Cloning and Expression Analyses of Pig Genes

Understanding the molecular bases of xenograft rejection is one of the highest priorities in the xenotransplantation field. Furthermore, the identification of physiological incompatibilities in the xenogeneic setting is also necessary for developing the appropriate strategies to have a long-term functioning xenograft. As the pig is the species of choice for the development of xenogeneic applications, the cloning of pig genes or cDNA is a key step to elucidate the interactions of pig and human molecules. It also provides the necessary information for assessing the level of mRNA expression of relevant proteins in tissues and organs of interest for xenotransplantation. In most cases, the cloning of the cDNA is sufficient to attain these goals. Thus, we describe a basic cloning method that comprises total RNA extraction, reverse transcription (RT), and polymerase chain reaction (PCR) amplification. We also include some links for databases and bioinformatic tools available in the Internet for the subsequent analyses and predictions. Finally, we recommend and explain the procedures of northern blotting and quantitative RT-PCR for conducting the mRNA expression studies.