Team:Tongji China/Animal check in

Animal Check in

We apply Mus musculus in our project, and we had submitted a series of check-in forms before.
In the following paragraphs, I will demonstrate the necessity and importance of using these organisms.

First, we are concerning about the necessity.

Our product depends a lot on the Type III secretion system (T3SS) and its interaction between several systems in mammalian species. Animal experiment is the only way for us to fully test the product. We need to use the wild type mice to prove that the method we use to deliver the antigens into immune system can work in the condition of mammals' gut, which is the core point of our whole project. So, we first need to do the frozen tissue section immunofluorescence staining experiment to prove the delivery capacity of our T3SS in mammals' gut and then do the ELISA to test whether the antigens which are successfully delivered to the T cells have the ability to activate the T cells and guide them to attack the tumor cells.
Immunity in our gut is too complex to mimic in vitro, so it is necessary to conduct our experiment also in vivo.
Nevertheless, we do not intend to skip the in vitro experiment. Before the experiment performed in mice, we will run Western Blot to make sure that the peptide (our antigen) will be secreted properly. Also, experiments on cells will be performed. We will use HeLa cells to check the ability to deliver the peptide into other cells. After that, for we can get onefold intestinal wall cells by cell primary culture and we need to not only test the secretion ability but also the ability to trigger the immune reactions.
But the HeLa cell we use to prove the delivery capacity of the T3SS cannot prove that the engineered bacteria also have the delivery capacity when orally take into the human gut, for the type of cells are totally different. And as we all know, the pH and the chemical environment in human gut are different from the cell culture environment, and all the differences might cause interference to the delivery capacity and even the survival of the bacteria.
As a result, we decide to test the ability of triggering immune reactions in a complete mammalian immune system in the animal experiments.

Then, importance is ought to be described.

The alternative way is to use the mammalian cells or other accessible organisms. We had used the HeLa cells to detect the delivery capacity at first, while this experiment is not sufficient enough to clarify whether the bacterium can alter mammalian immune system through intestinal way. So, we can't skip in vivo experiments which are actually in need of sacrificing laboratory animals. Moreover, another kind of mammals we can easily access is rabbit, but it's more appealing to us to use mice whichever in economic or convenience consideration.

Finally, what welfare we can supply for these organisms to facilitate their life.

Our university has special place to keep the experimental animals at SPF level. Specialists are employed to keep the animals. Within our experiment, we will check the mice every day to ensure they are in good condition and have enough food and water. As for our experiment, before killing the mice, we will have them narcotized so that they won't feel any pain.
Also, we will use as few number of animals as we can to meet our goals in project. The fewest animals we use is 24 mice. Our plan is to immune the mice and then do the ELISA to find out if it generates the antibody. We have 6 target antigens and 6 control. And we will also need to do the frozen tissue section immunofluorescence staining experiment, which also needs 6 mice as the experimental group and 6 mice as the control group. All these experiment designs come from the Experiment design assistant (EDA).
Our animal check-in form had already been adopted by iGEM organizing committee, and we also finished our experiments in vivo a few days ago.