Kobe J. Med. Sci., Vol. 54, No. 6, pp. E272-E278, 2008 Phone: +81-78-382-5600 Fax: +81-78-382-5619 E-mail: aiba@med.kobe-u.ac.jp E272 Generation of L7-tTA Knock-in Mice RUKA ECHIGO 1 , KAZUKI NAKAO 2 , MASAHIRO FUKAYA 3 , MASAHIKO WATANABE 3 , and ATSU AIBA 1* Division of Molecular Genetics, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan 1 ; Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Developmental Biology, Kobe 650-0047, Japan 2 ; Department of Anatomy and Embryology, Hokkaido University Graduate School of Medicine, Sapporo 060-8638, Japan 3 Received 19 December 2008/ Accepted 24 December 2008 Key Words: L7, tTA, knock-in mouse, cerebellum, Purkinje cell We generated a versatile mouse line, L7-tTA knock-in mouse, in which tetracycline-responsive transcriptional activator (tTA) gene was introduced into exon 2 of L7 locus. Since L7 is specifically expressed in cerebellar Purkinje cells, we expected Purkinje cell-restricted expression of tTA gene in the knock-in mice. In situ hybridization analysis exhibited that tTA mRNAs in those mice were expressed only in Purkinje cells. Introduction of transgene consisting of tetracycline-responsive element that is a binding site for tTA and ideal cDNAs into L7-tTA knock-in mouse would result in specific expression of cDNA encoding proteins in Purkinje cells and its expression could be controlled by doxycycline administration. L7-tTA knock-in mice would provide us with opportunity to elucidate the role of specific genes in cerebellar Purkinje cells. Tetracycline-responsive transcriptional activator (tTA) is a transcriptional factor which binds tetracycline-responsive element (TRE), and then activates transcription of a gene downstream of TRE. tTA is inactivated by antibiotic tetracycline or its analogue doxycycline (DOX), and activated by withdraw of DOX. This tetracycline-regulated system is very useful because a gene expression downstream of TRE can be controlled by DOX administration at specific developmental stage (2, 5,10). The Purkinje cell (PC) is an only output neuron in cerebellar cortex. Proximal dendrites of each PC are innervated by a single climbing fiber (CF) in adult brain and form glutamatergic synapses. PC is innervated by multiple CFs during early postnatal development, but establishes mono innervation by the end of the third postnatal week in mice (9, 16). Distal dendrites of PCs are innervated by parallel fibers (PFs), another glutamatergic input. To explore the molecular mechanism of motor learning, synaptic plasticity and neural circuit refinement that require PC function, we need the mice expressing the specific genes exclusively in PCs. L7 gene is expressed in PCs and retinal bipolar cells (3, 4). So we planed to insert tTA gene into L7 locus to accomplish tTA expression in PCs. The postsynapses of CF-PC and PF-PC synapses contain metabotropic glutamate receptor 1 (mGluR1) and -amino-3 hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor (6, 13, 15). PF-PC synapses, but not CF-PC synapses, have glutamate receptor delta 2 (GluR2) (11). These glutamate receptors are involved in cerebellar motor learning (6, 7,