Reporter cell line

Balaguer, R, Fenet, H., and Georget, V. et al. (2000). Reporter cell lines to monitor steroid and antisteroid potential of environmental samples. Ecotoxicology 9, 105-114. 

Balaguer P, Francois F, Comunale F, Fenet H, Boussioux AM, Pons M, Nicolas JC, Casellas C (1999) Reporter cell lines to study the estrogenic effects of xenoestrogens. Sci Total Environ 233 47-56... 

Ringerike, T. et al., Detection of immunotoxicity using T-cell based cytokine reporter cell lines ( Cell Chip ), Toxicology, 206, 257, 2005. 

Emter R, Ellis G, Natsch A (2010) Performance of a novel keratinocyte-based reporter cell line to screen skin sensitizers in vitro. Toxicol Appl Pharmacol 245 281-290... 

Wunder F, Tersteegen A, Rebmann A et al (2005) Characterization of the first potent and selective PDE9 inhibitor using a cGMP reporter cell line. Mol Pharmacol 68 1775-1781... 

Figure 2.5. A schematic of a p-lactamase reporter cell line that is coupled through a seven transmembrane G-protein—coupled receptor whose second messenger signaling pathway operates through cyclic adenosine monophosphate (cAMP). The G-protein, Gas, activates adenyl cyclase, increasing the concentration of cAMP, which stimulates the production of p-lactamase through cAMP response element.

Figure 2.7. Demonstration of a j3-lactamase reporter cell line. In a final volume of 0.02 mL, 5000-10,000 cells were seeded overnight at 37 C before being stimulated with excess agonist or antagonist for 4 h. The resultant /3-lactamase activity was measured after a 1 -h incubation with CCF2 (AuroraBio-sciences) at the indicated wavelengths in an LJL Analyst fluorometer. (a) The unstimulated cels, (b) the cells incubated with agonist, and (c) cells treated with agonist and excess antagonist. See color insert.

The evidence for the role of Toll-like receptors 4 or 2 as receptors for OxPLs is based on impaired responses to OxPLs or mmLDL in knockout animal models or after knockdown of TLRs in cultured cells [49-54], OxPLs do not demonstrate canonical activation of TLRs in cells naturally expressing these receptors or in reporter cell lines [55-57]. It can be hypothesized that this discrepancy is explained by the need of additional soluble or membrane-associated proteins that present OxPLs to TLRs [51,52]. The role of TLRs as candidate receptors mediating proinflammatory action of OxPLs is discussed in more detail in Chapter 11 in this book. 

CRE, NFKB, and CAGA12 reporter cell lines were activated with WntSa-conditioned medium, Forskolin, TNEa (tumor necrosis (actor alpha), and TGFp, respectively, and treated with 12-point dilutions of XAV939 or LDW643 (inactive analog). The corresponding reporter activity for each... 

This retinoid reporter cell assay has been successfully used to determine the distribution of retinoids in embryonic tissues (7,15,16). Retinoid reporter cell lines similar to those described here have been constructed by other laboratories (17,18). 

The reporter construct used to establish stable reporter cell lines is shown in Fig. 1. The construct consists of a reporter gene, in this case lacZ or luciferase, driven by a minimal promoter containing a TATA motif and a RARE. The RARE/TATA promoter is derived from the human p-retinoic acid receptor gene (8) and functions as an inducible enhancer that responds to all known RAR subtypes (19). Immediumtely 5 to the RARE is a silencer cassette... 

The two reporter cell lines used in these studies are derived from F9 teratocarcinoma cells and L cells. The F9 reporter cell line was established by transfection with a lacZ reporter gene construct and the L cell reporter line was established with luciferase as reporter gene. After selecting for the highest responders to retinoic acid (5 x 10" M), clonal cell lines were established and maintained under G418 selection. 

F9 and L cell reporter cell lines are normally passaged in T-75 flasks. The F9 cells require gelatinized flasks. 

In certain instances, it may be critical to know the relative amount of retinoid released from individual tissues or dissected regions of the embryo. To obtain a quantitative measure of retinoids released from tissues, we developed a reporter cell line using luciferase as the reporter molecule. When luciferase oxidizes its substrate luciferin, a photon is emitted that can be detected and quantified by use of a luminometer or scintillation counter. By relating known concentrations of retinoids to the amount of luciferase activity that they induce, the concentration of retinoids released by tissue samples can be determined. For quantifying retinoid release, a standard curve relating known concentrations of released retinoids to induced luciferase activity must be established. Known concentrations of released retinoids can be achieved by the use of ion-exchange beads that are loaded with retinoic acid and act as a point source of retinoid released onto reporter cell monolayers. 

F9 cells were transfected according to the method of Espeseth et al. (9) L cells were transfected as described (7). After selection in G418-containing medium, single colonies were grown up and tested for their response to RA. The highest responders were established as continuous reporter cell lines. The use of stably transfected reporter cell lines in place of transiently transfected cells provides a degree of stability and uniformity in the response of these cells that allows for a reproducible and reliable reporter assay... 

The luciferase reporter cell line may also be used to measure retinoids in tissue homogenates. The luciferase reporter cells can detect as little as 0.1 fmol of RA, and their response is linear up to 10 fmol of RA (7). 

I would like to thank Dr. Thomas Jessell (Howard Hughes Medical Institute, Columbia University) for his support during the generation and use of these reporter cell lines in his laboratory. I also thank Barbara Han for expert technical assistance in the production of these reporter cells and Ira Schieren for his expertise in computer-generated graphics. 

Cover illustration Fig. 1 from Chapter 3, Detection and Measurement of Retinoic Acid Production by Isolated Tissues Using Retinoic Acid-Sensitive Reporter Cell Lines, by Michael Wagner. 

By day 14 of differentiation, MYH6 levels should already be elevated and should be visible as a fluorescent signal when using a reporter cell line. We however keep the cultures for 4 more days to allow a further increase of the MYH6 signal, which results in an improved dynamic range for quantification. 

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