Efficient enrichment of homozygous bi-allelic knockout microminiature porcine cells using a novel selection system and production of genome-edited cloned piglets

2017 Fiscal Year Final Research Report

• 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
• Keywords: α-Gal epitope / GAAT1 / CRISPR/Cas9 / targeted toxin / genome editing / isolectin IB4 / LDLR

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..

Free Research Field

応用動物科学