Overview
In order to protect ourselves and the environment when conducting experiments, safety management through the proper use of equipment and reagents is important. We also believe that it is the responsibility of researchers to consider the safety of their collaborators. Below are the details of the practices we have implemented for the iGEM project.
Lab safety
GMO and Lab Safety Courses
We were required to take safety courses before conducting experiments at The Institute of Medical Science, The University of Tokyo. In addition, Wet Lab members were required to take and pass a safety course on microorganisms for research and a university-wide course on genetically modified organisms through the university's course management website.
Wet Lab Equipments
We used the following safety-related equipment.
- Safety cabinet
- Autoclave
Figure 1. Autoclave
Use of SYBR Green Ⅰ and SYBR Green Ⅱ
We used SYBR Green Ⅰ and SYBR Green Ⅱ as staining reagents to detect nucleic acids by qPCR. We understood that these reagents are potentially mutagenic due to the nature of their binding to nucleic acids, and that the pigment could penetrate into tissues because they are DMSO solutions, so gloves were always worn during use 1 When the reagent adhered to the gloves, they were promptly discarded.
Tear fluid collection
Our project aims to amplify miRNAs in tear fluid and quantify the amplified nucleic acids. We needed to validate our technology and to confirm that we can amplify specific miRNAs from actual human tear fluid. Artificial tear fluid cannot reproduce the foreign substances in human tear fluid, so actual human tear fluid must be used. For this reason, we planned to collect tear fluid from research collaborators.
Interviews with Experts
We interviewed research ethics experts and eye doctors to learn about what experimenters should do when handling human tear fluid samples (e.g., how to explain to the PUBLIC, how to consider the need, preparedness, etc.).
For more information, see Human Practices.
Ethical Review Application for IMSUT Researcher
The “Guidance on Ethical Guidelines for Life Sciences and Medical Research Involving Human Subjects" states that all parties involved in life sciences and medical research involving human subjects must undergo review by an ethical review committee 2. In accordance with this, we submitted an ethics application to the Ethics Review Committee of the Institute of Medical Science, The University of Tokyo, to which we belong, for review of the scientific and ethical validity of medical research on human subjects, and obtained approval for our research.
In our submission we primarily included the following
- Obtain informed consent from the patient and confirm consent.
- Personal information collected, such as history of ophthalmological diseases, must be kept in accordance with the office's regulations.
- Complying with withdrawal when consent is withdrawn by the patient after collection of lacrimal fluid.
- In the event of inflammation or other problems that may occur during the collection of tear fluid, the patient should be examined by an eye doctor affiliated with our laboratory.
Check-in Form
We saw that conducting laboratory experiments using human samples is not on the White List 3, so we filled in a check-in form immediately.
We found that there is a possibility of damaging the eyeball when collecting tear fluid. Therefore, we decided to practice using Schirmer strips before collecting tear fluid from research collaborators or to ask eye doctors to collect tear fluid. In the event of damage to the eye, an eye doctor in our laboratory would check the patient's eye condition and, in some cases, refer to another eye doctor.
Use of Similar sequences of the SARS-CoV-2 N gene
Our project aims to amplify miRNA in tear fluid and quantify the amplified nucleic acid using CRISPR-Cas3 or CRISPR-Cas12a. CRISPR-Cas3 and CRISPR-Cas12a used in this study are technologies developed by the Mashimo Laboratory, Division of Animal Genetics, The Institute of Medical Science the University of Tokyo, for application to SARS-CoV-2 detection kits, and recognize part of the SARS-CoV-2 Nucleocapsid Protein gene (N gene) 4. In experiments to measure the activity of CRISPR-Cas3 and CRISPR-Cas12a, we needed to purchase similar sequences of the N gene of the SARS-CoV-2 virus from a synthesis company to conduct experiment using CRISPR-Cas3 or CRISPR-Cas12a.
We saw that these sequences are not on the White List, so filled in a check-in form immediately. Also, we found that contamination of these sequences carries the risk of false positives in PCR tests for COVID-19, so asked the synthesis company to confirm that the above risks were eliminated in the sequences we used.
Safety Concerns on Future Application
We initially assumed that users would use Schirmer strips to collect tear fluid. However, Schirmer strips can damage the eye if used incorrectly. To improve this problem we are considering the use of strip meniscometry tubes. The strip meniscometry tube is a tear fluid suction tube that realizes the strip meniscometry method, a method of quantifying the tear fluid meniscus 5. Strip meniscometry can be performed without touching the eyelid or ocular surface, minimizing irritation 6. In addition, strip meniscometry can be performed in 5 seconds, whereas it takes 5 minutes to collect tear fluid with Schirmer strips. Therefore, the strip meniscometry tube would allow users to collect tear fluid in a safer and less burdensome manner.
References
TaKaRa-Bio Co,. Ltd. (n.d.). SYBRTMGreen I Nucleic Acid Gel Stain SYBRTMGreen Ⅱ Nucleic Acid Gel Stain 説明書 [SYBRTMGreen I Nucleic Acid Gel Stain SYBRTMGreen Ⅱ Nucleic Acid Gel Stain manual]. https://catalog.takara-bio.co.jp/PDFS/5760a_5761a_5770a_5771a_j.pdf
Ministry of Health, Labour and Welfare. (2023). Hito wo taisyo to suru seimei kagaku/igaku kei kenkyu ni kansuru rinri sshishin gaidansu [Ethical guidelines and guidance for life science and medical research involving human subjects]. https://www.mhlw.go.jp/content/001237478.pdf
iGEM. (n.d.). White List. iGEM Responsibility. https://responsibility.igem.org/guidance/white-list
Yoshimi, K., Takeshita, K., Yamayoshi, S., Shibumura, S., Yamauchi, Y., Yamamoto, M., Yotsuyanagi, H., Kawaoka, Y., & Mashimo, T. (2022). CRISPR-Cas3-based diagnostics for SARS-CoV-2 and influenza virus.iScience 25, 103830, 1-13. https://doi.org/10.1016/j.isci.2022.103830
Echo Electricity Co,.Ltd. (n.d.). sutorippu menisukometori ho niyoru ruieki tyoryuryo hyoka [Quantification of the volume of tear meniscus by strip meniscometry method]. https://www.echo-mf.jp/wp-content/uploads/2021/12/SMTube_brochure_ja-20170625.compressed.pdf
Dogru, M., Ishida, K., Matsumoto, Y., Goto, E., Ishioka, M., Kojima, T., Saeki, M., & Tsubota, K. (2006). Strip meniscometry: a new and simple method of tear meniscus evaluation. Invest Ophthalmol Vis Sci 47. 1895-1901. https://doi.org/10.1167/iovs.05-0802
