GENERATING GREEN FLUORESCENT MICE BY GERMLINE TRANSMISSION OF GREEN FLUORESCENT ES CELLS

Green fluorescent protein (GFP) and its variants currently represent t he only non-invasive markers available for labeling mammalian cells in culture or in a multicellular organism through transgenesis. To date this marker gene has been widely used in the study of many organisms, but as yet has not found large-scale application in mammals due to pro blems encountered with weak fluorescence and instability of the wild-t ype protein at higher temperatures. Recently, though, several mutants have been made in the wild-type (wt) GFP so as to improve its thermost ability and fluorescence. EGFP (enhanced GFP) is one such wtGFP varian t. As a first step in assessing the use of EGFP in ES cell-mediated st rategies, we have established a mouse embryonic stem (ES) cell lines e xpressing EGFP, which can be propagated in culture, reintroduced into mice, or induced to differentiate in vitro, while still maintaining ub iquitous EGFP expression. From the results presented we can suggest th at: (1) possible improvements in the efficiency of transgenic regimes requiring the germline transmission of ES cells by aggregation chimera s can be made by the preselection chimeric embryos at the blastocyst s tage; (2) the expression of a noninvasive marker, driven by a promoter that is active during early postimplantation development, allows acce ss to embryos during a window of embryonic development that has previo usly been difficult to investigate; (3) the behavior of mutant ES cell s can be followed with simple microscopic observation of chimeric embr yos or adult animals comprising green fluorescent cells/tissues; and ( 4) intercrosses of Fl mice and subsequent generations of animals show that progeny can be genotyped by UV light, such that mice homozygous f or the transgene can be distinguished from hemizygotes due to their in creased fluorescence. (C) 1998 Elsevier Science Ireland Ltd. All right s reserved.