Part:BBa_K2538008
Six Transmembrane Epithelial Antigen of Prostate 4 promoter (pSteap4)
pSteap4 (Six Transmembrane Epithelial Antigen of Prostate 4 promoter) is regulating the expression of Six Transmembrane Epithelial Antigen of Prostate 4 gene which is highly expressed in A1 reactive astrocyte cells. [1,2]
Usage and Biology
In ALS disease, under gliosis conditions, astrocyte cells transform from resting to reactive form, this transformation includes the induction of several genes including Six Transmembrane Epithelial Antigen of Prostate 4 gene by pSteap4.[1,2]
We will use this promoter to specifically express CRISPR dCas9 VP64 system in order to activate the synthetic minimal adenovirus major late promoter (pMLPm) that will express reverse Caspase3, by doing so we will be able to induce apoptotic death only in reactive astrocytes and not in the resting cells. By reducing the amount of reactive astrocytes we will prevent motor neuron death and prolong ALS patient survival.
Characterization
Reactivity test by immunostaining of astrocyte cell-line C8-D30 (Conducted by: Dinorah Friedmann-Morvinski's lab)
Aiming for expression under pSteap4 promoter specifically in reactive astrocytes, we wanted to validate rather the cell line that is used in our experiments, C8-D30, is indeed a good model for A1 reactive astrocyte. Reactivity validation was achieved via immune-staining to known markers (Fig 1).
High expression of GFAP and Nestin proteins is a specific feature of reactive astrocytes, usually appear after various brain injuries [3]. As can be seen in Figure 1, C8-D30 astrocytes cells displayed strong immunostaining signal for both GFAP and Nestin. Therefore, it is possible to conclude that the chosen cell line of C8-D30, is indeed that of reactive astrocytes. It can serve as an accurate model for testing this project hypothesis, and to examine the specificity of pSteap4 promoter.
Gene expression due to pSteap4 activity
This part was used by BGU 2018 team in the pSynt construct.
In this construct pSteap4 regulates the expression of gRNA. In our design the chosen gRNA spacer is complementary to three different loci in the pMLPm promoter.
C8-D30 cells were co-transfected with a Lenti-viral vector: pTimp1-dCas9-VP64 and pSynt. dCas9-VP64-gRNA complex targets and activates the pMLPm promoter in our design, otherwise pMLPm promoter remains non-active [4]. This activation has driven pMLPm to regulate the expression of exogenous reverse caspase3 fused to mCherry fluorophore by T2A peptide (Fig 2).
mCherry expression in C8-D30 reactive astrocyte cells was visible. Demonstrating the activity of our dCas9 editing approach and verifying expression of reverse caspase3. In fluorescent microscope we were able to detect mCherry fluorophore in several cells.
By demonstrating mCherry expression we were able to demonstrate both the specificity of our chosen promoter as well as the ability of our system to drive the expression of caspase3 (through the mCherry marker) we have designed into our plasmid.
References
1. Liddelow, Shane A., et al. "Neurotoxic reactive astrocytes are induced by activated microglia." Nature 541.7638 (2017): 481.
2. Zamanian J, Xu L, Foo L, et al. Genomic Analysis of Reactive Astrogliosis. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2012;32(18):6391-6410. doi:10.1523/JNEUROSCI.6221-11.2012.
3. Sosunov, Alexander A., et al. "Phenotypic conversions of “protoplasmic” to “reactive” astrocytes in Alexander disease." Journal of Neuroscience 33.17 (2013): 7439-7450.
4. Farzadfard, F., Perli, S. D., & Lu, T. K. (2013). Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. ACS synthetic biology, 2(10), 604-613.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
//promoter
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