Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ERT and Cre-ERT2 recombinases

TLDR

Conditional DNA excision between two LoxP sites can be achieved in mice using Cre‑ERT, a fusion of a mutated estrogen receptor ligand‑binding domain and Cre recombinase that is activated by 4‑hydroxy‑tamoxifen (OHT) but not natural ER ligands. The study aims to compare the in vivo efficiency of Cre‑ERT and Cre‑ERT2 for temporally controlled somatic mutations. Transgenic mice were engineered to express a floxed reporter and either Cre‑ERT or Cre‑ERT2 under the bovine keratin 5 promoter, which is specifically active in epidermal basal cells. In vitro, Cre‑ERT2 was ~4‑fold more efficiently induced by OHT than Cre‑ERT, and in vivo no background recombination was detected while Cre‑ERT2 was ~10‑fold more sensitive to OHT, with recombination induced in basal keratinocytes upon OHT administration.

Abstract

Conditional DNA excision between two LoxP sites can be achieved in the mouse using Cre-ERT, a fusion protein between a mutated ligand binding domain of the human estrogen receptor (ER) and the Cre recombinase, the activity of which can be induced by 4-hydroxy-tamoxifen (OHT), but not natural ER ligands. We have recently characterized a new ligand-dependent recombinase, Cre-ERT2, which was ∼4-fold more efficiently induced by OHT than Cre-ERT in cultured cells. In order to compare the in vivo efficiency of these two ligand-inducible recombinases to generate temporally-controlled somatic mutations, we have engineered transgenic mice expressing a LoxP-flanked (floxed) transgene reporter and either Cre-ERT or Cre-ERT2 under the control of the bovine keratin 5 promoter that is specifically active in the epidermis basal cell layer. No background recombinase activity could be detected, while recombination was induced in basal keratinocytes upon OHT administration. Interestingly, a dose-response study showed that Cre-ERT2 was ∼10-fold more sensitive to OHT induction than Cre-ERT.