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High-level endothelial expression of the human complement regulatory factor CD59 has been shown to protect transgenic mouse hearts from human complement-mediated injury in an ex vivo perfusion model. In this study we examine whether co-expression of CD55 provides additional protection. CD55/CD59 double-transgenic mice were generated by co-injection of CD55 and CD59 expression constructs driven by the human intercellular adhesion molecule 2 (ICAM-2) promoter. A line was established from one mouse that exhibited strong expression of CD55 and CD59 on vascular endothelium in the heart and other transplantable organs. An ex vivo perfusion model was used to compare hearts from these CD55/CD59 mice with hearts from a previously established line, which expressed CD59 at a similar level to the double transgenic line. CD59 hearts displayed prolonged survival compared to wild-type hearts during perfusion with 40% human plasma and maintained approximately 20% maximum work after 60 min. CD55/CD59 hearts were further protected, with work maintained at 35% of the maximum level after 60 min. The data demonstrate that high-level endothelial co-expression of CD55 and CD59 provides greater protection from human complement-mediated injury in this model than expression of CD59 alone.