Purification of mouse primordial germ cells by Nycodenz

Abstract

Primordial germ cells are important cells for the study of germ cell lineage. It has proved difficult to obtain highly purified primordial germ cells for preparation of a specific antibody. In the present study, a new method for purifying mouse primordial germ cells was developed using a Nycodenz gradient. Furthermore, the polyclonal anti-mouse primordial germ cells IgG derived from mouse primordial germ cells was prepared. As this IgG reacted only with primordial germ cells obtained at day 12.5 after mating, this antibody appeared to recognize the stage-specific antigen of primordial germ cells. One reason that a continuous primordial germ cell marker has not been obtained is because the purity of the primordial germ cells used has been too low to prepare the antibody. This new method represents a significant improvement in the purification of primordial germ cells; it is simpler than previous methods, and produced mouse primordial germ cells with a purity of more than 95%. In addition, the separation reagent Nycodenz is non-toxic and achieved separation of primordial germ cells without attachment of antibodies against the primordial germ cell membrane surface. This new purification method and stage-specific antibody will be useful for the analysis of the mechanisms of primordial germ cell migration.