26105,



72535cm

200

4500g

77 

58777TALEN mRNA
7634Platinum

ssODN
444.7%

6417.6%4350-500mg/dl
4373200-250mg/dl

7	 2.

473737Zinc Finger Nuclease mRNA

84CEL-1
33324413

26105747358 CRISPR/Cas

7PAM

7781mer

14sgRNA

ssODN

NGG

sgRNA

,

Cas9 mRNA

571461412321CRIPSR/Cas
48±7.4%450%

1.8% (1/574PAMsgRNA7744.7%4764617.6%

Platinum TALEN mRNA

734350-500mg/dl
43Platinum TALEN

ssODN4ng/ul

717 Platinum TALEN
734NGG

3CRIPSR/CasmRNA)

37200-250mg/dl
7743

,43,

37Hi-Fi Zinc Finger Nuclease

Finger NucleasemRNA

8 ng/µl

90% N2 	 

ISM-1

5% CO2, 5% O2,10µl

Surveyor mutation detection

7PCR

PCR

kit (Transgenomic, 706025)
33PCR

CEL-1

Hi-Fi ZFN

eHi-Fi ZFN

ZFN

88PCR

eHi-Fi32

82bp
3334450bp
2314eHi-Fi Zinc

Subject No.

26105Title

Establishment of diabetic marmoset model for preclinical research

Researchers

Tadashi Okamura, Erika Sasaki

Key word

Diabetic model, Marmoset, Preclinical research, Gene-editing

Abstract

In biomedical researches, laboratory animals bridge the gap between in vitro studies andclinical medicine. Although the most popular laboratory animal is rodents, rodent models are insufficient as
diabetic models for preclinical research to evaluate the efficacy and safety of autologous and xeno-islet celltransplantations. Non-human primates(NHPs) also play important roles in preclinical research in the biomedicalsciences. The common marmoset is a small New World primate that is native to the Atlantic coastal forests innortheastern Brazil in South America. Adult marmosets have an average height of 25-35cm, and weight200-500g. Marmosets take 15months to mature sexually, carry babies for 5months and often give birth to 2-3
young. The aim of our project is to establish diabetic marmoset model to introduce gene mutation for diabetes
gene by genome-editing technique using CRISPR/Cas system or TALEN. Because NHPs must be maintained by
non-sibling mating, dominant-negative mutation of the modified gene would be suitable to generate NHPs
genetically modified diabetic models to express phenotype.

1. Validation of diabetes gene mutation in mice (Tadashi Okamura)
Given that the ethical concerns regarding use of NHPs, it is important to validate the modified gene using
other laboratory animals. Platinum TALEN mRNAs for diabetes gene and ssODN which introduce diabetes
mutation were co-injected into the male pronucleus of single cell mouse embryo. We found that 44.4%(34/76) ofthe pups carried the mutations and 6 of them (17.6%) carried diabetes mutation. Blood glucose levels of diabetic
mice carrying heterozygous mutation for diabetes gene were markedly higher than those of control mice, even at
4weeks of age. Insulin tolerance test revealed that diabetic mice have a normal glycaemic response to exogenous
human insulin.

2. Method for estimation of mosaicism in the genome edited marmoset embryos (Erika Sasaki)
The genome editing technologies enable to produce target gene knock out (KO) non-human primates (NHP).
Recently, generations of target gene KO NHP have been reported. However, most likely due to the highmosaicism of mutant genes in NHPs, mutant animal phenotypes have not yet been observed. In this study, wehave developed methods for estimation of mosaicism before start target gene KO animal production. In vitro
synthesized ZFN mRNA was injected into the cytoplasm of pronuclear stage embryos and were cultured up tothe 8-cell stage. Then, 8-cell stage embryos were split in to each blastomere to be analyzed. After PCR, CEL-1
assays were performed by Surveyor mutation detection kit (Transgenomic, 706025). As the results of the CEL-1
assays showed that the genetic modification rate estimated from the number of mutant blastomeres was 37.5%(3/8) for HiFi-ZFN and 100% (8/8) for eHiFi-ZFN. Furthermore, sequence analysis revealed that eHiFi-injected
embryos contained only two types of mutant genes. These results suggest that genome modification in
eHiFi-ZFN-derived embryos occurred at the one-cell stage for female embryos and at the two-cell stage for maleembryos. Using this method, we were able to estimate the mosaicism of the target gene KO marmoset before
proceed animal production.

PVZ^# ¤h/s£I¤MÉ¿Í ¤rssODN

Plutinum TALEN mRNA¸ÉÏÇÑÉ¼½À¥2¯

Â²µ-g, Ç²·Î¶Å¼Í
E"ÄÁ³aa+T}'%R)¸Ï»ÀË·À±ÇÑÉ¼½À
FC`cfw£Ç²·Î²Ïº´·¹ÊÏ²Ï»ÌÏ
P/R]"g,\GScf

Ô"a+TÇÑÉ¼½ÀÉ¿ÍQOL¤>!

Sasaki, E. et al, 2009Ð}')8n[_¯ÐudxlM54ÃÀ£yÐË½À¡¦§& ¯©l¤9«\Gcf?*¢¨ÐSTZ£¬¯a+TtW£<:4

a+T}'%R±)¯¡£¬®i­ma± ¯g,\G¤{3UÐU"Ô"a+TÐÔ"a+TÐ64gLH26
1 CRISPR/Cas

Construct

Mutants/Total
Mice Tested(%)

SNP-KI (%)

SNP

1/57 (1.8)

0/1 (0)	
0.75IU/Kg)[mg/dl]Plutinum TALEN
2 Platinum TALEN

Construct

Mutants/Total
Mice Tested(%)

SNP-KI (%)

SNP

34/76 (44.7)

6/34 (17.6)
06

Platinum TALEN mRNA

4ng/ul30

60

90

120 [min]H26.0¤7D2ÓOJ¤}'%R±(¯@K±W

Method for estimation of mosaicism in the genome edited marmoset embryos

Culture embryos
until 8 cell stage

Microinjection
HiFi-ZFN1

2

3 4 5 6 7

37.5% (3/8)

8M

M

1

Split theblastomeres
eHiFi-ZFN

2 34 5 6 7 8

100% (8/8)

PCR&CEL-1 assay
ÇÑÉ¼½À`±Q
%R)N¸Ï»ÀË·À±|;

a+TÇÑÉ¼½ÀpN¤

Wild Type

ACTTGGTGCAGTACCGGACTGACTGGGACCACACCTGGACTGTGAGTGACTAGGATGTGA

HiFi Bm3

ACTTGGTGCAGTACCGGACTGACTGG-ACGGACTGACCTGGACTGTGAGTGACTAGGATG
ACTTGGTGCAGTACCGGACTGACT-----------------GTGAGTGACTAGGATGTGAACTTGGTGCAGTACCGGACTGACTGG-ACGGACTGACCTGCACTGTGAGTGACTAGGATG
ACTTGGTGCAGTACCGGACTGACTGG-ACGGACTGACCTGGACTGTGAGTGACTAGGATG

HiFi Bm4
HiFi Bm8-1bp,+4bp:1/9-17bp:2/9-1bp,+4bp:1/9-1bp,+4bp:1/9

eHiFi Bm1-8 ACTTGGTGCAGTACCGGACTGACTGGGATACCACACCTGGACTGTGAGTGACTAGGATGT +2bp:15/32-------------------------------------------GAGTGACTAGGATGTGA -50bp:17/32

研究発表及び特許取得報告について

課題番号：

２６指１０５

研究課題名： 糖尿病マーモセットを用いた前臨床試験システムの構築
主任研究者名：岡村匡史

論文発表
論文タイトル

著者

掲載誌

掲載号

The thymic cortical epithelium
determines the TCR repertoire ofIL-17-producing cdT cells.

Nitta, T., Muro, R., Shimizu, Y.,
Nitta, S., Oda, H., Ohte, Y.,
Goto, M., Yanobu-Takanashi, R.,
EMBO reports
Narita, T., Takayanagi, H.,
Yasuda, H., Okamura,T., Murata,
S. and Suzuki, H.

16

2015

The histidine transporter SLC15A4
coordinates mTOR-dependent
inflammatory responses andpathogenic antibody production.

Kobayashi T, Shimabukuro-DemotoS, Yoshida-Sugitani R, FuruyamaTanaka K, Karyu H, Sugiura Y,Immunity
Shimizu Y, Hosaka T, Goto M, KatoN, Okamura T, Suematsu M,
Yokoyama S, Toyama-Sorimachi N.

41

2014

Marmoset Neuroscience.

Tokuno H, Watson C, Roberts A,
Sasaki E, Okano H.

Neurosci Res.

93

2015

Neurosci Res93

2015

9(4)

2014

Prospects for genetically modified
non-human primate models,
Sasaki E.
including the common marmoset.

Takahashi T, Hanazawa K, Inoue T,
Birth of healthy offspring
Sato K, Sedohara A, Okahara J,
following ICSI in in vitro-matured
Suemizu H, Yagihashi C, Yamamoto PLoS One
common marmoset (Callithrix
M, Eto T, Konno Y, Okano H,jacchus) oocytes.
Suematsu M, Sasaki E.

年

学会発表
タイトル

発表者

学会名

場所

年月

高梨（矢延）理絵子、新井哲也、後CRISPR/Casシステムによるドミナント
藤元人、新田剛、中野堅太、清水有
ネガティブ型遺伝子変異の同定
紀子、岡村匡史

第4回ゲノム編集
広島研究会

2014年10月

CRISPR/Casシステムを利用したナイー
岡村匡史、高梨理絵子、後藤元人、
ブT細胞減少（TN）マウスの原因遺伝清水有紀子、鈴木春巳、新田剛子の同定

第61回日本実験
動物科学技術20142014年5月

札幌

その他発表(雑誌、テレビ、ラジオ等)
タイトル

発表者

発表先

場所

年月日

特許権者(申請者）（共願は全記登録日(申請日)

出願国

該当なし
特許取得状況について
発明名称※出願申請中のものは()記載のこと。
登録番号

該当なし
※該当がない項目の欄には「該当なし」と記載のこと。
※主任研究者が班全員分の内容を記載のこと。