Overview

We regard safety as a critical element of this project. Therefore, it is essential that any equipment or materials used or produced during the development process be handled safely. This section outlines the measures we have implemented to ensure the safe development and use of materials within the MOVE project.

Product Safety

The membrane vesicles (MVs) used in this project are not biological organisms and do not proliferate upon infection. Additionally, the RNA encapsulated within the MVs was carefully selected using NCBI’s BLAST to check for sequence homology with other organisms, ensuring a high degree of specificity. As a result, the reactions exhibit high specificity, minimizing toxicity to non-target organisms. As a specific example, the shRNA_alb1 designed against the rice blast pathogen Pyricularia oryzae (P. oryzae) can be highlighted (link to Parts). This shRNA targets the sequence GTGCTCGACGGCTGCTTAAAT (21 bp). A BLASTn[1] search of this target sequence yields the results shown in the following figure. From these results, it is evident that while the query cover for P. oryzae is 100%, the sequence similarity with other species does not exceed 85%. This indicates that at least 3 out of the 21 base pairs of the target sequence do not match in other species. According to Mocellin et al., siRNAs with a single nucleotide mismatch can function as negative controls in RNAi experiments[2], suggesting that shRNA_alb1 exhibits high specificity and is unlikely to exert significant effects on non-target species.

Figure 1. The 10 most homologous sequences when the target sequence of shRNA_alb1 is run through BLASTn

Laboratory Safety

Laboratory Safety Equipment

The experiments conducted as part of this project are classified under P-1 level. The laboratory adheres to the following constraints[3]:

  1. The laboratory must be equipped with the necessary structure and facilities required for conducting biological experiments.
  2. When conducting experiments involving genetically modified organisms (GMOs), the following precautions must be observed:

(1) Waste containing GMOs (including waste liquids) must be inactivated before disposal.

(2) Equipment, machinery, and tools contaminated with GMOs must be decontaminated before disposal or reuse (including prior washing, where applicable).

(3) Laboratory benches must be decontaminated after the completion of daily experiments or immediately if contaminated with GMOs.

(4) Laboratory doors must remain closed, except when entering or exiting the room.

(5) Windows must be kept closed or otherwise secured to prevent insect entry.

(6) Aerosol generation must be minimized during all procedures.

(7) If GMOs need to be removed from the laboratory or inactivated outside the lab, they must be contained in sealed, leak-proof containers.

(8) Personnel handling GMOs must take necessary precautions, such as handwashing, to prevent contamination or infection.

(9) Measures must be taken to prevent unauthorized persons from entering the laboratory.

In the laboratories of Professor Yoshihisa Matsumoto and Associate Professor Yoichi Tagawa, experiments are conducted under the supervision of graduate students, and access outside of working hours is prohibited.

Waste Disposal [4]

All waste in the laboratory is categorized as follows:

  • A-1: Small amounts of chemicals
  • A-2: Filter materials and other waste not exposed to hazardous chemicals
  • A-3: Filtration waste exposed to hazardous substances
  • A-4: Waste polyethylene tanks
  • A-6: Water waste containing difficult-to-filter precipitates
  • B-1: Glass
  • B-2: Silica gel
  • C-1: Needles and syringes
  • C-2: Gels, agar plates, etc.

Each type of waste is disposed of in dedicated bins, and biohazardous waste is sterilized before being collected by a specialized company.

Liquid waste is collected in dedicated polyethylene tanks, and each tank is accompanied by a “laboratory liquid waste separation and storage record sheet.” Waste is documented each time it is added to a tank. Before disposal, the contents of the liquid waste, as well as any leaks or contaminants, are inspected in each laboratory to ensure safety.

Waste Management

All waste is sorted into general waste (such as packaging and paper towels not contaminated by chemicals or biological substances) and waste contaminated by biological materials (such as gels and pipette tips). Biohazardous waste is properly sterilized before being collected by a contractor.

Safety Education [5], [6]

At the Tokyo Institute of Technology, the “Safety Management Regulations for Experiments Involving Genetic Modification” has been established in accordance with the Cartagena Act and Type II Ordinance. The Safety Committee provides guidance, and students involved in experiments are required to attend this training. The content includes:

  • Precautions when conducting experiments involving genetic modification (including guidelines on experiment locations and containment measures in accordance with the Cartagena Act).
  • Review of genetic modification experiments.

Participants are issued an experimenter number valid for 3.5 years (for students) as proof of attendance. Before beginning any experiment, participants must input this number in the “Experimenter” section of the experimental plan and pass the university’s review process. Furthermore, our institute has made courses on bioethics and regulations mandatory for students enrolled in life sciences departments who entered or will enter the university from the previous academic year onwards. This ensures that even students not directly involved in experiments receive appropriate ethical education.

Reference

[1]Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schäffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402.

[2] Mocellin, S., Provenzano, M. RNA interference: learning gene knock-down from cell physiology. J Transl Med  2 , 39 (2004). https://doi.org/10.1186/1479-5876-2-39

[3] Ministry of the Environment Japan Biosafety Clearing-House. 研究開発等に係る遺伝子組換え生物等の第二種使用等に当たって執るべき拡散防止措置等を定める省令. accessed: 2024/9/15

[4] Tokyo Institute of Technology Office of Campus Management General Safety Management Section Environmental Safety Promotion Office (2018). Guide to Appropriate Laboratory Waste Control . Accessed: 2024/08/16

[5] Tokyo Institute of Technology (2023/04/01). 国立大学法人東京工業大学遺伝子組換え実験等安全管理規則 . Accessed: 2024/08/16

[6] Tokyo Institute of Technology Research Promotion Department (2023). 遺伝子組換え実験について . Accessed: 2024/08/16