International Mammalian Genome Society

The 14th International Mouse Genome Conference (2000)

I12. Cell Type Specific Developmental Analysis of Mouse Primordial Germ Cells

Kanae Mitsunaga1, Koichiro Miike1, Kazuyuki Ohbo1, Minoru S.H. Ko,2 and Kuniya Abe1
1Inst. Mol. Emb. & Genet., Kumamoto Univ., Japan,
2National Institute on Aging, NIH, Baltimore, MD

In developing embryos of multicellular organisms, distinct sets of differentiated cells are derived from a few stem cell populations. The specification and expansion of diverse cell lineages from pluripotent stem cells are fundamental problems in developmental biology. However, there are technical difficulties to tackle these problems. For example, cells derived from different cell lineages are intermingled within developing embryos, which hampers direct characterization of each differentiating cell type at molecular level. The primordial germ cell (PGC) is a first cell type appeared during germ cell lineage, which gives rise to totipotent embryonic germ cells or gametes capable of making a genetic contribution to the next generation. PGC is thus considered as a key cell type for analysis of germ-soma differentiation as well as totipotency at the molecular level. PGC can be found at the base of allantois of the gastrulating embryos, then migrate toward genital ridges, in which they differentiate into gametes. Our knowledge on the molecular events underlying the germ line development is still limited, because, for one reason, attempts to study gene expression in PGC have been confounded by the difficulty in obtaining both sufficient quantities and purity of PGCs. We have overcome this problem using a novel combination of molecular and transgenic approaches. Two kinds of transgenic mice have been generated in which the cells of the germ lineage express reporter genes such as lacZ or GFP (Macgregor et al., 1996; Yoshimizu et al., 1999 ). PGCs were purified from these embryos using FACS sorting of the reporter expressing cells ( Abe et al., 1996 ). The ability to isolate highly pure populations of PGCs enables many interesting experiments to illustrate cellular and molecular characteristics of PGC with currently available, sophisticated tools. We have used cDNA derived from purified PGCs of various stages to construct gridded cDNA libraries using PCR-based techniques ( K. Abe, 1992; Abe et al., 1998 ). These cDNAs can be used to test the expression of known genes in PGC, or to monitor gene expression during germ line development. Analysis of e11.5 and e13.5 male and female PGC libraries revealed relative abundance and sequence information of ~5000 ESTs. Global gene expression profile in PGC is now being analyzed by high density cDNA array.

Information derived from these 'cell type specific developmental' analyses will constitute an important resource for future functional studies to understand the biological process of germ line stem cell development.

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