Efficient enrichment of homozygous bi-allelic knockout microminiature porcine cells using a novel selection system and production of genome-edited cloned piglets
| Project/Area Number |
15K07695
|
|---|
| Research Category |
Grant-in-Aid for Scientific Research (C)
|
| Allocation Type | Multi-year Fund |
|---|
| Section | 一般 |
|---|
| Research Field |
Animal production science
|
|---|
| Research Institution | Kagoshima University |
|---|
Principal Investigator |
SATO Masahiro 鹿児島大学, 医用ミニブタ・先端医療開発研究センター, 教授 (30287099)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
三好 和睦 鹿児島大学, 農水産獣医学域農学系, 教授 (70363611)
|
|---|
| Project Period (FY) |
2015-04-01 – 2018-03-31
|
| Project Status |
Completed (Fiscal Year 2017)
|
| Budget Amount *help |
¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000)
Fiscal Year 2017: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2016: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2015: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
|
|---|
| Keywords | α-Gal epitope / GAAT1 / CRISPR/Cas9 / targeted toxin / genome editing / isolectin IB4 / LDLR / マイクロミニブタ / 胎仔性繊維芽細胞 / ゲノム編集 / ＬＤＬＲ / 動脈硬化 / 体細胞核移植 |
|---|
| Outline of Final Research Achievements |
Somatic cell nuclear transfer (SCNT) has been employed as one of the efficient tools for the production of genetically modified (GM) pigs. The GM cell isolation used for SCNT is often difficult due to occasional contamination of untransfected cells. We here used a novel approach for enrichment of porcine cells after introduction of CRISPR/Cas9 components. A single guide RNA targeted to GAAT1 gene, involved in the synthesis of cell-surface α-Gal epitope, is a prerequisite. When the transfected cells were reacted with toxin-labeled IB4 for to eliminate α-Gal epitope-expressing cells, the surviving clones lacked α-Gal epitope expression and were highly expected to exhibit induced mutations at another target loci. SCNT using these cells as donors successfully resulted in the production of cloned blastocysts with genome-edited nuclei. Thus, this novel system will be useful for SCNT-mediated acquisition of GM cloned piglets..
|
Report
(4 results)
Research Products
(50 results)
-
[Journal Article] Isolation and characterization of lymphoid enhancer factor-1-positive deciduous dental pulp stem-like cells after transfection with a piggyBac vector containing LEF1 promoter-driven selection markers.2017
Author(s)
Murakami T, Saitoh I, Sato M, Inada E, Soda M, Oda M, Domon H, Iwase Y, Sawami T, Matsueda K, Terao Y, Ohshima H, Noguchi H, Hayasaki H
-
Journal Title
Archives of Oral Biology
Volume: 81
Pages: 110-120
DOI
Related Report
Peer Reviewed / Open Access / Acknowledgement Compliant
-
-
-
-
-
-
-
-
-
-
-
[Journal Article] The piggyBac-Based Gene Delivery System Can Confer Successful Production of Cloned Porcine Blastocysts with Multigene Constructs.2016
Author(s)
Sato M, Maeda K, Koriyama M, Inada E, Saitoh I, Miura H, Ohtsuka M, Nakamura S, Sakurai T, Watanabe S, Miyoshi K.
-
Journal Title
Int J Mol Sci.
Volume: 17
Issue: 9
Pages: 1424-1424
DOI
NAID
Related Report
Peer Reviewed / Open Access
-
-
-
-
-
-
[Journal Article] One-step generation of multiple transgenic mouse lines using an improved Pronuclear Injection-based Targeted Transgenesis (i-PITT)2015
Author(s)
Ohtsuka M., Miura H., Mochida K., Hirose M., Hasegawa A., Ogura A., Mizutani R., Kimura M., Isotani A., Ikawa M., Sato M. and Gurumurthy C. B.
-
Journal Title
BMC Genomics
Volume: 16
Issue: 1
Pages: 274-274
DOI
NAID
Related Report
Peer Reviewed / Open Access
-
-
-
-
-
[Journal Article] Choice of feeders is important when first establishing iPSCs derived from primarily cultured human deciduous tooth dental pulp cells.2015
Author(s)
Saitoh I, Inada E, Iwase Y, Noguchi H, Murakami T, Soda M, Kubota N, Hasegawa H, Akasaka E, Matsumoto Y, Oka K, Yamasaki Y, Hayasaki H, Sato M
-
Journal Title
Cell Medicine
Volume: 8
Issue: 1-2
Pages: 9-23
DOI
Related Report
Peer Reviewed / Open Access
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
[Presentation] The genetic engineering-based isolation of lymphoid enhancer-binding factor-1 (LEF1) positive stem-like cells from human deciduous tooth cell-derived iPSCs.2016
Author(s)
Murakami T, Saitoh I, Sato M, Inada E, Soda M, Iwase Y, Sawami T, Suzuki A, Ohshima H, Hayasaki H
Organizer
10th Biennial conference of the pediatric dentistry association of Asia in conjunction with 54th annual conference of the Japanese society of pediatric dentistry
Place of Presentation
Tokyo Dome Hotel (Tokyo, Japan)
Year and Date
2016-05-26
Related Report
Int'l Joint Research
-
-
-
-
-
-
-
-
