Brief expression of a GFP cre fusion gene in embryonic stem cells allows rapid retrieval of site-specific genomic deletions

Abstract

The Cre DNA recombinase of bacteriophage P1 has become a useful tool for precise genomic manipulation in embryonic stem (ES) cells that have been gene modified by homologous recombination. We have re-engineered the cre gene to allow ready identification of living Cre+cells by constructing a functional fusion between Cre and an enhanced green fluorescent protein from Aequorea victoria (GFPS65T). The GFP cre fusion gene product rapidly targeted the nucleus in the absence of any exogenous nuclear localization signal. Moreover, GFPCre catalyzed efficient DNA recombination in both a mouse 3T3 derivative cell line and in murine ES cells. Fluorescence- activated cell sorting (FACS) of transiently GFP cre -transfected ES cells not only allowed rapid and efficient isolation of Cre+cells after DNA transfection but also demonstrated that a burst of Cre expression is sufficient to commit cells to Cre-mediated 'pop-out' of loxP -tagged DNA from the genome. Thus, GFP cre allows rapid identification of living cells in which loxP - flanked DNA sequences are destined to be removed from the genome by Cre-mediated recombination without reliance on recombinational activation or inactivation of a marker gene at the target locus. In addition, the GFP cre fusion gene will prove useful in tracing tissue-specific Cre expression in transgenic animals, thereby facilitating the generation and analysis of conditional gene knockout mice.

Nociceptor-specific gene deletion reveals a major role for Nav1.7 (PN1) in acute and inflammatory pain

“…By driving Cre with a tissue-specific promoter, gene deletion can be effected in particular tissues. This refinement of null mutant technology has proved useful for functional studies (15)(16)(17). To elucidate the physiological roles of Na v 1.7, we generated ''floxed'' mice in which loxP sites were inserted in introns flanking exons 14 and 15, which encode most of domain II.…”

Peripheral Thy1+ lymphocytes rearranging TCR‐γδ genes in LAT‐deficient mice

“…Correctly recombined ES cell clones were electroporated with a Cre-EGFP fusion expression vector [33] to remove the floxed Neo r cassette and to invert the segment of the Lat gene that is located between the 5 0 and 3 0 loxP511 sites. One day after transfection, enhanced green fluorescence protein positive ES cells were sorted by flow cytometry and cloned by limiting dilution.…”

“…Proper recombination at the 5 0 end was further verified by Southern blot www.eji-journal.eu using a 600-bp 5 0 internal BamHI-XbaI probe on KpnI-digested genomic DNA, generating an additional 6.8-kb band. Finally, a neomycin probe was used to ensure that non-homologous recombination events had not occurred in the selected clones.Correctly recombined ES cell clones were electroporated with a Cre-EGFP fusion expression vector [33] to remove the floxed Neo r cassette and to invert the segment of the Lat gene that is located between the 5 0 and 3 0 loxP511 sites. One day after transfection, enhanced green fluorescence protein positive ES cells were sorted by flow cytometry and cloned by limiting dilution.…”

Predetermined chromosomal deletion encompassing the Nf-1 gene

“…ES cells were cultured in DMEM supplemented with 15% FCS and 1000 U/ml ESGRO (Gibco BRL) on gelatinized plates. The day before transfection with the GFPcre expression vector pBS500 (Gagneten et al, 1997), ES cells were split and seeded at about 4610 5 cells per 3.5 cm plate. The next day, cells were transfected with 1 mg pBS500 complexed with 3 ml Fugene 6 Transfection Reagent (Boehringer Mannheim) in 2 ml medium according to the manufacturer's protocol.…”

“…Cell line D7-2 ( Figure 2a) carries both insertions on one chromosome, whereas in F8-1 cells, the integrations occurred on di erent chromosomes. To achieve the desired intrachromosomal deletion, D7-2 cells were transfected with an expression plasmid encoding a GFP-cre fusion protein (Gagneten et al, 1997).…”