Generating green fluorescent mice by germline transmission of green fluorescent ES cells.
Green fluorescent protein (GFP) and its variants currently represent the 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 problems encountered with weak fluorescence and instability of the wild-type protein at higher temperatures. Recently, though, several mutants have been made in the wild-type (wt) GFP so as to improve its thermostability and fluorescence. EGFP (enhanced GFP) is one such wtGFP variant. As a first step in assessing the use of EGFP in ES cell-mediated strategies, we have established a mouse embryonic stem (ES) cell lines expressing EGFP, which can be propagated in culture, reintroduced into mice. or induced to differentiate in vitro, while still maintaining ubiquitous EGFP expression. From the results presented we can suggest that: 1) possible improvements in the efficiency of transgenic regimes requiring the germline transmission of ES cells by aggregation chimeras can be made by the preselection chimeric embryos at the blastocyst stage: (2) the expression of a noninvasive marker, driven by a promoter that is active during early postimplantation development, allows access to embryos during a window of embryonic development that has previously been difficult to investigate (3) the behavior of mutant ES cells can be followed with simple microscopic observation of chimeric embryos or adult animals comprising green fluorescent cells/tissues. and (4) intercrosses of F1 mice and subsequent generations of animals show that progeny can be genotyped by UV light, such that mice homozygous for the transgene can be distinguished from hemizygotes due to their increased fluorescence.
Hadjantonakis
A K
AK
Samuel Lunenfeld Research Institute, Mount Sinai Hospital, 600 University Avenue, Toronto, ON M5G 1X5, Canada.
Gertsenstein
M
M
Ikawa
M
M
Okabe
M
M
Nagy
A
A
D000818:Animals; D002678:Chimera / Q000235:genetics; D004171:Diploidy; D004624:Embryo Transfer / Q000379:methods; D004622:Embryo, Mammalian; D047108:Embryonic Development / Q000235:genetics; D005260:Female; D018507:Gene Expression Regulation, Developmental; D005821:Genetic Techniques; D005854:Germ Cells; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000032:analysis* / Q000235:genetics* / Q000378:metabolism; D008297:Male; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics* / Q000254:growth & development; D011123:Polyploidy; D011247:Pregnancy; D012641:Selection, Genetic; D013234:Stem Cells / Q000502:physiology*; D019076:Transgenes
D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins