Genome Toxicity and Impaired Stem Cell Function after Conditional Activation of CreERT2 in the Intestine

Abstract

Summary: With the tamoxifen-inducible CreERT2 system, genetic recombination can be temporally controlled in a cell-type-specific manner in intact animals, permitting dissection of the molecular underpinnings of mammalian physiology. Here we present a significant drawback to CreERT2 technology for analysis of intestinal stem cells. Using the intestine-specific Villin-CreERT2 mouse strain, we observed delayed intestinal regeneration post irradiation. Villin-CreERT2 activation was associated with DNA damage and cryptic loxP site cleavage. Analysis of stem cell-specific CreERT2 strains showed that the genome toxicity impairs function of crypt base columnar stem cells, resulting in loss of organoid initiating activity. Importantly, the stem cell impairment is short-lived, with return to normal by 7 days post tamoxifen treatment. Our findings demonstrate that mouse genetic experiments that utilize CreERT2 should consider the confounding effects of enhanced stem cell sensitivity to genome toxicity resulting from CreERT2 activation. : Samuelson and colleagues demonstrate that activation of CreERT2 in the mouse intestine leads to intestinal stem cell (ISC) toxicity. Impaired stem cell function was shown by reduced organoid-forming ability in ISC-CreERT2 strains. Also, Villin-CreERT2 mice exhibited impaired crypt regeneration after ISC injury induced by γ-irradiation. DNA damage due to cryptic loxP site cleavage suggested a mechanism of ISC genotoxicity. Keywords: intestinal stem cell, organoid, CreERT2, loxP, genotoxicity, tamoxifen, mouse, crypt regeneration