Regulatory
pSteap4+mu

Part:BBa_K2538007

Designed by: Mor Pasi   Group: iGEM18_BGU_Israel   (2018-09-03)


Six Transmembrane Epithelial Antigen of Prostate 4 promoter (pSteap4) with a point mutation

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]

During PCR amplification from mouse genomic DNA a random point mutation occurred that may affect the promoter activity. (nucleotide #770 was replaced from G to A)


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 in order to activate pMLPm promoter 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 wheather the cell-line that is used in our experiments, C8-D30, is indeed a good model for A1 reactive astrocytes. Reactivity validation was achieved via immune-staining to known markers (Fig 1).

Figure 1: C8-D30 cells stained with DAPI (blue), Nestin (green) and GFAP (Red). Conducted for us on our cells by: Dinorah Friedmann-Morvinski's lab


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.

Promoter assay In order to assess the strength of pSteap4 promoter, we performed a promoter assay in C8-D30 cell-line. We examined the promoter by quantifying the amount of enzyme produced by the Luciferase gene (Fig 2).

Figure 2: The activity of pSteap4 promoter in reactive astrocyte cell line (C8-D30). Luciferase reporter assay demonstrating transcriptional activation mediated by pSteap4 promoter in C8-D30 cells. The pSteap4 promoter showed 8.14-fold increased activity, as compared to the control. Relative luciferase expression results are presented after normalization to Renilla luciferase activity and represent the means ± standard deviation of three independent experiments.

The pSteap4 value is very high when compared to the negative controls of empty vector, which suggest that our promoter is active in our reactive astrocyte C8-D30 cell-line.

According to the literature and this experiment, pSteap4 promoter is indeed expressed strongly in our reactive astrocytes. Therefore, while working with the cell line we can rely on these promoter to express our constructs specifically in reactive astrocytes. However, further experiments with a resting astrocyte sample must be performed in order to further confirm that these two promoters are active only in reactive astrocytes, but not in resting ones.

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.‏




Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//awards/part_collection
//promoter
Parameters
None