To control for reaction efficiency, kanamycin cassette control RNA (Promega) was added to the extracted RNA mixture and assayed as above with primers, 5' GGC TCG CGA TAA TGT CGG G and 5' GAT GGT CGG AAG AGG C. Quantitated kanamycin cDNA levels were used to normalise viral cDNA levels.
For the first PCR reaction, the maC46 transgene cassette was amplified from M87o-RRE [9] using the oligonucleotides M87o-for (5' GGG GGA TCC CCC GGG CTG CAG GAA TTC GCC CTT CTC TAG CGC TAC CGG TCG CCG C 3') and M87o-rev (5' TCC TCG CCC TTG CTC ACC ATG CAT GCG GGC TCC AGC TCC AGG CGC T 3').
Two separate ligation reactions were performed for every sample, one reaction for each linker cassette using 15 U T4 ligase (30 U T4 ligase HC from Thermo Scientific).
Genomic DNA isolated from a single colony was tested by multiplex real-time polymerase chain reaction (PCR) for staphylococcal cassette chromosome mec (SCC mec) elements, accessory gene regulator (agr) group, Panton Valentine leukocidin (PVL), and toxic shock syndrome toxin-1 (TSST) [ 7].
These integrases may be better suited for cassette exchange reactions as compared with Cre or FLP [18].
Alternatively and also possible, the cassette exchange reactions might have occurred already at the transient episomal stage where RMCE was initiated between the two cassettes still present as episomal plasmids.
IntI can catalyze a reversible reaction by which gene cassettes can be inserted into, or excised from, an integron via recombination between the attI site and a 59-be or between two 59-be sites [ 5, 6].
In particular the unidirectional activity has made them valuable for the promotion of DNA integration by recombinase-mediated cassette exchange reactions and for the development of iterative recombination approaches [ 4- 7].
Another problem is this technique's complicated joining reaction in which a promoter cassette, the purified V gene fragment and a terminator cassette must be assembled in a specific order based on their short homology overlaps.
The appearance of blue colonies was dependent on inclusion of the lacZ cassette in the reaction (Table 2) and on the inclusion of Flp in the reaction (data not shown).
