Revision as of 01:41, 1 November 2017

HRE (Hypoxia Response Element) pTal (Minimal Promoter)

Figure 1: Signaling pathway of HIF1A.

Overview

Hypoxia induced factors (HIFs) are transcription factors responding to decreased oxigen levels in the cellular environment. Hypoxia induced factor 1 alpha (HIF1A) is a protein constantly expressed in mammalian cells. Under normoxic conditions HIF1A is hydroxylated and is marked by the E3 ubiquitin ligase which leads to the degradation by the proteasome. However, in hypoxic conditions HIF1A is stabilized and can heterodimerize with HIF1B. Also HIF1A transcription is often significantly upregulated under hypoxic conditions. Under hypoxic conditions HIF1 can then bind to hypoxia response elements (HREs) (BBa_K2295003) in the nucleus and lead to the expression of the gene of interest (Ziello et al., 2007).

Characterization

Freiburg 2017

Construct and Conditions

Gene expression specific to low oxygen concentrations (hypoxia) as given in the tumor microenvironment was tested with promoters containing hypoxia response elements (HREs) (Schödel et al., 2011). However, it is difficult and expensive to cultivate mammalian cells under hypoxic conditions. The transcription factor hypoxia inducible factor 1 alpha (HIF1A), which binds to HREs, is regulated by proline hydroxylase domain proteins (PHDs). Like in the absence of oxygen, modification of HIF1A by ΡΗDs can also be inhibited by cobalt dichloride (CoCl2), which was therefore used to mimic hypoxia (Epstein et al. 2001).

Jurkat and HEK293T cell lines with stable integration of the TATA-like minimal promoter (pTal; Mahindhoratep et al., 2014), modified to contain quadruple HRE, expressing eCFP were tested. Alternatively, single enhancer element promoters were introduced into CHO-K1 by PEI transfection. For analysis, cells were incubated with CoCl2 for 24 h prior to measurement by flow cytometry. Concentrations used vary between the cell lines due to different onset of toxicity.

Data and Discussion

Flow cytometry of Jurkat 4xHRE-pTal:eCFP cells shows increasing eCFP fluorescence correlating with moderate rising treatment (Fig. 1 a). Promoter activity in this most relevant cell line is strongest at 80 μM CoCl2, decrease of signal at greater levels of induction may stem from toxicity of CoCl2. Overall low expression is likely due to weak transcriptional activity inherent in this cell line (Michel et al., 2017).

Figure 2: Flow cytometry of hypoxia response element promoter tests.

a) Jurkat, b) HEK293T cells stably transduced with 4xHRE-pTal:eCFP, and c) HRE-pTal:eCFP or d) 4xHRE-pTal:eCFP PEI transfected into CHO-K1 cells. For analysis cells were incubated 24 h with indicated concentrations of CoCl₂. Significant differences were determined using one-tailed student’s t-test (Excel 2017) followed by Bonferroni-Hoch correction; * p ** p non-significant and decreasing differences are not marked.

Cloning

Figure 3: BBa_K2295003 and multiple enhancer elements

Multiple Enhancer Elements

Tuning the amount of enhancer elements, downstream transcription can be regulated. By introducing a BglII restriction site between the HRE and the pTal promoter in BBa_K2295003, an easy cloning strategy to change the amount of multiple enhancer elements is offered. Using compatible end cloning, multiple enhancer elements can be increased exponentially as shown in Figure 3.

Sequencing Results

Figure 4: Sequencing Result

Sanger sequencing was done at GATC with BBa_G00100. Geneious was used to map result to the expceted sequence. Line one: coverage, line 2: expected sequence, line 3: sequencing result

The original sequence of the pTal promoter was modified by one basepair due to a EcoRI restriction side which is not allowed in RFC10. Sequence and Features

References

This part can be seen as an improvement of BBa_K1456004 and BBa_K1720002