DNA RESEARCH 20, 255–262, (2013) doi:10.1093/dnares/dst007 Advance Access publication on 29 March 2013 Development and Characterization of cDNA Resources for the Common Marmoset: One of the Experimental Primate Models } SHOJI Tatsumoto1,†,NAOKI Adati1,‡,YASUSHI Tohtoki1, ,YOSHIYUKI Sakaki1,§,THORSTEN Boroviak2, SONOKO Habu3,HIDEYUKI Okano4,HIROSHI Suemizu5,ERIKA Sasaki6, and MASANOBU Satake7,* RIKEN Genomic Sciences Center, Suehiro-cho 1-7-22, Tsurumi-ku, Yokohama 230-0045, Japan1; Centre for Stem Cell Research, Wellcome Trust and Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK2; Department of Immunology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421, Japan3; Department of Physiology, School of Medicine, Keio University, Shinano-machi 35, Shinjyuku-ku, Tokyo 160-8582, Japan4; Biomedical Research Department, Central Institute for Experimental Animals, Tonomachi 3-25-12, Kawasaki-ku, Kawasaki 210-0821, Japan5; Department of Applied Developmental Biology, Central Institute for Experimental Animals, Tonomachi 3-25-12, Kawasaki-ku, Kawasaki 210-0821, Japan6 and Institute of Development, Aging and Cancer, Tohoku University, Seiryo-machi 4-1, Aoba-ku, Sendai 980-8575, Japan7 *To whom correspondence should be addressed. Tel. þ81-22-717-8477. Fax. þ81-22-717-8482. Email: [email protected] Edited by Dr Toshihiko Shiroishi (Received 9 May 2012; accepted 1 March 2013) Abstract The common marmoset is a new world monkey, which has become a valuable experimental animal for biomedical research. This study developed cDNA libraries for the common marmoset from five different tissues. A total of 290 426 high-quality EST sequences were obtained, where 251 587 sequences (86.5%) had homology (1E2100) with the Refseqs of six different primate species, including human and marmoset. In parallel, 270 673 sequences (93.2%) were aligned to the human genome. When 247 090 sequences were assembled into 17 232 contigs, most of the sequences (218 857 or 15 089 contigs) were located in exonic regions, indicating that these genes are expressed in human and marmoset. The other 5578 sequences (or 808 contigs) mapping to the human genome were not located in exonic regions, suggesting that they are not expressed in human. Furthermore, a different set of 118 potential coding sequences were not similar to any Refseqs in any species, and, thus, may represent unknown genes. The cDNA libraries developed in this study are available through RIKEN Bio Resource Center. A Web server for the marmoset cDNAs is available at http://marmoset.nig.ac.jp/index.html, where each marmoset EST sequence has been annotated by reference to the human genome. These new libraries will be a useful genetic resource to fa- cilitate research in the common marmoset. Key words: common marmoset; cDNA; gene resource 1. Introduction † Present address: National Institute of Genetics, Yata 1111, Mishima 411-8540, Japan. The mouse is a widely used and well-studied model ‡ Present address: Research Equipment Center, Hamamatsu animal for biomedical research. Many techniques for University School of Medicine, Handayama 1-20-1, Higashi- ku, Hamamatsu 431-3192, Japan. sophisticated genetic manipulations to model dis- } Present address: Division of Cancer Genomics, National Cancer eases have been established and researchers hope Center Research Institute, Tsukiji 5-1-1, Chuo-ku, Tokyo 104- that the results obtained can be extrapolated to 0045, Japan. § Present address: Toyohashi University of Technology, humans. Although this assumption is true in some Hibarigaoka 1-1, Tenpaku-cho, Toyohashi 441-8580, Japan. cases, there are several areas of biomedical research # The Author 2013. Published by Oxford University Press on behalf of Kazusa DNA Research Institute. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by- nc/3.0/), which permits non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected]. 256 cDNA Resources for the Common Marmoset [Vol. 20, where this proves to be more difficult. These areas 2. Materials and methods include neuroscience, behavioural research, toxicol- ogy, drug development, and infectious diseases.1 To 2.1. RNA extraction and library construction overcome these limitations, efforts have been made Cytoplasmic RNA was extracted from the liver (MLI), to carry out biomedical studies in non-human brain and spinal cord (MSC), spleen (MSP), testis primate model organisms, as the latter are more (MTE), and embryonic stem (ES) cells (MES) of the closely related to humans. common marmoset using Trizol reagent. Marmoset One of these established non-human primate ES cells were cultured as described previously.11 Full- model organisms is Callithrix jacchus (common mar- length cDNA libraries were constructed from the total moset). The marmoset is a small new world monkey RNAs of the aforementioned tissues/cells using a and offers many advantages as an experimental vector-capping method.12 cDNAs generated from MLI, animal over other non-human primates. It is small MSC, MSP, MTE, and MES were ligated into pGCAP1, in size, which makes it comparatively easy to handle. pGCAPzf3, pGCAPzf3, pGCAP10, and pGCAP10 vectors, Furthermore, it has been bred in captivity and its respectively. Colonies of Escherichia coli transformants progeny have been maintained for .30 years in la- were picked randomly, inoculated into 384-well plates boratory environments. Also, it does not harbour or using a Flexys colony picker (Genomic Solutions Ltd., transmit hazardous infectious agents. Therefore, the Cambridgeshire,UK),andstoredat2808C. common marmoset is increasingly used in biomedical research worldwide. For example, models of auto- immune diseases involving the central nervous 2.2. EST sequencing system have been developed in the common marmo- Colonies were picked from 99 plates for MSC; 200 set and it has been used extensively as a primate plates each for MES, MSP, and MTE; and 201 plates model.2 – 4 More recently, genetically modified for MLI (Supplementary Table S1). Sequencing templates common marmosets have been produced successfully were prepared using a TempliPhi DNA Amplification Kit and their transgenes have been transmitted through (GE Healthcare UK Ltd., Buckinghamshire, UK). The se- the germ line.5 In the future, it would be very useful quencing reactions for the 50-end directional ESTs were to develop transgenic marmosets as models of conductedusingaBigDyeTerminatorv3.1Cycle human diseases. Sequencing Kit (Applied Biosystems, Inc., CA, USA). The Intensive efforts have been made to develop re- sequence primers used for pGCAP1, pGCAP10, and search tools for using the common marmoset as an pGCAPzf3 were 50-AGGCCTGTACGGAAGTGT-30,50-AGG experimental animal. For instance, several lines of CCTGTACGGAAGTGT-30,and50-CAAGGCGATTAAGTT monoclonal antibodies have been prepared, which GGGT-30, respectively. The sequencing reaction products are directed against immunity-related antigens of were purified by ethanol precipitation and loaded onto the marmoset.6 – 8 Many, but not all anti-human 3730 DNA Analyzers (Applied Biosystems Inc.). antigen antibodies cross-reacted with the corre- sponding marmoset antigens, so it was necessary to establish marmoset-specific antibodies.9 Apilot 2.3. Selection of high-quality EST data gene analysis study reported cDNA sequencing of im- The raw sequence data were basecalled using the munity-related genes.10 Based on genome-wide ana- KB basecaller program, which identified 345 600 lyses, a draft sequence of the common marmoset, sequences. A cross-match program was applied to known as caljac3, was produced and made available the raw data to remove sequences derived from to the public via the genome browser of the vectors and those added as caps during plasmid University of California Santa Cruz (http://genome. construction (2minimach 10, 2minscore 20). ucsc.edu/). Low-quality sequences [quality value (QV) ¼ one for The current study describes the preparation of each nucleotide + 3 neighbouring nucleotides meas- cDNA libraries for the common marmoset using uring ,105 in length] were masked by N (average five different cell types/tissues, which resulted in QV . 15). If N was longer than 10 nucleotides, the identification of 290 426 high-quality EST sequences located 30 of these N were removed. sequences. These sequences were characterized by Finally, only high-quality sequences longer than 100 comparison with the sequences of six primate nucleotides were selected and used in further ana- species, including humans. Overall, the EST lyses (see the high quality column, Supplementary sequences transcribed in the marmoset shared Table S1). There were 290 426 high-quality ESTs, many common features with those from humans, which represented 84% of the raw sequence data. whereas a small fraction was found to be unique High-quality EST sequences were obtained frequently to the marmoset. and were relatively abundant (93%) in MES and No. 3] S. Tatsumoto et al. 257 MTE, whereas their frequencies were comparatively assembled to estimate how many genes/transcrip- low (72–80%) in MLI, MSC, and MSP. tional units were read as ESTs (Table 1). Overlapping The sequence length was higher in MES and MTE sequences were clustered within the longest sequence (702–706 nucleotides), than in MLI, MSC, and MSP using