Verification of gene-edited pig production method from Japan
Project/Area Number |
21K19527
|
Research Category |
Grant-in-Aid for Challenging Research (Exploratory)
|
Allocation Type | Multi-year Fund |
Review Section |
Medium-sized Section 55:Surgery of the organs maintaining homeostasis and related fields
|
Research Institution | Osaka University |
Principal Investigator |
宮川 周士 大阪大学, 微生物病研究所, 招へい研究員 (90273648)
|
Co-Investigator(Kenkyū-buntansha) |
前田 晃 大阪大学, 大学院医学系研究科, 招へい教員 (00319708)
|
Project Period (FY) |
2021-07-09 – 2024-03-31
|
Project Status |
Granted (Fiscal Year 2022)
|
Budget Amount *help |
¥6,500,000 (Direct Cost: ¥5,000,000、Indirect Cost: ¥1,500,000)
Fiscal Year 2023: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Fiscal Year 2022: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
Fiscal Year 2021: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
|
Keywords | GGTA1 / CMAH / CRISPR/Cas3 / ブタ線維芽細胞 / Hanganutziu-Deicher抗原 / ブタ内在性レトロウイルス / 再生医療 / バイオ人工臓器 / ノックアウト / CRISPR |
Outline of Research at the Start |
バイオ人工臓器(異種移植)の分野では、当初、相同組み替え法は困難を極めたが、新法によるknockout(KO)法が我々も含めブタでも成功している。しかし、CRISPR/Cas9法には特許の問題が絡んでおり、今回研究協力者の真下知士グループが開発したCRISPR/Cas3法を応用してKO-ブタの作出を進める。現時点でブタ細胞での有効性はまだ確立されていない。ブタの繊維芽細胞、等を使って、先ずはこのCas3法で既にCas9法でKO経験のあるブタGalTと4GalNT2のKOを比較しながら行う。次に、SLA ClassI,II、とPERV(内在性レトロウイルス)-CのKOの有効性の検証を行う。
|
Outline of Annual Research Achievements |
異種移植の開発に国産のCRISPR/Cas3法を検討している。既にCas9法でknockout(KO)経験のあるブタCMAHやb4GalNT2のKOと比較しながら、GalT(GGTA1)のKOをCas3法で進めた。まず、ブタでのgenome情報をもとにcrRNAをGGTA1の機能ドメインであるexon 9の上下に置いた(crRNA=#45と#86)。 次に、4群、①群Control (No Cascade)、②群 Cascade+Cas3+#45、③群 Cascade+Cas3+#86、④群 Cascade+Cas3+#45+#86を置いた。<Cascade:pX-puro/Cascade (=bpNLS-Cascade)、Cas3:BPNLS-hCas3、crRNA=45:pBS-U6-crRNA=45、crRNA=86:pBS-U6-crRNA=86 > これらをブタ線維芽細胞に遺伝子導入を行い、48時間後の細胞を回収し、GSIB4を使ったFACS解析、およびゲノム抽出PCRにて解析した。 FACSの結果としては、欠損は②群、④群で多くみられた。遺伝子変異に関しては、④群で一番多く認めた。より大きなサイズの変異はこの方法では確認できなかったが、CRISPR/Cas3によるKOでブタ線維芽細胞において確かな変異(del:294 – 754bp)が確認できた。 次に、この④群 のCas3と#45-crRNA及び#8-crRNAをそれぞれpCXNに組み、pCXN/Cas3+#45-crRNA及びpCXN/Cas3+#45-crRNを作成し、これらを用いて、今度はブタ血管内皮細胞(SEC)に遺伝子導入を行い、SEC/GGTA1-KOの作出を試みた。同時にpCXN-GFPを導入し遺伝子効率を考慮しながら、再三に渡って試みるも、slectionに難渋しcloneの確立には至ってはいない。
|
Current Status of Research Progress |
Current Status of Research Progress
3: Progress in research has been slightly delayed.
Reason
前年度と同じになるが、CRISPR/Cas3法のブタ細胞での困難性。遺伝子導入した結果として、小さなdelしか出来ないCas9法に比して、Cas3法では、多くの場合約300bp以上のdelが発生しており、細胞の核に対してもダメージを与えていることが考えられる。
|
Strategy for Future Research Activity |
CRISPR/Cas3法を継続し、ブタ細胞を使ってGGTA1遺伝子のKO細胞等を作出する。ブタ内在性レトロウイルス(PERV)を含む、他の遺伝子にも挑戦する。
|
Report
(2 results)
Research Products
(19 results)
-
[Journal Article] A study of the mechanisms responsible for the action of new immunosuppressants and their effects on rat small intestinal transplantation2022
Author(s)
Shuji Miyagawa, Tasuku Kodama, Rei Matsuura, Pei-Chi Lo, Rieko Sakai, Chiyoshi Toyama, Yuichi Takama, Yoshiyuki Ihara, Yoichi Kakuta, Kazuaki Yamanaka, Katsuyoshi Matsunami, Hiroshi Eguchi, Akira Maeda, Hiroomi Okuyama
-
Journal Title
Transpl Immunol .
Volume: 70
Pages: 101497-101497
DOI
Related Report
Peer Reviewed
-
[Journal Article] Immunosuppressive effect of a C5a receptor antagonist via macrophage regulation of an intestinal transplant in a rat model2022
Author(s)
Chiyoshi Toyama, Akira Maeda, Shuhei Kogata, Koki Takase, Tasuku Kodama, Kazunori Masahata, Masafumi Kamiyama, Takehisa Ueno, Yuko Tazuke, Hiroshi Eguchi, Katsuyoshi Matsunami, Shuji Miyagawa, Hiroomi Okuyama
-
Journal Title
Transpl Immunol.
Volume: -
Pages: 101559-101559
DOI
Related Report
Peer Reviewed / Open Access
-
-
[Journal Article] Complement regulation in the Triple-KO era,2022
Author(s)
Miyagawa S, Maeda A, Toyama C, Kogata S, Okamatsu C, Yamamoto R, Masahata K, Kamiyama M, Eguchi H, Watanabe M, Nagashima H, Ikawa M, Matsunami K, Okuyama H.
-
Journal Title
Frontiers in Immunol
Volume: -
DOI
Related Report
Peer Reviewed
-
[Journal Article] Suppression of macrophage-mediated xenogeneic rejection by the ectopic expression of human CD177.2022
Author(s)
Kogata S, Lo PC, Maeda A, Okamatsu C, Sato K, Yamamoto R, Haneda T, Yoneyama T, Toyama C, Eguchi H, Masahata K, Kamiyama M, Okuyama H, Miyagawa S.
-
Journal Title
Transpl Immunol.
Volume: -
Pages: 101663-101663
DOI
Related Report
Peer Reviewed
-
[Journal Article] Suppression of xenogeneic innate immune response by a membrane-type human surfactant protein-A.2022
Author(s)
Toyama C, Maeda A, Kogata S, Yamamoto R, Masahata K, Ueno T, Kamiyama M, Tazuke Y, Eguchi H, Okuyama H, Miyagawa S.
-
Journal Title
Exp Ther Med.
Volume: -
Issue: 3
DOI
Related Report
Peer Reviewed
-
[Journal Article] Study of the CRISPR/Cas3 System for Xenotransplantation2022
Author(s)
Miyagawa S, Watanabe M, Nagashima H, Sato K, Kogata S, Toyama C, Masahata K, Kamiyama M, Yamamoto R, Eguchi H, Maeda A, Yoshimi K, Mashimo T, Okuyama H.
-
Journal Title
Transplant Proc
Volume: 54
Issue: 2
Pages: 522-524
DOI
Related Report
Peer Reviewed
-
-
-
[Presentation] The suppression of macrophage-mediated intestinal transplant rejection by C5a receptor antagonist.2022
Author(s)
Chiyoshi Toyama, Akira Maeda, Shuhei Kogata, Koki Takase, Tasuku Kodama, Kazunori Masahata, Takehisa Ueno, Masafumi Kamiyama, Yuko Tazuke, Hiroshi Eguchi, Katsuyoshi Matsunami, Shuji Miyagawa, Hiroomi Okuyama
Organizer
The Transplantation Society 2022
Related Report
-
[Presentation] Effect of a C5a receptor antagonist on macrophage function in an intestinal transplant rat model2022
Author(s)
Chiyoshi Toyama, Akira Maeda, Shuhei Kogata, Koki Takase, Tasuku Kodama, Kazunori Masahata, Takehisa Ueno, Masafumi Kamiyama, Yuko Tazuke, Hiroshi Eguchi, Katsuyoshi Matsunami, Shuji Miyagawa, Hiroomi Okuyama
Organizer
The Transplantation Society 2022
Related Report
-
-
[Presentation] T Human CD177 suppresses the neutrophil-mediated xenocytotoxicity2021
Author(s)
Tomohisa Yoneyama, Akira Maeda, Shuhei Kogata, Chiyoshi Toyama, Kazunori Masahata, Masafumi Kamiyama, Chizu Okamatu, Yuko Tazuke, Takehisa Ueno, Hiroshi Eguchi, Hiroomi Okuyama, Shuji Miyagawa
Organizer
CAST 2021
Related Report
Int'l Joint Research
-
[Presentation] T A membrane-type surfactant protein A (SP-A) suppresses macrophage-mediated cytotoxicity in swine endothelial cells2021
Author(s)
Chiyoshi Toyama, Akira Maeda, Shuhei Kogata, Kazuki Sato, Chizu Okamatsu, Hiroshi Eguchi, Masafumi Kamiyama, Yuko Tazuke, Takehisa Ueno, Hiroomi Okuyama, Shuji Miyagawa
Organizer
CAST 2021
Related Report
Int'l Joint Research
-
[Presentation] Testing the CPISPR / Cas3 system for the xenotransplantation field.2021
Author(s)
Shuji Miyagawa, Masahito Watanabe, Kazuki Sato, Shuhei Kogata, Chiyoshi Toyama, Kazunori Masahata, Masahumi Kamiyama, Hiroshi Eguchi, Akira Maeda, Kazuto Yoshimi, Tomoji Mashimo, Hiroomi Okuyama, Hiroshi Nagashima
Organizer
CAST 2021
Related Report
Int'l Joint Research
-
[Presentation] CPISPR / Cas3 system for pig Gal-T (GGTA12021
Author(s)
Shuji Miyagawa, Masahito Watanabe, Kazuki Sato, Shuhei Kogata, Chiyoshi Toyama, Kazunori Masahata, Masafumi Kamiyama, Hiroshi Eguchi, Akira Maeda, Kazuto Yoshimi, Tomoji Mashimo, Hiroomi Okuyama, Hiroshi Nagashima
Organizer
The 16th International Xenotransplantation Association
Related Report
Int'l Joint Research
-
[Presentation] Human CD177 Suppress Macrophage-Mediated Xenogeneic Rejection2021
Author(s)
Shuhei Kogata, Akira Maeda, Pei-Chi Lo, Chizu Okamatsu, Kazuki Sato, Riho Yamamoto, Tomohisa Yoneyama, Chiyoshi Toyama, Hiroshi Eguchi, Yuko Tazuke, Hiroomi Okuyama, Shuji Miyagawa
Organizer
The 16th International Xenotransplantation Association
Related Report
Int'l Joint Research
-
-