Team:TECHNION-ISRAEL/delay mechanism
Our goal is to delay the membrane expression of allergy or autoimmune disease-causing epitopes until the HSCs engraft within the bone marrow. Engraftment occurs through a “Homing” mechanism (a phenomenon whereby cells migrate to a specific location with the aid of cytokines). This is done to prevent an immunological reaction while the engineered cells are still within the bloodstream.
We considered using various induction schemes such as Tet, Cumate, and Biotin based systems. Some of them are toxic while others have not yet been fully characterized. Finally, after consulting with several experts in the field we decided to use a Tet based system, the most commonly used inducible expression system for research in eukaryote cell biology. [1] Due to the nature of our proposed treatment, wherein protein expression is only meant to be temporarily repressed, we decided to use the Tet-Off system which allows for minimal usage of the inducer (Figure 1) [2] .
Figure 1: Tet-Off system mechanism
In the Tet-Off system, a tetracycline-controlled transactivator protein (tTA), which is composed of the Tet repressor DNA binding protein (TetR) from Escherichia coli fused to the strong transactivating domain of VP16 from Herpes simplex virus, regulates expression of a target gene that is under transcriptional control of a tetracycline-responsive promoter element (TRE).
The TRE is made up of Tet operator (tetO) sequence concatemers fused to a minimal promoter (commonly the minimal promoter sequence derived from the human cytomegalovirus (hCMV) immediate-early promoter). In the absence of Dox, tTA binds to the TRE promoter and activates transcription of the target gene. In the presence of Dox, tTA cannot bind to the TRE, and expression from the target gene remains inactive [2] .
We tested the Tet-Off system in our model cells and showed a strong and effective induction curve .
Lastly, it is important to note that the Tet-Off system is not applicable to our final design as it is considered toxic in adolescents. We decided to continue with this system in our research as it is well characterized and will allow for effective and simple usage in animal testing. There are constantly new induction systems being developed and we hope that a suitable and efficient option will be available in the near future.
- Gossen, Manfred, and Hermann Bujard. "Tight control of gene expression in mammalian cells by tetracycline-responsive promoters." Proceedings of the National Academy of Sciences 89.12 (1992): 5547-5551.
- Agha-Mohammadi, Siamak, et al. "Second‐generation tetracycline-regulatable promoter: repositioned tet operator elements optimize transactivator synergy while shorter minimal promoter offers tight basal leakiness." The journal of gene medicine 6.7 (2004): 817-828.
Department of Biotechnology & Food Engineering
Technion – Israel Institute of Technology
Haifa 32000, Israel
igem.technion.2017@gmail.com
