02% sodium azide (Sigma) and 1% FCS (Invitrogen). Subsequently, a double immunofluorescence staining, performed in microtiter plates, was carried out to stain live cells. Turkey lymphocytes were stained indirectly using a cross-reactive anti-chicken CD8 monoclonal antibody (undiluted supernatant from mouse hybridoma 11–39, IgG1; kindly obtained from
Vainio)  and an anti-mouse IgG1 find more phycoerythrin-labelled conjugate (Molecular Probes, Invitrogen) (30 min, 1/100 in staining medium). The anti-CD8 monoclonal antibody recognizes CD8+αβ and CD8+α and does not recognize CD4+CD8+ cells . Cells were subsequently incubated for 15 min with 10% mouse serum and finally stained directly with a cross-reactive fluorescein-labelled monoclonal antibody (30 min, 1/100 in staining medium) generated against chicken CD4 (KUL04, IgG1, kindly provided by Goddeeris) . All incubations were performed on ice and cells were washed three times in between incubations using staining medium (4 °C, 5 min, 1000 rpm). Staining controls consisted of directly (CD4) and
indirectly (CD8) stained cells, cells stained with an irrelevant monoclonal JNK inhibitor chemical structure antibody (IgG1) and cells incubated with the conjugate solely. Ten thousand living cells were analyzed using FACSCanto flow cytometry (BD Biosciences). Dead cells were eliminated based on their light scatter characteristics. Non-parametric Kruskal–Wallis and Mann–Whitney tests were used for all statistical analyses. Results were considered significantly different at the level of p < 0.05. The presence of the ompAopt gene (1061 bp) however and the EGFP gene (720 bp) in pcDNA1, was verified by PCR clone analysis and DNA sequencing using SP6 and T7 primers.
The PCR product (1781 bp) was visualised on an ethidium bromide stained agarose gel. A DNA fragment of approximately 1800 bp could be observed which indicates that the fusion gene ompAopt–EGFP was successfully cloned into pcDNA1. Sequencing of the PCR product indicated the correct DNA sequence of both genes and showed that the EGFP gene was cloned in the exact reading frame. Following transfection of DF-1 cells using Polyfect®, co-localisation of MOMPopt and EGFP could be clearly demonstrated ( Suppl. Fig. 1). Successful codon-optimisation was shown by the increased red fluorescence for MOMPopt when compared to MOMP ( Suppl. Fig. 1) and confirmed by the increased CAI from 0.698 for ompA to 0.981 for ompAopt in chicken and from 0.606 for ompA to 0.948 for ompAopt in turkeys. Lipoplexes and polyplexes were characterised by measuring their size and zeta potential. In general, particle sizes decreased and the zeta potential of especially polyplexes increased with increasing ratio (data not shown). The former is probably due to the higher condensation of the pDNA, while the latter is due to an excess of the cationic polymers protruding at the surface of the polyplexes.