205

Laboratory Animal Research Center

Division of Animal Genetics
先進動物ゲノム研究分野
Professor
Senior Assistant Professor
Assistant Professor
Project Assistant Professor

Tomoji Mashimo, Ph.D.
Kazuto Yoshimi, Ph.D.
Saeko Ishida, D.V.M., Ph.D.
Tomoaki Fujii, Ph.D.

教 授
講 師助 教
特任助教

博士（人間・環境学） 真博士（医科学）
吉博士（医学）
石博士（理学）
藤下見田井知 士
一 人紗恵子智 明

Genome engineering technologies achieve a "revolution" in life science andmedical science. These techniques allow us to manipulate genes of interest forseveral purposes. Using those technologies, we have developed many usefulstrains in mice and rats. We are now focusing on generating "humanized animals"
or "immunodeficient animals". These valuable animals can be used forxenotransplantation of human cells/tissues including human HSCs and IPSCs. We
are also developing therapeutic strategies such as cell therapy and gene therapy
with genome editing tools.

Characterization of several severe combined immunodeficiency rats for humanized models
Ryuya Iida, Kousuke Hattori, Alejandro Soto-Gutierrez2, Kazuki Takeishi3, Kazuto Yoshimi, Saeko
Ishida, Tomoji Mashimo
1, Institute of Experimental Animal Sciences, Graduate School of Medicine, Osaka University, Osaka
565-0871, Japan2, Department of Surgery and Science, Graduate
School of Medical Sciences, Kyushu University,Fukuoka, Japan3, Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA
The immunodeficiency animals are valuable experimental models, not only in the studies of immunodeficiency related diseases, they also have goodperformances in the application of grafting varioustissues. Therefore, the immunodeficiency animals
have been widely applied in generation of humanizeanimals, regeneration medical, tumor researches, etc.
By utilizing the CRISPR/Cas9 genome editing tool,
we generated a Severe Combined Immunodeficiency(SCID) rat model, which carry homozygous mutationin both Il2rg and Rag2 gene. These combined mutations caused the retard of both T cell and B cell development, as well as the deﬁciency of functional NKcells and cytokines secretion, providing favorable invivo environment for the subsistence and proliferation of exogenous cells or tissues. Other than the immunodeficiency animals that generated by combining the mutations from different rat strains, our SCID
rats have a clear genetic background of F344 rats. OurSCID rats has been set up a Bio-recourse project, andprovided to institutes and researchers all over theworld. In the following studies, we devote to modifying other genes in these SCID rats, to improve the efficiency of xenograft and alleviate acute xenogeneic
graft-versus-host disease (GVHD) in the recipient
SCID rats.
Efficient and precise gene disruption with CRISPR-Cas3 in human T cells
Tomoaki Fujii, Kazuto Yoshimi, Kohei Takeshita1,Kazumasa Yokoyama2, Koji Tamada3, Tomoji
Mashimo
1 Advanced Photon Technology Division, RIKEN206
SPring-8 Center
2 C4U Corporation
3 Department of Immunology, Yamaguchi University Graduate School of Medicine
Chimeric Antigen Receptor (CAR)-T cell therapy
is promising cancer immunotherapy. Conventionally,
CAR-T cells are produced from autologous T cells,
but this can be high costs, long manufacturing periods, and difficulties in ensuring uniform quality ofcell sources. We reported that genome editing using
CRISPR-Cas3 system is possible in human cells. Thenovel genomic editing system can produce fewer
off-target and mosaic mutations compared to CRISPR-Cas9, which is the most widely used genome editing tools. Our research aims are to use CRISPR-Cas3
to generate a safe and effective CAR-T therapy. To
overcome the limitations of producing autologous
CAR-T cells, we investigated whether CRISPR-Cas3
system induces genetic modifications on genes involved in graft-versus-host disease and immune rejection in Jurkat cells, a human acute T cell leukemia
cell line. As a result of this experiment, it caused lossof function of the target genes and we found that
there were no off-target mutations observed in itscells. In addition, this system can also generate targeted deletions of the target genes in human primary Tcells. These results suggest that the CRISPR-Cas3 system could be a powerful genetic tool for generating
allogenic CAR-T cells.
Generation of several genetically engineered miceand rat models via genome editing technologies
Kosuke Hattori, Yuko Yamauchi, Natsuki Matsushita1, Kazuto Kobayashi2, Saeko Ishida, KazutoYoshimi, Tomoji Mashimo

1, Division of Laboratory Animal Research, Aichi
Medical University School of Medicine
2, Department of Molecular Genetics, Institute ofBiomedical Sciences, Fukushima Medical University School of Medicine
CRISPR-Cas9 systems have been widely used forgene targeting in mice and rats. The non-homologous
end joining (NHEJ) repair pathway, which is dominant in zygotes, efficiently induces insertion or deletion (indel) mutations as gene knockouts (KOs) at targeted sites, whereas gene knock-ins (KIs) via
homology-directed repair (HDR) are difficult to generate.
We have developed the two KI strategies withCRISPR/Cas9 for the large genomic regions in rodents. One is the long single strand DNA (lssDNA)-mediated KI method. Microinjection and electroporation of originally synthesized lssDNAs withgRNA and Cas9 mRNA could produce several typesof KI mice and rats with a good efficiency such as
GFP-tagging, floxed and repeat sequence replacement. In addition, we have also used a double-stranded DNA (dsDNA) donor template with Cas9 and two
single guide RNAs (sgRNAs), one designed to cut thetargeted genome sequences and the other to cut boththe flanked genomic region and one homology arm ofthe dsDNA plasmid, resulting in 20%–50% KI efficiency among G0 pups. This combinational methodof NHEJ and HDR mediated by the CRISPR-Cas9 system, named Combi-CRISPR, facilitates the efficientand precise KIs of plasmid DNA cassettes in mice andrats.
We have established genetically-modified miceand rats via these genome editing strategies with several collaborators.

Publications
1.		Junki Kawakami, Satoshi Hisanaga, Yuki Yoshimoto, Tomoji Mashimo, Takehito Kaneko, NaotoYoshimura, Masaki Shimada, Makoto Tateyama,
Hideto Matsunaga, Yuto Shibata, Shuntaro Tanimura, Kosei Takata, Takahiro Arima, [ ... ], TakeshiMiyamoto		Remnant tissue enhances early postoperative biomechanical strength and infiltration of Scleraxis-positive cells within the grafted tendon in a rat
anterior cruciate ligament reconstruction model		PLOS ONE https://doi.org/10.1371/journal. pone.
02939442.		Natsuki Matsushita, Shigeki Kato, Kayo Nishizawa, Masateru Sugawara, Kosei Takeuchi, Yoshiki
Miyasaka, Tomoji Mashimo, *Kazuto KobayashiP rotocol for highly selective transgene expression
through the flip-excision switch system by using aunilateral spacer sequence in rodents.

S TAR protocols 4(4) 102667-102667 2023
3.		Hiroki Furuie, *Yuka Kimura, Tatsuhiro Akaishi,
Misa Yamada, Yoshiki Miyasaka, Akiyoshi Saitoh ,Norihiro Shibuya, Akiko Watanabe, Naoki Kusunose, Tomoji Mashimo, Takeo Yoshikawa, Mitsuhiko Yamada, Kazuho Abe, Hideo Kimura		Hydrogen sulfide and polysulfides induce GABA/glutamate/D-serine release, facilitate hippocampal LTP, and regulate behavioral hyperactivity		Sci Rep 2023 31;13(1):17663.
4.		Yaguang Luo, Shinya Akatsuka, Yashiro Motooka,
Yingyi Kong, Hao Zheng , Tomoji Mashimo,Tatsuhiko Imaoka, *Shinya Toyokuni		BRCA1 haploinsufficiency impairs iron metabolism to promote chrysotile‐induced mesothelioma
via ferroptosis resistance		Cancer Sci. 2023, 114(4): 1423–1436.
5.		Masato Kinboshi, Saki Shimizu, Kentaro Toku-207
dome, Tomoji Mashimo, Tadao Serikawa, Hidefumi Ito, Ryosuke Takahashi, Akio Ikeda, *Yukihiro Ohno		Imbalance of glutamatergic and GABAergic neurotransmission in audiogenic seizure-susceptible
Leucine-rich glioma-inactivated 1 (Lgi1)-mutantrats		Heliyon 9(7) e17984-e17984 2023
6.		Victoria Morley, Karamjit Singh Dolt, Carlos J Alcaide-Corral, Tashfeen Walton, Christophe Lucatelli, Tomoji Mashimo, *Adriana A S Tavares,*Tilo Kunath		In vivo18F-DOPA PET imaging identifies a dopaminergic deficit in a rat model with a G51D α-synuclein mutation		Frontiers in Neuroscience 2023 24:17:1095761.
7.		Masayuki Mori, Jian Dai, Hiroki Miyahara, Ying
Li, Xiaojing Kang, Kazuto Yoshimi, Tomoji Mashimo, Keiichi Higuchi, Kiyoshi Matsumoto		Cyba and Nox2 mutant rats show different incidences of eosinophilia in the genetic backgroundand sex-dependent manner		Exp Anim. 2023 May 17;72(2):233-241.