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Blood, Vol. 96 No. 2 (July 15), 2000:
pp. 719-726
Insertion of enhanced green fluorescent protein into the lysozyme
gene creates mice with green fluorescent granulocytes and macrophages
Nicole Faust,
Florencio Varas,
Louise M. Kelly,
Susanne Heck, and
Thomas Graf
From the Albert Einstein College of Medicine, Bronx, NY; Artemis
Pharmaceuticals, Cologne, Germany; the European Molecular Biology
Laboratory, Heidelberg, Germany; and the Department of Molecular and
Cellular Biology, Ciemat, Madrid, Spain.
Pluripotent hematopoietic stem cells have been
studied extensively, but the events that occur during their
differentiation remain largely uncharted. To develop a system that
allows the differentiation of cultured multipotent progenitors by
time-lapse fluorescence microscopy, myelomonocytic cells were labeled
with green fluorescent protein (GFP) in vivo. This was achieved by knocking the enhanced GFP (EGFP) gene
into the murine lysozyme M (lys) locus and using a targeting
vector, which contains a neomycin resistant (neo) gene flanked
by LoxP sites and "splinked" ends, to increase the frequency of
homologous recombination. Analysis of the blood and bone marrow of the
lys-EGFP mice revealed that most myelomonocytic cells,
especially mature neutrophil granulocytes, were fluorescence-positive,
while cells from other lineages were not. Removal of the neo
gene through breeding of the mice with the Cre-deleter strain
led to an increased fluorescence intensity. Mice with an inactivation
of both copies of the lys gene developed normally and were fertile.
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