Verification of gene-edited pig production method from Japan

2023 Fiscal Year Final Research Report

• 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: MIYAGAWA Shuji 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
• Keywords: CISPR/Cas3 / Knockout / GGTA1 / PERV-C / Xenotransplantation

Outline of Final Research Achievements

In order to apply the CISPR/Cas3 method to pig cells, we attempted knockout (KO) of GGTA1 and porcine endogenous retrovirus (PERV)-C.
Focusing on exon 9 of GGTA1, we prepared target plasmids and introduced them into pig fibroblast cells. First, the expression of Gal-epitope was analyzed by FACS, and then nested PCR was performed. Furthermore, the PCR products of each group were transformed into E. coli, and a large number of colonies were checked by PCR, Sanger method . The results demonstrated a deletion of 294-754bp was produced in GGTA1 gene.
Next, we focused on PERV-C and attempted to KO it using just the same as the Cas3 method for GGTA1. However, it was not possible to analyze the deletion of PERV-C in E.coli colonies, because of sit number of PERV. However, in conclusion, we confirmed the effectiveness and feature of the CRISPR/Cas3 method in xenotransplantation.

Free Research Field

Xenotransplantation

Academic Significance and Societal Importance of the Research Achievements

この研究で、異種移植分野の（ブタを使った）臓器開発での遺伝子のtargeting法で、一般に普及しているCRISPR/Cas9を使わずに、日本で開発されている新たな方法であるCRISPR/Cas3を使うことが可能になれば、特許の問題を気にせずに遺伝子編集が可能になる。これは単に異種移植分野だけでなく、他の医学・畜産、等の分野に大きく影響をもたらす。