BRIEF EXPRESSION OF A GFPCRE FUSION GENE IN EMBRYONIC STEM-CELLS ALLOWS RAPID RETRIEVAL OF SITE-SPECIFIC GENOMIC DELETIONS

The Cre DNA recombinase of bacteriophage P1 has become a useful tool f or precise genomic manipulation in embryonic stem (ES) cells that have been gene modified by homologous recombination. We have re-engineered the ore gene to allow ready identification of living Cre(+) cells by constructing a functional fusion between Cre and an enhanced green flu orescent protein from Aequorea victoria (GFPS65T), The GFPcre fusion g ene product rapidly targeted the nucleus in the absence of any exogeno us nuclear localization signal, Moreover, GFPCre catalyzed efficient D NA recombination in both a mouse 3T3 derivative cell line and in murin e ES cells, Fluorescence-activated cell sorting (FAGS) of transiently GFPcre-transfected ES cells not only allowed rapid and efficient isola tion of Cre(+) cells after DNA transfection but also demonstrated that a burst of Cre expression is sufficient to commit cells to Cre-mediat ed 'pop-out' of loxP-tagged DNA from the genome. Thus, GFPcre allows r apid identification of living cells in which loxP-flanked DNA sequence s are destined to be removed from the genome by Cre-mediated recombina tion without reliance on recombinational activation or inactivation of a marker gene at the target locus, In addition, the GFPcre fusion gen e will prove useful in tracing tissue-specific Cre expression in trans genic animals, thereby facilitating the generation and analysis of con ditional gene knockout mice.