Establishment of prion deficient cells in cattle

• Project/Area Number: 14560281
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: 生物資源科学
• Research Institution: KYOTO UNIVERSITY
• Principal Investigator: SHINKAI Yoichi Kyoto University, Institute for Virus Research, Professor, ウイルス研究所, 教授 (20211972)
• Co-Investigator(Kenkyū-buntansha): SENDAI Yutaka Kyoto University, Research Institute for Functional Peptides, Senior Researcher, 研究員
• Project Period (FY): 2002 – 2003
• Project Status: Completed (Fiscal Year 2003)
• Budget Amount: ¥4,300,000 (Direct Cost: ¥4,300,000); Fiscal Year 2003: ¥1,200,000 (Direct Cost: ¥1,200,000); Fiscal Year 2002: ¥3,100,000 (Direct Cost: ¥3,100,000)
• Keywords: PRION / CATTLE / GENE TARGETING / BSE / クローニング / ノックアウト

Research Abstract

Animal cloning techniques have enabled gene disruption in several species. Here, we report the first successful disruption of the prion gene in cattle. METHODS : The prion gene of the Japanese Black cow (JBC) was used to construct PrP-5. 1 and PrP-5. 2, the targeting vectors for the bovine prion gene, and these constructs were introduced into the fetal fibroblast cell line JBC906 by the lipofection method. Ten polymerase chain reaction (PCR)-positive colonies were obtained from 833 G418-resistant colonies, and Southern blot analysis revealed successful homologous recombination at the prion locus in these colonies. CONCLUSIONS : Heterozygous knockout of the prion gene was performed in JBC, and production of a homozygous prion knockout JBC by a second round of targeting 906htGT is currently in progress.