Tools for molecular studies in xenotransplantation

Understanding the molecular bases of xenograft rejection is one of the highest priorities in the xenotransplantation field. This information is needed for the successful development of genetic modifications of the animal source of xenogeneic cells and organs that prevent rejection. Furthermore, the identification of physiological incompatibilities in the xenogeneic setting is also necessary for developing the appropriate strategies that allow long-term xenograft function. As the pig is the species of choice for the development of the majority of xenogeneic applications, the cloning of pig genes or cDNA is a key step to elucidate the interactions of pig and human molecules. In addition, there are currently multiple bioinformatic tools which facilitate the study in silico of protein structures, molecular interactions, and docking sites. Thus, we describe a basic cloning method that comprises total RNA extraction, reverse transcription (RT), and polymerase chain reaction (PCR) for cDNA amplification and include some links for databases and bioinformatics tools available on the Internet for the subsequent analyses and predictions. Finally, some procedures of protein expression and analysis of protein interactions by surface plasmon resonance (SPR) are described for elucidating the molecular mechanisms of xenograft function and rejection.