Difference between revisions of "Part:BBa K2295005"

Revision as of 23:12, 1 November 2017

CTLA4 (cytotoxic T-lymphocyte-associated Protein 4)

cytotoxic T-lymphocyte-associated Protein 4

Cloning

Figure 3: BBa_K2295003 and multiple enhancer elements

Characterization of the CTLA4 promoter

The CTLA4 promoter (pCTLA4), which is <a href="http://2017.igem.org/Team:Freiburg/Design">VEGF-A</a> responsive, was characterized using <a href="http://2017.igem.org/Team:Freiburg/Cell_culture">Jurkat and HEK293T cells</a>. Stable cell lines were generated containing pCTLA4 with quadruple NFAT binding sites (NFATbs) and the TATA like minimal promoter <a href="http://2017.igem.org/Team:Freiburg/Cloning">pTal</a> (Mahindhoratep et al., 2014) expressing eCFP as reporter gene. The VEGF receptor 2 (VEGFR-2) was added by PEI transfection for HEK293T cells, which do not express this gene (Liu et al., 2014). In order to characterize pCTLA4, transfected cells were induced with different concentrations of VEGF-A for 24 h. To investigate if the promoter can be activated via artificially increasing the intracellular Ca²⁺ concentration, cell were induced with ionomycin causing influx of Ca²⁺ into the cells (Bittinger et al., 2004). Fluorescence was measured by flow cytometry after 24 h of treatment.

Sequence and Features