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Direct Hematological Toxicity and Illegitimate Chromosomal Recombination Caused by the Systemic Activation of CreER^T21

Atsuko Yoshioka Higashi^*, Tomokatsu Ikawa^¶, Masamichi Muramatsu^||, Aris N. Economides^#, Akira Niwa, Tomohiko Okuda, Andrew J. Murphy^#, Jose Rojas^#, Toshio Heike, Tatsutoshi Nakahata, Hiroshi Kawamoto^¶, Toru Kita^* and Motoko Yanagita^2,

^* Department of Cardiovascular Medicine, Department of Pediatrics, COE Formation, Career-Path Promotion Unit for Young Life Scientists, Graduate School of Medicine, Kyoto University, Kyoto, Japan; ^¶ Laboratory for Lymphocyte Development, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan; ^|| Department of Molecular Genetics, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan; and ^# Regeneron Pharmaceuticals, Tarrytown, NY 10591

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.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This study was supported by Grants-in Aid from the Ministry of Education, Culture, Science, Sports, and Technology of Japan (Wakate 177090551, Ho-ga 19659219); a Center of Excellence grant from the Ministry of Education, Culture, Science, Sports, and Technology of Japan; a research grant for health sciences from the Japanese Ministry of Health, Labor, and Welfare; a grant from the Astellas Foundation for Research on Metabolic Disorders; a grant from the Novartis Foundation for the promotion of science; a grant from Kato Memorial Trust for Nambyo Research; a grant from Hayashi Memorial Foundation for Female Natural Scientists; a grant from Japan Foundation for Applied Enzymology; and in part by a grant-in-aid for Research on Biological Markers for New Drug Development, Health and Labour Sciences, Research Grants from the Ministry of Health, Labour and Welfare of Japan.

A.Y.H., T.I., A.N., T.O., and H.K. performed experiments; A.N.E., A.J.M., and J.R. generated R26CreER^T2 mice; M.M., T.H., T.N., T.K., and M.Y. analyzed results and made the figures; and M.Y., M.M., and H.K. designed the research and wrote the paper.

² Address correspondence and reprint requests to M. Yanagita, Career-Path Promotion Unit for Young Life Scientists, Graduate School of Medicine, Kyoto University, Kyoto, Japan. E-mail address: motoy{at}kuhp.kyoto-u.ac.jp

³ Abbreviations used in this paper: ER^T2, mutated ligand-binding domain of the estrogen receptor; TM, tamoxifen; WT, wild type; 4-OHT, 4-hydroxytamoxifen.

⁴ The online version of this article contains supplemental material.

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				A. Uhmann, K. Dittmann, J. Wienands, and H. Hahn Comment on "Direct Hematological Toxicity and Illegitimate Chromosomal Recombination Caused by the Systemic Activation of CreERT2" J. Immunol., September 1, 2009; 183(5): 2891 - 2891. [Full Text] [PDF]

				M. Yanagita, A. Higashi, T. Ikawa, M. Muramatsu, A. N. Economides, and H. Kawamoto Response to Comment on "Direct Hematological Toxicity and Illegitimate Chromosomal Recombination Caused by the Systemic Activation of CreERT2" J. Immunol., September 1, 2009; 183(5): 2891 - 2892. [Full Text] [PDF]