Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase

Abstract

The ability to generate specific genetic modifications in mice provides a powerful approach to assess gene function. When genetic modifications have been generated in the germ line, however, the resulting phenotype often only reflects the first time a gene has an influence on - or is necessary for - a particular biological process. Therefore, systems allowing conditional genetic modification have been developed (for a review, see [1]); for example, inducible forms of the Cre recombinase from P1 phage have been generated that can catalyse intramolecular recombination between target recognition sequences (loxP sites) in response to ligand [2] [3] [4] [5]. Here, we assessed whether a tamoxifen-inducible form of Cre recombinase (Cre-ERTM) could be used to modify gene activity in the mouse embryo in utero. 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 was ligand dependent, rapid and efficient. The results demonstrate that tamoxifen-inducible recombination can be used to effectively modify gene function in the mouse embryo.

Endothelial Notch1 Is Required for Proper Development of the Semilunar Valves and Cardiac Outflow Tract

“…SHF‐derived endothelial cells are also known to populate the pulmonary trunk and pulmonary valve, suggesting that the effect of Notch1 may still be limited to the endothelial cell layer 22. Because other publications have implicated the role of neural crest cells in OFT formation, we obtained neural crest–specific Wnt1‐Cre mice to ascertain the role of Notch1 specifically in the neural crest 23. We did not observe any cardiac phenotypes in Notch1 fl/wt ;Wnt1‐Cre +/− ;Nos3 −/− mice.…”

“…Nos3 −/− and Notch1 +/− ;Nos3 +/− mice were bred to obtain Notch1 +/− ;Nos3 −/− mice (n=49) and littermate controls (n=216) and were genotyped, as described previously 20. For lineage‐specific deletions of Notch1 (using Tie2‐Cre,21 Mef2C‐Cre,22 Wnt1‐Cre23), Nos3 −/− ;Cre +/− male mice were bred with Notch1 flox/wt ;Nos3 +/− female mice to obtain Notch1 flox/wt ;Tie2‐Cre +/− ;Nos3 −/− (n=6) mice, Notch1 flox/wt ;Mef2C‐Cre +/− ;Nos3 −/− (n=7), Notch1 flox/wt ;Wnt1‐Cre +/− ;Nos3 −/− (n=4), and control littermates (n=4, n=4, n=4, respectively). SHF lineage tracing was completed by breeding Mef2C‐Cre +/− male mice with ROSA26 mT/mG female mice 24…”

Kinetics of tamoxifen‐regulated Cre activity in mice using a cartilage‐specific CreERT to assay temporal activity windows along the proximodistal limb skeleton

“…Tamoxifen (Sigma, T-5648) was administered by IP injection as previously described (Danielian et al, 1998) with a few modifications. To bring the drug more rapidly into solution, tamoxifen was first dissolved at a concentration of 100 mg/ml in 100% ethanol and then diluted to a final concentration of 10 mg/ml into sterile vegetable oil for IP injection.…”

Endogenous bone morphogenetic protein antagonists regulate mammalian neural crest generation and survival

“…Embryonic day 0.5 is defined as noon of the day after plug detection. Embryos bearing Wnt1-Cre (Danielian et al, 1998) and R26R (Soriano, 1999) alleles, which together mark neural crest lineages, were generated as previously described (Chai et al, 2000;Stottmann et al, 2004).…”