Implementation of the modified-SHIRPA protocol for screening of dominant phenotypes in a large-scale ENU mutagenesis program Hiroshi Masuya, 1 Maki Inoue, 1 Yumiko Wada, 1 Aya Shimizu, 1 Junko Nagano, 1 Akiko Kawai, 1 Ayako Inoue, 1 Tomoko Kagami, 1 Taeko Hirayama, 1 Ayako Yamaga, 1 Hideki Kaneda, 1 Kimio Kobayashi, 1 Osamu Minowa, 1 Ikuo Miura, 1 Yoichi Gondo, 2 Tetsuo Noda, 1 Shigeharu Wakana, 1 Toshihiko Shiroishi 1 1 Functional Genomics Research Group, RIKEN Genomic Sciences Center, 3-1-1 Kouyadai, Tsukubai, Ibaraki, 305-0074, Japan 2 Population and Quantitative Genomics Team, RIKEN Genomic Sciences Center, 1-7-22 Suehiro-cho, Tsurumi-ku, Yokohama City, Kanagawa, 230-0045, Japan Received: 22 July 2004 / Accepted: 26 July 2005 Abstract SHIRPA is a three-stage protocol for the compre- hensive assessment of primarily mouse behavior. The first stage consists of high-throughput pheno- typing of 33 behavioral observations and 7 metabolic or disease observations. We modified this part of the protocol by integrating new morphologic observa- tions into the initial phenotype assay of behavior and dysmorphology. Behavioral observations assessed by this protocol, now referred to as the ‘‘modified- SHIRPA,’’ are compatible with the original ‘‘SHIR- PA’’ protocol. Using modified-SHIRPA, we screened dominant phenotypes of more than 10,000 G 1 prog- eny generated by crossing DBA/2J females with ENU-treated C57BL/6J males. To date, we have ob- tained 136 hereditary-confirmed mutants that ex- hibit behavioral and morphologic defects. Some independent mutant lines exhibited similar pheno- types, suggesting that they may represent alleles of the same gene or mutations in the same genetic pathway. They could hold great potential for the unraveling of the molecular mechanisms of certain phenotypes. Mouse mutants generated by gene-driven or pheno- type-driven mutagenesis are powerful tools for exploring and elucidating gene function. In general, a comprehensive, standardized, and high-throughput protocol is essential for sketching out phenotypic appearance before specific in-depth analysis of the phenotype. SHIRPA is a three-stage protocol that primarily allows behavioral characterization. The first stage consists of up to 40 separate observational profilings, including gait, balance, various reflection assays, and startle response. All behaviors are scored to provide a semi-quantitative assessment of each parameter. It gives a rough behavioral profile of the animal. It provides indications about specific behav- ioral anomalies resulting from muscular, lower motor neuronal, spinocerebellar, sensory, neuropsychiatric, and autonomic defects. The second stage involves basic phenotype tests that provide slow output data about spontaneous locomotor activity, food intake, and pathology. The third stage uses more sophisti- cated tests for the analysis of neurologic mutants. SHIRPA allows the detection of physiologic, behav- ioral, and neurologic changes in phenotypes by screening the huge number of mice produced in large- scale ENU-mutagenesis projects (Rogers et al. 1997; Nolan et al. 2000). It is has also been used for the behavioral characterization of various mouse strains and specific mutants (Rafael et al. 2000; Hatcher et al. 2001; Rogers et al. 2001; Norreel et al. 2001). We conducted a large-scale mouse ENU-muta- genesis project in RIKEN Genomic Sciences Center (GSC). At the initial stage of the project, we established a screening system for the detection of early-onset dominant phenotypes by observing 812-week-old mice (Inoue et al. 2004; Masuya et al. 2004). In order to expand the effectiveness of the first stage of phenotype analysis by the original SHIRPA protocol, we added new morphologic observation tests and named the protocol ‘‘modified-SHIRPA.’’ Correspondence to: Toshihiko Shiroishi; E-mail: tshirois@lab.nig. ac.jp DOI: 10.1007/s00335-005-2430-8  Volume 16, 829837 (2005)  Ó Springer Science+Business Media, Inc. 2005 829 Original Contributions