Safety and Security

Our project aims to develop a novel viral defense mechanism that actively induces cell death in influenza virus-infected cells, thereby preventing viral amplification within animal hosts. The ultimate goal is to create and implement GM chickens equipped with this system for societal application.

To achieve this project and goal, we developed both social implementation and laboratory experimental plans, establishing and executing safety measures for each.

Creating GM animals presents significant ethical and legal barriers. Therefore, we did not produce GM chickens. Instead, we developed a comprehensive implementation plan for GM chicken production and social integration, incorporating a robust safety framework. This safety plan incorporated input from multiple stakeholders and underwent iterative refinement. For detailed information on the safety plan, please refer to the Entrepreneurship page.

Safety of Transport between Labs

We properly designed experimental protocols and conducted experiments across three laboratories. This involved transporting genetically modified DNA, RNA, and animal cells between two laboratories. We followed iGEM Safety Policies[1], ensuring all materials were appropriately packaged and sealed to prevent environmental release during transport. We consulted with principal investigators at each laboratory to ensure safe and accurate transportation procedures.

1.DNA Working

Biocontainment

The microorganisms used were E. coli DH5α and BL21 (provided by the National Institute of Genetics), both classified as Risk Group 1. Therefore, they pose minimal risk of causing human disease and are included in the iGEM Safety microorganism whitelist. All experiments related to this project were conducted in laboratories at Kyoto University's Institute for Life and Medical Sciences. Waste containing genetically modified organisms was clearly segregated to prevent accidental spillage and disposed of by autoclaving based on PI guidance, or appropriately treated with ethanol and chlorine bleach when necessary. Additionally, all tips, tubes, and other paper and plastic waste were properly sorted and disposed of as laboratory waste. Furthermore, all laboratory equipment that contacted genetically modified organisms was autoclaved before disposal to prevent release of viable genetically modified organisms.

Safety Measures

Before beginning any experiments, we received hands-on safety training from our PI, who carefully explained how to use laboratory equipment and avoid serious risks such as injury or fatal accidents. This instruction covered the safe handling of hazardous tools and procedures, including the use of liquid nitrogen, open flames from gas burners, autoclaves, and centrifuges. We also discussed the risks posed by potentially harmful chemicals. In our work, the only hazardous chemical we used was Ethidium Bromide (EtBr), a substance suspected of being carcinogenic, which we handled under direct supervision and instruction from our PI. No other highly dangerous substances—such as strong acids or bases, corrosive reagents, heavy metals, mutagens, neurotoxic compounds, or explosive materials—were involved in our experiments. In preparation for our genetic modification work, we completed a DNA modification training course at Kyoto University. This program introduced us to the legal framework and regulations governing genetically modified organisms (GMOs), as well as their safe handling and disposal. We also studied past incidents of improper GMO use and learned about emergency protocols and response strategies. Our PI, who has extensive experience working with GMOs, including E. coli, directly instructed us in the safe practices for handling genetically modified E. coli in the laboratory.

2.Animal Cell Lines Experiments

Biocontainment

Many animal cells are designated as Risk Group 2 by iGEM[2]. Therefore, we submitted a Check-In Form and obtained approval from the Safety Committee. The animal cell lines used were HEK293 (Human Embryonic Kidney 293) and DF-1 (chicken fibroblast cells). Both cell lines are commonly used in standard laboratories and have high safety profiles.
This experiment serves as a safe, virus-free proof of concept by externally introducing double-stranded RNA (dsRNA) into cells to mimic viral stimulation. This approach enables us to simulate key aspects of viral infection without using actual viruses, thereby establishing a foundational demonstration of our system. To achieve this, the animal cells used in the experiment have been engineered to express the designed fusion protein through three distinct delivery methods.

1. Transient Transfection: Introducing plasmids encoding the fusion protein.
2. Stable Cell Lines: Creating cell lines that constitutively express the fusion protein.
3. Direct Protein Introduction: Purifying the fusion protein from E. coli and delivering it into the cells, reproducing the methods from the reference publication.

Our system is composed of designed fusion proteins that link a dsRNA sensor to an apoptosis inducer.

• dsRNA Sensors: RIG-I or PKR
• Apoptosis Inducers: APAF1 or iCaspase9

All of these proteins are derived from Homo sapiens, Gallus gallus, or Anas platyrhynchos. Importantly, none of them are toxic to humans.
To validate the dsRNA-dependent function, we will observe for apoptosis after introducing dsRNA (made by in vitro transcription) to cells that have received the fusion protein. We will prepare the dsRNA for our experiment according to the following publication[1].
All components are non-toxic to humans. The safety of this approach is supported by: A prior publication confirming the function of a similar PKR-APAF1 fusion (Reference2). Use of pre-approved parts, including the iGEM safe part HIV TAT (BBa_K1202006) and a modified RIG-I with its interferon-inducing domain removed.

Safety Measures

All experiments involving animal cells (and not viruses) will be directly supervised by our team's instructor, Dr. Shinnosuke Honda. All experiments will be conducted in Dr. Shinnosuke Honda's laboratory, which is part of the Graduate School of Agriculture at Kyoto University and is highly proficient in genetically manipulating animal cells.
Furthermore, only members who have been instructed by Dr. Shinnosuke Honda and have learned how to handle animal cells will be directly involved in the experiments.

3.Virus Infection Experiments

Biocontainment

Influenza viruses are not on the iGEM White List[3]. Therefore, we submitted a Check-In Form and obtained approval from the Safety Committee to conduct our activities. Two types of influenza viruses were used: Human Influenza Virus and Low Pathogenic Avian Influenza Virus. The cells infected with these viruses were those described in the Animal Cell Lines Experiments section.
The specific viral strains used are as follows.

Human Influenza Virus

• A/WSN/33 (H1N1)
• A/California/04/2009 (H1N1pdm)
• A/Victoria/361/2011 (H3N2)

Low Pathogenic Avian Influenza Virus

• A/duck/Hokkaido/8/1980 (H3N8)
• A/swan/Hokkaido/481102/2017 (H4N6)
• A/duck/Hokkaido/95/2014 (H8N4)

These viruses are commonly used in laboratories, have minimal pathogenicity, and pose little risk of infection or adverse health effects.Therefore, members with BSL2 virus handling training can safely conduct these experiments without requiring specialized virology expertise.

Additionally, to conduct infection experiments with these viruses, we needed to thoroughly understand two critical aspects: the rationale for BSL2 classification of our viruses and the potential risks of BSL escalation to BSL3. We investigated Japanese legal regulations regarding these matters and demonstrated that our experiments could be safely conducted at BSL2 level within the scope of activities permitted by iGEM. Based on this comprehensive analysis, we successfully obtained approval from the Safety Committee.

First, Japanese domestic regulations specify BSL requirements for each pathogen. Influenza viruses are classified by serological subtype, with all subtypes except H5 and H7 permitted for BSL2 handling[4]. Therefore, all influenza viruses included in this application can be handled in BSL2 laboratories. Furthermore, within the scope of our planned experiments, Japanese legal regulations do not require BSL escalation during experimental procedures, allowing us to consistently conduct all experiments in BSL2 facilities.

Generally, viral BSL classification is elevated when experiments increase viral pathogenicity. According to [5], BSL2 viruses may be reclassified as BSL3 if they develop any of the following pathogenicity criteria (①-③), excluding equine influenza and pandemic influenza viruses:

1. ① IVPI (Intravenous Pathogenicity Index representing pathogen virulence obtained through intravenous inoculation tests) exceeding 1.2 in 6-week-old chickens.
2. Mortality rate of 75% or higher when intravenously inoculated into chickens aged 4-8 weeks.
3. In addition to the requirements in ① and ②, serological subtypes H5 or H7 with multiple basic amino acids at the hemagglutinin cleavage site, and amino acid sequences estimated to be similar to those confirmed as pathogens meeting criteria ① or ②.

However, BSL escalation from BSL2 to BSL3 will not occur in our experiments for the following four reasons:

• Experiments avoid conditions that induce viral virulence enhancement through interspecies infection.
• No artificial viral mutagenesis is performed.
• No animal infections or large-scale cultivation are conducted.
• Non-H5/H7 influenza viruses do not increase pathogenicity in cell-level experiments.

Therefore, we conclude that our experiments pose zero risk of inducing significant viral virulence enhancement.

Safety Measures

All viral infection experiments will be conducted at Dr. Takeshi Noda's laboratory at Kyoto University's Institute for Life and Medical Sciences.

This laboratory is certified by Kyoto University's safety committee for BSL2 experiments and maintains operational BSL3 facilities for highly pathogenic viruses.

Two team members participated in infection experiments: one experienced in DNA work and animal cell handling, and one in DNA work. Both completed BSL2 training at the Noda laboratory on August 18-20, 2025, covering equipment handling, aseptic techniques, and virus safety protocols specific to Kyoto University students. All experiments will be conducted under continuous supervision by BSL2-experienced instructors, with dangerous procedures performed by supervisors rather than team members to ensure safety.

When including multiple viral strains in our experimental design, we implemented the following precautions:

We will not co-infect single cells with two different viral strains. To prevent unintended viral contamination during infection experiments, clean benches will be thoroughly cleaned between each experiment, samples will be properly isolated, and we will never handle two viral strains simultaneously.

Furthermore, handling multiple viral strains independently is routinely performed at the host laboratory of Professor Noda, and we will proceed cautiously under their strict guidance. These experiments have been approved by the Kyoto University Recombinant DNA Experiment Safety Committee and comply with the Kyoto University Regulations Regarding Safety Control of Recombinant DNA Experiments, etc. and its Enforcement Rules[6],[7].

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