Transfection gene-activated

Keywords. Polyamidoamine dendrimers. Activation of PAMAM dendrimers. Transfection, Gene transfer, DNA-dendrimer complex... 

We utilize three independent methods to assess a compound s effect in vivo on translation—metabolic 35S-methionine/cysteine labeling, polysome profile analysis, and effects on transfected reporter gene activity. 

OECD. 2006e. Stably transfected transcriptional activation TA) assay for detecting estrogenic activity of chemicals - The human estrogen receptor alpha mediated reporter gene assay using HeLa-hER-9903 cell line. Version October 2006. Paris OECD, http //www.oecd.org/dataoecd/49/55/37531918.pdf... 

Lin et al. [149] have recently reported the synthesis of 2-(4,5-dimethoxy-2-nitrobenzyl)-20-hydroxyecdysone (6-6), which itself has a very low affinity for ecdysteroid receptors expressed in mammalian 293T cells, but can be photolysed at 348 nm to release 20E, which then activates the receptor-regulated reporter gene. With this system, it will be possible not only to control the temporal activation of transfected genes, but also their spatial activation. The authors recognise that the effectiveness of this system can be considerably improved in future by derivatising an ecdysteroid (poA or muA) which is more active in ecdysteroid-responsive mammalian cells. 

Incubate cells with transfection complex for 24 h. Test cell viability using 0.4% Trypan Blue to stain cells and count viable cells at seeding, day of transfection, and just prior to harvest. Harvest cells and assay for reporter gene activity Spin down cells and lyse cells to assay for luciferase activity. Figures 1 and 2 illustrate typical transfection results, observed visually as lacZ stained cells (Fig. 1) or Relative Light Units (RLUs) when luciferase activity is measured (Fig. 2). 

In a mammalian system, transcriptional repression has also been ascribed to PBC-HOX heterodimers. Multimers of a PBX-HOX co-operative binding site (TGATTGAT) decrease reporter gene activity in transfected cells by comparison to an otherwise identical reporter bearing HOX monomer binding sites (Saleh et al., 2000b). However, at least some of this repression could be due to PBX homodimers and PBX MEIS or PBX-PREP heterodimers that are likely to bind multimerized PBC-HOX sites. Two instances of EXD-dependent but HOX-independent repression could be mediated by comparable EXD homodimers or EXD HTH heterodimers (Rauskolb and Wieschaus, 1994 Pinsonneault et al., 1997). 

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