Team:Freiburg/Demonstrate
Proof of Concept
The CARTELTM AND gate is a genetic circuit constructed of two promoters interconnected through a protein, to integrate two inputs into one output. For input one, two alternatives were evaluated. As a proof of concept we characterized all the promoters in mammalian cell lines
In order to characterize the promoters we generated stable cell lines, containing the promoters for the inputs driving a reporter protein. During the summer we generated stable cell lines with multiple enhancer elements for the inputs: pH, VEGF and hypoxia in Jurkat and HEK293T cells. Additional transient experiments were performed with HEK293T and CHO-K1 cells. These cells were exposed to low pH, high VEGF concentration and hypoxia and the output was measured via flow cytometry (Fig. 1).
Figure 1: Flow cytometry of hypoxia response element and cAMP response element promoter analysis. a) Jurkat stably expressing 4xHRE-pTal:eCFP. Cells were incubated 24 h with indicated concentrations of CoCl2. A significant increase in eCFP positive cells can be observed for 80 µM CoCl2. b) Jurkat stably expressing 4xCRE-pTal: eCFP. Cells were incubated 24 h in pH adjusted RPMI 1640 and induced with forskolin (10 µM) and IBMX (10 µM). The highest amount of eCFP positive cells can be observed for pH 6.5. Data points are mean values of triplicates, error bars represent the standard deviation. Significant differences were determined by using one-tailed student’s t-test (Excel 2017) followed by Bonferroni-Hoch correction (a) and by using ANOVA (b); * p < 0.05, ** p < 0.01, *** p < 0.001, non-significant and decreasing differences are not marked.
The CARTELTMAND gate requires the knockdown or knockout of the endogenous gene coding for hypoxia-inducible factor 1 alpha (HIF1A) to ensure exclusive control over HIF1A by the introduced AND gate. We generated several stable HIF1A knockdown Jurkat and HEK293T cells. Transient expression of HA tagged HIF1A in knockdown cells showed it behaves as the endogenous HIF1A, without being affected by the siRNA used to generate the knockdown (Fig. 2, 3).
Figure 2: Transient introduction of artificial HIF1A in HIF1A shRNA1 knockdown HEK293T cell line.
Induction of the overexpressed HIF1A-HA can be observed in the last lane. Endogenous and artificial HIF1A were detected with HIF1A antibody; reintroduced HIF1A was detected via an HA-tag; GAPDH served as a loading control. Wild type (WT) and knockdown (HIF1A shRNA1) cells were transfected with CMV:HIF1A-HA one day prior to induction with CoCl2 to simulate hypoxia. Cells were harvested 24 hours after induction. Immunoblot assay was performed using antibodies against HIF1A, HA-tag and GAPDH.
Figure 3: Transient introduction of artificial HIF1A in HIF1A shRNA2 knockdown HEK293T cell line.
Induction of the overexpressed HIF1A-HA can be observed in the last lane. Endogenous and artificial HIF1A were detected with HIF1A antibody; reintroduced HIF1A was detected via an HA-tag; GAPDH served as a loading control. Wild type (WT) and knockdown (HIF1A shRNA2) cells were transfected with CMV:HIF1A-HA one day prior to induction with CoCl2 to simulate hypoxia. Cells were harvested 24 hours after induction. Immunoblot assay was performed using antibodies against HIF1A, HA-tag and GAPDH.
As an additional safety feature for the CARTELTM T cells we generated stable T cell lines with the kill switch gene thymidine kinase. In the event of uncontrolled actions by the T cells, they can be eliminated by the administration of the drug ganciclovir. It is only affecting cells containing the kill switch, unmodified cells remain intact. To evaluate the efficiency of our kill switch we performed an apoptosis assay with ganciclovir (Fig. 4).
Figure 4: Ganciclovir killing curve with Jurkat and HEK293T cells Depicted is the survival rate in percent after induction of the killing with ganciclovir. Once tested in a) Jurkat cells for 120 h and b) HEK293T cells for 72 h. Investigated was the killing of wild type (WT) and stable cell lines containing the suicide gene (SG). All experiments were performed in triplicates.
This summer we could demonstrate all parts of the CARTELTM AND gate are functional. After optimization and improvement of individual parts the system needs to be tested in primary T cells and may then proceed to pre-clinical studies with animal models. To learn more about possible improvements and future applications visit our outlook page!
Results
Applied Design
