Supplementary description Title:

1. Supplementary description Title: A novel mouse model for Down syndrome that harbor a single copy of human artificial chromosome (HAC) carrying a limited number of genes from human chromosome 21 Journal name: Transgenic Research Authors: KenichiMiyamoto1, Nobutaka Suzuki2,Kosuke Sakai1, Shuichi Asakawa3, Tsuneko Okazaki2,JunKudoh1, Masashi Ikeno1,2, Nobuyoshi Shimizu4 1Laboratory of Gene Medicine, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan, 2Chromo Research Inc., 1212 Shihongi, Midori-ku, Nagoya, Aichi 458-0039, Japan, 3Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, 4Advanced Research Center for Genome Super Power, Keio University, 2 Okubo, Tsukuba, Ibaraki 300-2611, Japan. Corresponding author: Nobuyoshi Shimizu, Ph.D. Advanced Research Center for Genome Super Power, Keio University, 2 Okubo, Tsukuba, Ibaraki 300-2611, Japan Phone: +81- 29-865-1641 FAX: +81-29-865-1641 E-mail: smzgsp@dmb.med.keio.ac.jp

2. Miyamoto et al., Supplementary Fig. S1 Skeletal muscle Small intestine Adrenal grand No DNA Spleen Thymus Kidney Colon Testis Brain Lung Heart Liver Eye Human CBS Mouse Cbs Human CRYAA Mouse Cryaa Human U2AF1 Mouse U2af1 Mouse Gapdh

3. Miyamoto et al., Supplementary Fig. S2 Fetal Skeletal muscle Blood leukocyte Skeletal muscle Small intestine Fetal Thymus Fetal Kidney Fetal Spleen Fetal Heart Fetal Brain Fetal Lung Fetal Liver Pancreas Placenta Prostate No DNA Thymus Kidney Spleen Ovary Colon Testis Heart Brain Lung Liver hCBS hCRYAA hU2AF1 hGAPDH

4. Miyamoto et al., Supplementary table. S1 Supplementary table S1. Transmission rate of HAC in parental TMC mouse individuals.

5. Supplementary materials and methods Human Multiple Tissue cDNA panel analysis: Expression analysis of three genes (hCBS, hCRYAA and hU2AF1) was performed using the Human Multiple Tissue cDNA (MTC) panels (Clontech; I, II and fetal panels) containing cDNAs from 24 human tissues. The primers specific to human transcripts were the same as used in Fig 3 and were described in Materials and Methods of the manuscript. Approximately 100 pg of each cDNAs were used as template. PCR condition was as follows; 94ºC for 2 min, 35 cycles of 94ºC for 15 sec, 60ºC for 30 sec and 72ºC 1 min, and 1 cycle of 72ºC 5 min. Control PCRs were performed with the hG3PDH usingprimer pairs; Fwd 5’-TGAAGGTCGGAGTCAACGGATTTGGT-3’ and Rev 5’-CATGTGGGCCATGAGGTCCACCAC-3’. RT-PCR for expression analysis of human transgenes and orthologous mouse genes: Total RNA was extracted from the tissues of a 5 week-old TMC male mouse at fourth generation of backcross using TRIzol reagent (Invitrogen). Five mg of RNAs without DNase treatment were used as template for reverse transcription by Superscript III reverse transcriptase (Invitrogen), then PCRs were conducted using Platinum Taq polymerase (Invitrogen) under the following conditions; 95ºC for 2 min, 35 cycles of 95ºC for 15 sec, 60ºC for 30 min and 72ºC for 1 min, and 1 cycle of 72ºC for 7 min. The primers used were the same as used in RT-PCR shown in Fig. 2 and were described in Materials and Method of the manuscript.

6. Supplementary figure Legends Supplementary Fig. S1. Expression analysis of three human transgenes and orthologous mouse genes in various tissues of TMC mouse. RT-PCRs specific to transcripts from three human transgenes (hCBS, hCRYAA and hU2AF1) and mouse orthologs were performed using total RNAs from thirteen tissues extracted from a 5 week-old TMC male mouse at fourth generation of backcross. Human or mouse specific primers used were the same as those used in Fig. 3. RT-PCR consists of 35 cycles of reactions were performed. Supplementary Fig. S2. Expression analysis of three genes (hCBS, hCRYAA and hU2AF1) in human tissues. The expressions of hCBS, hCRYAA and hU2AF1 in various human tissues were examined by RT-PCR using Human Multiple Tissue cDNA (MTC) panel (Clontech). hGAPDH was used for loading control. Supplementary table S1. Transmission rate of HAC in parental TMC mouse individuals. The number of TMC mouse offspring from a TMC mouse was counted during back cross with C57BL/6J.