An approach for controlling the timing and order of engineered mutations in mice

Significant advances in our understanding of normal development and disease have been facilitated by engineered mice in which genes can be altered in a spatially, temporally, or cell type restricted manner using site specific recombinase systems like Cre‐loxP or Flp‐frt. In many circumstances it is important to understand how interactions between multiple genes influence a given phenotype. Robust approaches for precisely controlling multiple genetic alterations independently are limited, however, thus the impact of mutation order and timing on phenotype is generally unknown. Here we describe and validate a novel Gt(ROSA)26Sor targeted transgene allowing precise control over the order and timing of multiple genetic mutations in the mouse. The transgene expresses an optimized, Flp‐estrogen receptor fusion protein (Flpo‐ERT2) under the control of a loxP‐stop‐loxP cassette. In this system, genes modified by loxP sites are altered first upon expression of Cre. Cre also eliminates the loxP‐stop‐loxP cassette, permitting widespread expression of Flpo‐ERT2. Because of the estrogen receptor fusion, Flp activity remains inert until administration of tamoxifen, allowing genes modified by frt sites to be modified subsequently with controllable timing. This mouse transgene will be useful in a wide variety of applications where independent control of different mutations in the mouse is desirable.