Lineage-specific cell disruption in living mice by Cre-mediated expression of diphtheria toxin A chain Hiroyuki Matsumura a,1 , Hidetoshi Hasuwa b , Naokazu Inoue b , Masahito Ikawa b , Masaru Okabe a, * a Faculty of Pharmaceutical Sciences, Osaka University, Japan b Genome Information Research Center, Osaka University, Yamadaoka 3-1, Suita, Osaka 565-0871, Japan Received 15 May 2004 Abstract We have established a transgenic mouse line in which floxed neomycin resistant cassette was inserted between the CAG promoter and EGFP. When these transgenic mice were mated with Cre-expressing transgenic animals, the offspring obtained were fluorescent green. We then established a transgenic mouse line in which EGFP in the above construct was replaced by diphtheria toxin A chain (DT). When the latter transgenic mice were mated with mice expressing Cre restricted to germ cells, we obtained healthy but sterile offspring due to a disruption of germ line cells by DT expression. We predict that this strategy will be useful for the construction of new animal models for human diseases, featuring a variety of missing cell lineages produced by disruption with DT. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Cell lineage specific disruption; Transgenic mouse; Cre; LoxP; EGFP; Diphtheria toxin; Animal model for human disease Homologous recombination is used to analyze gene function in living animals. However, a disruption of cer- tain genes is lethal in the embryonic stage making analysis of gene function in adulthood impossible. A conditional gene recombination system has emerged to circumvent this problem. Target genes are flanked by LoxP (or FRT) sequences and excised with an organ-specifically ex- pressed Cre (or with FLP) recombinase to yield condition- al gene-disrupted animals [1,2]. In such a system, limited expression of the recombinase in specific cell lineages is important. We designed a transgenic mouse line in order to evaluate the organ-specific expression of Cre recombin- ase. In this line, the silent EGFP gene is activated by exci- sion of the floxed-stuffer sequence with Cre, leaving the recombined cells fluorescent green. It has been reported that diphtheria toxin A chain (DT) expression with an organ-specific promoter in transgenic animals results in organ-specific cell damage or loss [3–6]. However, resultant animals may display difficulty in sur- vival or breeding. Although they function as good animal models for human diseases, establishing and maintaining such transgenic mouse lines is clearly problematic. We de- vised a lineage-specific cell disruption system to express DT which is silent and harmless without the co-expression of Cre recombinase. In the present paper, we demonstrate that this system could provide versatile animal models for human diseases by breeding with various organ-specific, Cre-expressing mice. Materials and methods Generation of CAG-Cre and prm1-Cre transgenic mice. We produced pCXN-Cre to express Cre recombinase in all cell types in early em- bryonic stages to ensure that Cre-mediated recombination would occur throughout the body. The plasmid consisted of the CAG-promoter, Cre 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.06.139 * Corresponding author. Fax: +81-6-6879-8376. E-mail address: okabe@gen-info.osaka-u.ac.jp (M. Okabe). 1 Present address: Institute for Frontier Medical Sciences, Kyoto University, Japan. www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 321 (2004) 275–279 BBRC