Generation of Venus reporter knock-in mice revealed MAGI-2 expression patterns in adult mice Kan-ichiro Ihara a , Tomoki Nishimura a , Tomokazu Fukuda b , Tetsuya Ookura c , Katsuhiko Nishimori a,⇑ a Laboratory of Molecular Biology, Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, Miyagi 981-8555, Japan b Laboratory of Animal Breeding and Genetics, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, Miyagi 981-8555, Japan c National Food Research Institute, 2-1-12 Kan-nondai, Tsukuba, Ibaraki 305-8642, Japan article info Article history: Received 17 June 2011 Received in revised form 13 December 2011 Accepted 28 January 2012 Available online 15 February 2012 Keywords: MAGI-2 S-SCAM Venus Knock-in mice abstract The membrane-associated guanylate kinase inverted 2 (MAGI-2) protein, which is known to localize at the tight junction of epithelial cells, contains multiple copies of the PDZ and WW domains in its structure. Although the expression pattern of Magi2 mRNA in representative organs has been previously published, its detailed cellular distribution at the histological level remains unknown. Such detailed information would be useful to clarify the biological function of MAGI-2. Here, we report the generation of Venus reporter knock-in mice for Magi2 in which exon 6 of the gene was substituted by the Venus-encoding sequence. We detected the expression of the Venus reporter protein in kidney podocytes from these knock-in mice. We also detected Venus reporter protein expression in spermatids within the testes and within neurons in various regions of the brain. Detection of the reporter protein from these diverse locations indicated the endogenous expression of MAGI-2 in these tissues. Our data suggested a potential function of MAGI-2 in the glomerular filtration process and sperm cell maturation. These data indicate that the Venus reporter knock-in mouse for Magi2 is a useful model for the further study of Magi2 gene function. Ó 2012 Elsevier B.V. All rights reserved. Membrane-associated guanylate kinase inverted 2 (MAGI-2) is a multidomain scaffolding protein that contains multiple PDZ and WW domains. These domains function in binding to proline- and arginine-rich proteins. Studies have described the binding of MAGI-2 to the tumor suppressor phosphatase and tensin homolog (PTEN) protein, suggesting a possible function of MAGI-2 in its signaling pathway (Wu et al., 2000). Additionally, MAGI-2 was reported to control the protein interaction between PTEN and b-catenin, another important protein in the carcinogenic pathway (Subauste et al., 2005). In addition to its suggested roles in carcinogenic pathways, MAGI-2 was reported to have an important function in neuronal tissues. MAGI-2 (also referred to as Synaptic scaffolding molecule; S-SCAM) was localized to the postsynaptic density (PSD) area of neuronal membranes (Hirao et al., 1998). Supporting its putative role in neuronal function, MAGI-2 was reported to bind to the known N-methyl-D-aspartate glutamate receptor (NMDAR). Fur- thermore, localization of the MAGI-2 protein to both excitatory and inhibitory synapses was reported (Sumita et al., 2007). Attempts to elucidate the biological function of MAGI-2 in vivo have been reported using knockout mice. The mutant mice showed abnormal elongation of the dendritic spines, suggesting an important role of MAGI-2 during neuronal morphogenesis (Iida et al., 2007). During investigation of the role of MAGI-2, special consideration needs to be given to the existence of its three splicing variants, MAGI-2a, b, and c (Hirao et al., 2000). The re- ported MAGI-2 knockout mice described above showed functional loss of MAGI-2a only; therefore, the in vivo functions of the other splicing MAGI-2 forms remain largely unknown. To help determine MAGI-2 function, we inserted the Venus fluorescence reporter cassette into exon 6 of the mouse Magi2 gene (creating the Venus knock-in mouse for Magi2) using a homolo- gous recombination technique. Exon 6 of Magi2 is common to all three variants of the protein. As a result, insertion of the Venus re- porter cassette into exon 6 enabled visualization of the total distri- bution of all three MAGI-2 forms. The Venus protein is a mutant form of the enhanced yellow fluorescent protein (EYFP) that shows a 30-fold increase in fluorescence compared to that of the original EYFP (Nagai et al., 2002). Furthermore, the Venus protein can be detected by the green fluorescent protein (GFP) antibody by using Western blotting and immunohistochemistry. Due to this strong reporter activity, it is possible to determine the detailed tissue distribution of a specific gene product. For example, we previously 1567-133X/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.gep.2012.01.006 ⇑ Corresponding author. Tel.: +81 22 717 8770; fax: +81 22 717 8883. E-mail address: knishimori@m.tohoku.ac.jp (K. Nishimori). Gene Expression Patterns 12 (2012) 95–101 Contents lists available at SciVerse ScienceDirect Gene Expression Patterns journal homepage: www.elsevier.com/locate/gep