Assay, reporter system

Recent studies have further examined the iron stress response of pseudomonads using an iron-regulated, ice-nucleation gene reporter (inaZ) for induction of the iron stress response (17,18,84). This particular reporter system was developed by Loper and Lindow (85) for study of microbial iron stress on plant surfaces but was later employed in soil assays. In initial. studies, cells of Pseudomonas fluorescens and P. syringae that contained the pvd-inaZ fusion were shown to express iron-responsive ice-nucleation activity in the bean rhizosphere and phyllosphere. Addition of iron to leaves or soil reduced the apparent transcription of the pvd-inaZ reporter gene, as shown by a reduction in the number of ice nuclei produced. 

FIGURE 20.2 The involvement of RARE on apo-lO -lycopenoic acid-transactivated RARp expression. Upper panel Diagram of the RARp reporter vector with wild type and mutated R AREs. Lower panel HeLa cells transfected with the RARp reporter vector and an internal control vector were treated with 5 pmol/I. of apo-lO -lycopenoic acid or 1 pmol/L of all-trans retinoic acid for 24h. Luciferase activities were measured by dual-luciferase reporter system. Values are means of SEM of three replicate assays., statistically significantly different, as compared with control in the same group, P < 0.05. (Adapted from Lian, F. et al., Carcinogenesis, 28, 1567, 2007. With permission.)... 

The variety of assays reported here displays the great versatility of luminescent detection systems. As already described, in all luminescent systems the main advantages are the high sensitivity and specificity, which reduce to the minimum the sample treatment, and the ease of use of the reagents and the luminometer. Immobilized systems greatly reduce the cost per assay on the other hand, their preparation requires expertise, especially in the surface activation step on nylon tubes. 

Assays can be prepared with a reporter system containing, for example, the hrefly luciferase gene. The reporter cells are coupled to receptor genes. When a ligand binds to the receptor, a luminescent glow can be observed. In this way, the effects of the signaling events are evaluated. 

Persson, KP., Ekehed, S., Otter, C., Lutz, E.S., McPheaL J., Masimirembwa, C.M. and Andersson, T.B. (2006) Evaluation of human liver slices and reporter gene assays as systems for predicting the cytochrome p450 induction potential of drugs in vivo in humans. Pharmaceutical Research, 23, 56-69. 

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