic and lysogenic stages. DNA can be inserted or excised from the Escherichia coli host genome by recombination between specific sites, AttB (bacterial) and AttP (phage). This process is mediated by the λ proteins int (integrase) and xis (excisionase), and a host protein IHF (integration host factor). GATEWAY cloning technology uses this process to insert fragments of DNA directionally into specially adapted vectors. These vectors contain a negative selectable marker, the ccdB gene, to select against nonrecombinant clones. Promoter or gene fragments are made GATEWAY compatible with adapter primers and amplified by PCR. These fragments are used in a BP clonase reaction to create ENTRY clones. Usually the pDONR vector used to generate such ENTRY clones is chosen so that the antibiotic selection marker is different from that of the pDEST vector, which finally generates an expression clone. This favors the selection of the expression clone and selects against the pENTRY clone. Now that many pENTRY and pDEST vectors have been generated and made available in stock centers, the antibiotic resistance genes are predetermined and may not always be compatible with each other. This problem is frequently experienced by plant researchers, since many full-length cDNA libraries have been generated using the pDONR-TOPO, pDONR221, or pENTR1A vectors, which are all kanamycin resistant in bacteria, and many pDEST vectors have been adapted from conventional plant transformation vectors, which are also frequently kanamycin resistant in bacteria. The following protocol describes ways in which such difficult vector combinations can be used effectively to obtain the appropriate expression clone without having to convert the pENTRY clone or pDEST clone to vectors with compatible antibiotic resistances.