Fate-Mapping Technique: Grafting Fluorescent Cells into Gastrula-Stage Mouse Embryos at 7-7.5 Days Post-coitum

Abstract

INTRODUCTIONAnalysis of the developmental fate of cell populations in different parts of the embryo enables the construction of fate maps. These reveal the organization of the body plan and can presage the expression of molecular characteristics of cell lineages and the formation of body parts. The efficacy of fate-mapping techniques is critically dependent on their ability to track the cells and all their descendants without compromising the development of the embryo. Cell grafting involves isolating a population of genetically tagged cells from a transgenic embryo and grafting them to a defined site in a nontransgenic host embryo. Tissue colonization is analyzed using a genetic tag (e.g., a fluorescent protein that can be visualized noninvasively) that allows tracking of the transplanted cells and their descendants in the host embryo throughout development in culture. Alternatively, a lacZ transgene can be used to localize graft-derived cells histologically. Differentiation of the graft-derived cells can be studied by examining the expression of molecular markers by in situ hybridization of gene transcripts or immunohistochemical detection of lineage-specific proteins. This protocol describes how to graft cells isolated from a donor embryo into the germ layer of a wild-type host mouse embryo at 7-7.5 days post-coitum (dpc).

The Transcriptional and Functional Properties of Mouse Epiblast Stem Cells Resemble the Anterior Primitive Streak

“…EpiSCs were examined for their germ-layer differentiation potential by transplantation to the primitive streak of host ARC/s OB/EB-stage (E7.5) embryos (Franklin et al, 2007). EpiSC colonies in cultures were washed once with PBS followed by PBS containing 2 mM CM-DiI (in alcoholic solution, Molecular Probes, C7000).…”