Difference between revisions of "Part:BBa K2295005"

Revision as of 23:15, 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 VEGF-A responsive, was characterized using Jurkat and HEK293T cells. Stable cell lines were generated containing pCTLA4 with quadruple NFAT binding sites (NFATbs) and the TATA like minimal promoter pTal (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.

VEGF and Ionomycin combined induce pCTLA4 in Jurkat

In Jurkat cells, 4xNFATbs-pTal:eCFP expression decreases with increasing VEGF-A concentration (Fig. 19 a). Addition of ionomycin (5 µM) inverts the response on increasing VEGF-A concentration to the expected trend (Fig. 19 b). Generally low expression in Jurkat cells may arise due to transcriptional inactivity in these cells. The influence of ionomycin on the expression of 4xNFATbs-pTal:eCFP combined with VEGF-A can not be evaluated as the results do not allow conclusions without further experiments. Results indicate that VEGF-A is not an optimal input for our AND gate. Further experiments have to be performed as the CD4+ Jurkat cell line is not identical to primary T cells.

Sequence and Features