Pluripotent embryonal carcinoma cells are available for identifying cellular targets responsible for botulinum neurotoxins

2017 Fiscal Year Final Research Report

• Project/Area Number: 15K08476
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Bacteriology (including mycology)
• Research Institution: Fujita Health University
• Principal Investigator: Tsukamoto Kentaro 藤田保健衛生大学, 医学部, 講師 (80434596)
• Co-Investigator(Kenkyū-buntansha): 幸田 知子 大阪府立大学, 生命環境科学研究科, 助教 (80336809)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Keywords: ボツリヌス毒素 / 多能性胚性癌細胞 / P19 / ガングリオシド / CRISPR/Cas9

Outline of Final Research Achievements

In order to prove the utility of P19 cells for the study of the intracellular mechanism of botulinum neurotoxins, ganglioside-knockout neurons were generated by deletion of ganglioside synthase in P19 cells using CRISPR/Cas9 system. The sensitivity of the generated ganglioside-knockout P19 neurons to botulinum neurotoxin type C was decreased considerably, and the exogenous addition of the gangliosides GD1a, GD1b, and GT1b restored the susceptibility of P19 cells to botulinum neurotoxin type C. Therefore, the genome-edited P19 cells generated by the CRISPR/Cas9 system were useful for identifying and defining the intracellular molecules involved in the toxic action of botulinum neurotoxins. Moreover, we evaluated the endocytotic pathway for the uptake of botulinum neurotoxin type C into P19 cells. Consequently, we conclude that BoNT/C could enter cultured neuron independent of both synaptic vesicle recycling and clathrin-mediated endocytosis.

Free Research Field

細菌学