A SYSTEMATIC MOLECULAR-GENETIC APPROACH TO STUDY MAMMALIAN GERMLINE DEVELOPMENT

It is difficult to study gene expression in mammalian embryonic germ c ells as PGCs constitute only a minor proportion of the mouse embryo. W e have overcome this problem by using a novel combination of establish ed molecular and transgenic approaches. A line of mice has been genera ted in which the cells of the germ lineage express the p-galactosidase reporter gene during embryogenesis. Using this line, germ cells have been purified to near homogeneity from embryos at discrete stages duri ng germline development by use of a stain for P-gal activity and a flu orescence activated cell sorter. Subsequently, cDNA libraries have bee n constructed from each germ cell population using a modified lone-lin ker PCR strategy. These combined cDNA libraries represent genes expres sed in PGCs during mammalian germline development. To facilitate a mol ecular genetic approach to studying mammalian germline development, th ese cDNA libraries will be pooled to form an arrayed, addressed refere nce embryonic germ cell cDNA library. In parallel with large-scale cDN A sequencing efforts; genes that are differentially expressed in germ cells will be identified by screening the reference library with probe s generated by subtractive hybridization. Complementary DNAs identifie d using this approach will be analyzed by sequencing, database compari son, genomic mapping and in situ hybridization to ascertain the potent ial functional importance of each gene to germline development. In add ition to providing a wealth of novel information regarding patterns of gene expression during mammalian germline development, these results will form the basis for future experiments to determine the function o f these genes in this process.