title: Direct hematological toxicity and illegitimate chromosomal recombination caused by the systemic activation of CreERT2.
abstract: The CreER(T2) for conditional gene inactivation has become increasingly used in reverse mouse genetics, which enables temporal regulation of Cre activity using a mutant estrogen binding domain (ER(T2)) to keep Cre inactive until the administration of tamoxifen. In this study, we present the severe toxicity of ubiquitously expressed CreER(T2) in adult mice and embryos. The toxicity of Cre recombinase or CreER(T2) in vitro or in vivo organisms are still less sufficiently recognized considering the common use of Cre/loxP system, though the toxicity might compromise the phenotypic analysis of the gene of interest. We analyzed two independent lines in which CreER(T2) is knocked-in into the Rosa26 locus (R26CreER(T2) mice), and both lines showed thymus atrophy, severe anemia, and illegitimate chromosomal rearrangement in hematopoietic cells after the administration of tamoxifen, and demonstrated complete recovery of hematological toxicity in adult mice. In the hematopoietic tissues in R26CreER(T2) mice, reduced proliferation and increased apoptosis was observed after the administration of tamoxifen. Flow cytometric analysis revealed that CreER(T2) toxicity affected several hematopoietic lineages, and that immature cells in these lineages tend to be more sensitive to the toxicity. In vitro culturing of hematopoietic cells from these mice further demonstrated the direct toxicity of CreER(T2) on growth and differentiation of hematopoietic cells. We further demonstrated the cleavage of the putative cryptic/pseudo loxP site in the genome after the activation of CreER(T2) in vivo. We discussed how to avoid the misinterpretation of the experimental results from potential toxic effects due to the activated CreER(T2).
authors.affiliation: Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Atsuko Yoshioka
authors.initials: AY
authors.lastname: Higashi
authors.forename: Tomokatsu
authors.initials: T
authors.lastname: Ikawa
authors.forename: Masamichi
authors.initials: M
authors.lastname: Muramatsu
authors.forename: Aris N
authors.initials: AN
authors.lastname: Economides
authors.forename: Akira
authors.initials: A
authors.lastname: Niwa
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Okuda
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Murphy
authors.forename: Jose
authors.initials: J
authors.lastname: Rojas
authors.forename: Toshio
authors.initials: T
authors.lastname: Heike
authors.forename: Tatsutoshi
authors.initials: T
authors.lastname: Nakahata
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kawamoto
authors.forename: Toru
authors.initials: T
authors.lastname: Kita
authors.forename: Motoko
authors.initials: M
authors.lastname: Yanagita
mesh_terms: D000740:Anemia / Q000201:enzymology / Q000235:genetics* / Q000473:pathology; D000818:Animals; D002869:Chromosome Aberrations; D002875:Chromosomes / Q000235:genetics*; D018920:Coculture Techniques; D016326:Extracellular Matrix Proteins / Q000235:genetics; D005260:Female; D055879:Gene Knock-In Techniques; D006131:Growth Inhibitors / Q000235:genetics* / Q000633:toxicity; D006412:Hematopoietic Stem Cells / Q000276:immunology / Q000473:pathology*; D019426:Integrases / Q000235:genetics* / Q000378:metabolism / Q000633:toxicity; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D016254:Mutagenesis, Insertional; D008249:Protein-Lysine 6-Oxidase / Q000235:genetics; D011960:Receptors, Estrogen / Q000235:genetics* / Q000378:metabolism; D011995:Recombination, Genetic; D017154:Stromal Cells / Q000473:pathology
chemical_list: D016326:Extracellular Matrix Proteins; D006131:Growth Inhibitors; D011960:Receptors, Estrogen; C077112:Lox protein, mouse; D008249:Protein-Lysine 6-Oxidase; C045073:Cre recombinase; D019426:Integrases