PCR-Based Cloning and Differential Screening of RNAs from Xenopus Primordial Germ Cells: Cloning Uniquely Expressed RNAs from Rare Cells

Primordial germ cells (PGCs) are a small population of unique cells from which all germ cells arise in an organism. In this sense, PGCs can be considered the stem cells of the species. An important characteristic of PGCs is their ability to remain developmentally totipotent,whereas somatic cells become restricted in their fates. Understanding the genetic program that underlies the retention of totipotency is a major goal in the stem cell field. To accomplish this goal, methods must be considered for both the isolation of these cells and the purification of the RNAs they express. The isolation of PGCs from any organism presents certain challenges. Because PGCs arise outside the gonad during early embryogenesis, their exact location within a germ layer is unknown. In addition, PGCs are relatively rare in number compared to somatic cells (on the order of 0.05% or less). This chapter presents detailed procedures for isolating live PGCs from Xenopus laevis embryos and for cloning their expressed genes, should be applicable to other organisms that have PGCs rich in mitochondria.