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

Abstract

Conditional DNA excision between two LoxP sites can be achieved in the mouse using Cre-ER(T), 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-ER(T2), which was approximately 4-fold more efficiently induced by OHT than Cre-ER(T) 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-ER(T) or Cre-ER(T2) 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-ER(T2) was approximately 10-fold more sensitive to OHT induction than Cre-ER(T).

Genetic inducible fate mapping in mouse: Establishing genetic lineages and defining genetic neuroanatomy in the nervous system

“…(CreER T2 ), which has been shown to be between 4-and 10-fold more sensitive to 4-hydroxytamoxifen in vivo (Feil et al, 1997;Indra et al, 1999). 4-hydroxytamoxifen is a metabolite of tamoxifen that is generated in the liver and believed to be the active compound in binding the ER, although administration of either compound, at the same concentration, results in indistinguishable recombination efficiency (Kuhbandner et al, 2000).…”

Wnt Signaling Inhibition Deprives Small Intestinal Stem Cells of Clonogenic Capacity

“…The ERT2 partner immobilizes Cre enzyme in the cytoplasm until an antagonist of the estrogen receptor, tamoxifen [or its metabolite 4‐hydroxitamoxifen (4‐OHT)], is administered. Thus, the usage of CreERT2 allows excision of floxed DNA sequences in a timely manner (Indra et al ., 1999). MEFs isolated from Rosa26 CreERT2/tdTomato embryos at E13.5 were cultured with 4‐OHT or vehicle (ethanol) and genotyped by PCR to confirm the locus recombination and generation of the Rosa26 dnTCF4 allele (Fig.…”

Impaired cervical homeostasis upon selective ablation of RXRα in epithelial cells

“…4g,i,j) confirming cervical epithelial dedifferentiation. Tamoxifen dose-response of our CreER T2 floxed mice was 10-fold more sensitive than other systems such as CreER T for both nuclear translocation and recombinase activity (Indra et al, 1999). In addition, secondary Tamoxifen effects should be rapidly reversed a few days after treatment.…”

“…This became possible through conditional inactivation of RXRa using the Cre/loxP technology and transgenic mice selectively expressing the tamoxifen-inducible Cre-ER T2 recombinase in epidermal keratinocytes (Li et al, 2000;Metzger et al, 2003). The conditional RXRa ablation in epidermal keratinocytes of adult mice, induced by a mild Tam treatment (0.1 mg/mouse, for 5 days) (Indra et al, 1999), resulted in alopecia, hyperproliferation and aberrant terminal differentiation, and skin inflammatory reaction (Li et al, 2000).…”