Gene Trap Vector Screen for Developmental Genes in Differentiating ES Cells

Abstract

Publisher Summary The availability of mouse embryonic stem (ES) cells, together with the development of ‘‘entrapment vectors,’’ has made it possible to efficiently introduce insertional mutations in ES cells and to screen for candidate genes in ES cells and embryos derived from the ES cells. ES cells are undifferentiated, pluripotent cells derived from the inner cell mass (ICM) of blastocysts. This chapter describes an expression screen to identify genes that are expressed at specific stages of mouse development by using retroviral entrapment vectors. ES cell clones with entrapment vector integration into putative developmentally regulated genes can be selected by virtue of their stage-specific reporter gene expression and by colocalization with stage and lineage-specific markers upon in vitro differentiation into embryoid bodies (EB). Selected clones can be further examined in vivo , either by generating transgenic lines or ‘‘transient’’ chimeric embryos. Finally, genomic integration sites can be cloned and used to screen stage-specific embryonic cDNA libraries. This approach allows efficiently identifying and preselecting in vitro for insertions into genes that are restricted in their temporal and spatial distribution in the postimplantation embryo and that may not be expressed in pluripotent embryonal cells. This chapter focuses on the strengths of the in vitro differentiation screen to preselect for entrapment integrations into developmentally regulated genes.