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A transgenic reporter system can be used for recombination-mediated modulation of gene activity in the developing mouse embryo

A transgenic reporter system consisting of a tamoxifen-inducible form of Cre recombinase (Cre-ERTM) that can be used for recombination-mediated modulation of gene activity in the developing mouse embryo. Using the enhancer of the Wnt1 gene to restrict the expression of Cre-ERTM to the embryonic neural tube, we found that a single injection of tamoxifen into pregnant mice induced Cre-mediated recombination within the embryonic central nervous system, thereby activating expression of a reporter gene. Induction is ligand dependent, rapid and efficient. The invention demonstrates that tamoxifen-inducible recombination can be used to effectively modify gene function in the mouse embryo.

The construction of the system was performed as follows: To express cre within the WntI expression domain, the cDNA for Cre was placed under the control of the Wnt enhancer. Transgenic mouse strains that expressed this construct were mated with the R170 reporter line to determine whether Cre-mediated activation of the reporter could take place within the WntI expression domain. In embryos carrying both transgenes, b-galactosidase activity is detected within the midbrain by 8.5 post coitum (dpc) and the complete pattern of expression by 11.5 dpc. Next, Cre fusion proteins were generated using the hormone-binding domain of a mutant mouse estrogen receptor (Cre-ERTM) and placed under the regulation of the WntI enhancer. To determine if recombination occurred in the absence of ligand, we crossed stem cell line R170 and Cre-ERTM and stained for b-galactosidase activity at 11.5 dpc. All embryos determined by genotyping to be carrying both transgenes showed a staining pattern that was indistinguishable from embryos carrying only the reporter, whether pregnant mice were uninjected or injected with corn oil. When the pregnant mice from this intercross were injected intraperitoneally with 1.0 mg tamoxifen (TM) per day for four consecutive days (from 7.5 to 10.5 dpc) prior to collection at 11.5 dpc a strong and reproducible b-galactosidase staining was obtained in the WntI pattern in all embryos carrying both transgenes.

The reporter can be activated within the WntI domain, and activation - which requires intragenic recombination, transcription and translation - occurred in less than 12 hours and is active from 8.0 days dpc in the neural plate of the mouse embryo.

Intellectual Property Status: Patent(s) Pending

Applications

The ability to generate specific genetic modifications in mice provides a powerful approach to assess gene function. As such systems allowing conditional genetic modification have been developed; for example, inducible forms of the Cre recombinase from P1 phage have been generated that can catalyze intramolecular recombination between target recognition sequences (loxP sites) in response to ligand. However, when genetic modifications have been generated in the germ line, the resulting phenotype often only reflects the first time a gene has an influence on a particular biological process. As a consequence it is imperative to develop new models that can help understand better how these genes function.

Wnt1-Cre tamoxifen-inducible recombination can be used to modulate gene activity in developing mouse embryos to study neural development.

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