NJU-China gives high priority to safety and aims to take all necessary precautions to ensure no personal or environmental harm occurs.
Safe Project Design
Using chassis safe organisms
In our project, only non-pathogenic chassis model organisms are chosen: E.coli (DH5-Alpha), cell line (HEK293T), mouse(C57BL/6, primary cultured neurons) and recombinant Adeno Associated Virus 2 (AAV2).
E. coli is a Risk 1 type organism, which means it poses little to no threat to human safety. HEK293T is a safe type of cell line widely used in vitro. Besides, the usage safety of C57BL/6 mouse and AAV2 has been approved by the chairman of institutional safety and ethical committee. Using these organisms, all parts of HEK293T or AAV2 were ordered from commercial company. All of these organisms are commonly used in scientific research and synthetic biology.
Using safe parts
Harmful parts are not included in our project. Our elements contain 5'-UTR of TBEV and 3'-UTR of beta-actin. Cells' viability has been tested in order to confirm that our elements in test are safe. Our results clearly show that the element is very safe.
Meeting 3Rs Principle
In addition, we do our best to design the experiments meeting the high standards of principles of humane experiment such as 3Rs, The Animal Welfare Act(AWA), etc.
Replacement: What we do is about neuron targeting so we need neurons to see whether our elements could work. We have considered using N2a neuroblastoma cells. Compared with neurons isolated from mice, N2a cell has inconspicuous neurites thus it is not appropriate for our experiment. Also, since we hope our element could be used as drugs, it's necessary to test it in mice before it could be used further. For these reasons, we chose mice as our experimental animal.
Reduction: We do reduce the number of mice we use in the experiments. We have used 24 male adult mice and 13 pregnant mice in total.
As for the 13 pregnant mice, we cultured their fetus cortical neurons. One for cytotoxicity test and the other twelve are equally divided into 4 groups, each of them are transfected with one kind of our plasmid.
As for the 24 male adult mice, they are equally divided into 4 groups as well. Different mice, different conditions, we need at least 6 mice a group to fulfill the requirement in statistic.
Refinement: We do try our best to minimize animal suffering and improve their welfare. We strictly follow animal welfare principles when we use mice for experiments. The mice living environment is clean with air and water and food disinfection. A group of mice live together in one cage and the cage is big enough for their movement. There are boxes including food, water container, disinfected bits of wood. We change the wood bits regularly. We prevent other mice from hearing or seeing during we are sacrificing the mice after proposal anesthetization.
Personal safety is very important in the workplace, we ensure high standards are met.
All team members participating in lab work attend a mandatory safety training session where we learn biosafety rules in lab, such as fire and electrical safety, and acquire essential laboratory techniques, such as hands-on training on plasmid extraction. The team members also learn how to deal with the laboratory waste, such as cell culture medium and sharp objects. Before entering the laboratory, all team members systematically learn how to protect themselves if there is an accident such as gas leaking.
Regarding animal experiments, the chosen team members all have received specific training including Mandatory Animal Care and Use(MACU)Training and Rodent Survival Surgical(RSS)Training to gained certification before experiments.
During experiments, all team members wear long pants, closed-toed shoes basically. We use Personal Protective Equipment (PPE) including lab coats/disposable gown/surgical scrub shirt, goggles and gloves to prevent any physical contact or contamination with biological materials. We are also required to wear surgical masks and hair bonnets when conducting rodent survival surgery.
Our college has institutional safety and ethical committee. Before conducting our experiments, we need approval from the committee. There are experts who tell us the possible risks and how to avoid them in the project. And they won't approve our experiments if it's out of our handle. Also, we do strictly follow the safety requirements by our national biosafety regulation.
All team members are required to attend safety training to learn how to use lab equipment including biosafety cabinet, centrifuge, UV Spectrophotometer etc. When working with a new reagent or piece of equipment, a faculty lab manager or experienced member is always present to assist.
During the animal experiments, we comply with animal regulations, institutional policies, guidelines, and procedures we have learned in MACU. The C57BL/6 mice we use live in a standard environment and are not allowed to be taken to laboratory for more than 24 hours. Our operations are strictly held in a clean dedicated space and mice are euthanized after experiments and collected to burn in order to prevent spreading diseases. When operating them, we use gas anesthesia machines and analgesics to relax them, minimizing pain and distress. During the operation, we focus our mind to prevent being bitten and continue to assess their respiration or pinch their toes and tails to ensure enough anesthesia at the same time. Proper closure (wound clip) is done to help the cuts healing. When we conduct rodent survival surgery, a surgical technician is presented to offer guidance and oversight to the procedure. Therefore, the animal experiments are under the control of our instructor.
We follow the lab safety rules such as using a laminar hood and wearing gloves and lab coats when operating E.coli, 293T and AAV2. E.coli containers are autoclaved after experiment and 293T cells are also dealt with in a proper way after experiments. What's more, experiments using AAV are done by our teacher and he will always directly conduct us when we do such experiments on our hands. Finally, we make sure that we wash our hands before leaving the bench area.
All team members are required to clean up the equipment and the whole lab space after use. All disposables that have been contacted with biological material are disposed in biohazard waste. The lab also contains sharps containers, experimental waste containers and domestic waste containers for special disposal. All biohazard waste and experimental waste are specially treated (such as being autoclaved) and transported apart from normal domestic waste.
In the future, we expect to inject our exosomes or virus into nervous system and target to the neurites of neurons to cure some neurological diseases caused by a decrease of expression of certain proteins playing their roles in neurites.
Since the exosomes contain a lot of stuff we don't know, injection into the animal body could lead to severe consequences. There is a long way to go until its put into use. However, using AAV as the element package has a much lower risk level because we know what's in the capsid and the AAV we use cannot replicate. It is widely used among gene therapy researchers for its low pathogenicity and causing low immune response. Also, the amount of AAV injected into human can be controlled to avoid the problems of insufficient or excess of dose.
Only when the elements and therapy are guaranteed to be safe and ideally effective can we perform next step experiments in larger animals such as dogs. The further experiments will be performed in primates to ensure its safety. However, before putting into use, we will surely conduct more experiments to confirm the elements we use could work and are safe.