Chicken Embryo Transplantation of Melanoma Cells

The chicken embryo transplantation was performed as previously described [4 (link)]. Briefly, melanoma cells were treated with PAPPA-specific siRNAs or scrambled control siRNA as described and labelled with CM-DiO as per manufacturer's instructions (Invitrogen, USA). Cells were grown overnight in a hanging-drop fashion to allow the formation of aggregates. Fertile chicken eggs were incubated at 38°C for 48 hours prior to transplantation. Cell aggregates were harvested and carefully injected with a glass pipette into the trunk neural tube lumen of developing chicken embryos. The eggs were then sealed with adhesive tape and re-incubated for 2 days. After incubation, embryos were removed from the eggs and fixed with 4% paraformaldehyde and whole mounts or cross sections were analyzed for the localization of melanoma cells using a Lumar V12 Zeiss microscope.