Reporter gene expression

In vivo studies in animals suggest that endosulfan may disrupt normal reproductive hormone levels in male animals, but that it is not an endocrine disrupter in females. Persistent depressed testicular testosterone was seen in male rats after intermediate duration oral exposures to endosulfan. In ovariectomized female rats, orally administered endosulfan did not induce normal development of female reproductive tissues, and in female mice and immature female rats, acute parenteral exposure to endosulfan did not affect several endocrine-related end points. In vitro studies have evaluated endosulfan for estrogen receptor (ER) and cytosolic protein binding affinity, ER-mediated reporter gene expression, estrogenic induction of cell proliferation, and alteration of relative abundance of active estradiol metabolites. Overall, in vitro evidence in favor of endosulfan estrogenicity indicates relatively weak potency compared to 17[3-estradiol. Apparently contradictory results were reported in different... 

The test system was considerably less sensitive to endosulfan when mouse ER, rather than human ER, was used to mediate (3-gal activity (Ramamoorthy et al. 1997). In similar assays, endosulfan at 10 jM had no effect on (3-gal activity in yeast Saccharomyces) transfected with either the human or rainbow trout ER (Andersen et al. 1999). In addition, no effect was observed on transcriptional activation of HeLa cells transfected with plasmids containing an estrogen receptor as a responsive element (Shelby et al. 1996). Endosulfan also did not induce transient reporter gene expression in MCF-7 human breast cancer cells at an incubation concentration of 2.5 pM (Andersen et al. 1999). Maximum endosulfan-induced ER-mediated luciferase reporter gene expression occurred in vitro in a T47D human breast adenocarcinoma cell line at approximately 10 pM, while 50% expression of luciferase occurred at about 5.9 pM the maximum expression was approximately 59% of the effect from exposure to 0.03 nM estradiol (0.00003 pM) (Legler et al. 1999). Luciferase expression from combined treatment with endosulfan and dieldrin was additive over concentrations ranging from 3 to 8 pM. 

Ziady AG, Ferkol T, Dawson DV, Perlmutter DH, Davis PB (1999) Chain length of the polylysine in receptor-targeted gene transfer complexes affects duration of reporter gene expression both in vitro and in vivo. J Biol Chem 274 4908 1916... 

The administration of SPLP results in reporter gene expression at the tumor site (Fig. 7B). Injection of free plasmid or lipoplexes resulted in no detectable gene expression at the tumor site. However, transfection was observed in the limg, liver, and spleen. SPLP, on the other hand, did not show detectable levels of gene expression in these organs. 

Particles from cationic lipids may also be useful for antisense therapy of skin disease — a nontoxic increase in the oligonucleotide uptake by cultivated keratinocytes and a sebocyte cell line has been reported [66]. Moreover, cationic dendri-mers also efficiently transfer reporter gene DNA to human keratinocytes cultivated in vitro. In the skin of hairless mice, in vivo transfection was possible with complexes, yet reporter gene expression was localized to perifollicular areas. Transfection, however, failed with the naked plasmid. For prolonged contact, biodegradable membranes coated with dendrimer/DNA complexes were used [67]. This hints at a follicular uptake of these complexes and indicates that gene transfection also may be possible with human skin, which has a thicker stratum comeum compared with mouse skin (eight to ten vs. two to three layers [58]). 

Packer M (1992) The neurohumoral hypothesis a theory to explain the mechanism of disease progression in heart failure. J Am Coll Cardiol 20 248-254 Pepperl DJ, Regan JW (1993) Selective coupling of a2-adrenergic receptor subtypes to cAMP-dependent reporter gene expression in transiendy transfected JEG-3 cells. Mol Pharmacol 44 802-809... 

K.H. Lee, S.S. Byun, J.H. Choi, J.Y. Paik, Y.S. Choe, B.T. Kim, Targeting of lacZ reporter gene expression with radioiodine-labelled phenylethyl-beta-d-thiogalacto-pyranoside, Eur. J. Nucl. Med. Mol. Imaging 31 (2004) 433-438. 

Anti-androgenic effect. Sterol fraction of the dried fruit, in cell culture, was active on CA-PC3 and reduced androgen-induced reporter gene expression in transcriptional assay IC50 50 pg/mL . Administration to... 

The estrogenic and antiestrogenic activities of several PBDE congeners and three hydroxylated PBDEs were tested in vitro using human breast cell line assays based on estrogen receptor (ER)-dependent luciferase reporter gene expression (Mccrts et al. 2001). The hydroxylated PBDEs,... 

Mortimer et al., 1999). In contrast, the level of reporter gene expression was enhanced when cells were exposed to lipoplexes during or just before mitosis (Brunner et al., 2000). Finally, the relatively low rate of cell proliferation of primary cultures of ciliated human airway epithelia was found to be one of the determinants for their low transfectability by lipoplex (Fasbender et al., 1997). These results, however, do not preclude the possibility that DNA can be translocated through nuclear pore complex by a NLS-independent mechanism. 

Ross, G.F., Bruno, M.D., Uyeda, M., Suzuki, K., Nagao, K., Whitsett, J.A. and Korfhagen, T.R. (1998) Enhanced reporter gene expression in cells transfected in the presence of DMI-2, an acid nuclease inhibitor. Gene Then, 5, 1244-1250. 

DNA injection directly into mouse diaphragm has also resulted in luciferase expression and there appeared to be no damage to the diaphragm due to the DNA injections (Davis and Jasmin, 1993). In a related study, /3-galactosidasc ( /3-gal)-encoding pDNA injected into the articular space of rabbit knee joints resulted in /3-gal expression in the joints (Yovandich etal., 1995). In the same study, chloramphenicol acetyltransferase (CAT) encoding pDNA injected into rat knee joints also led to reporter gene expression, with peak expression 48 hours after injection and with no detectable activity 15 days later. 

Naked pDNA encoding luciferase has also been able to transfect rat stomach where reporter gene expression was found up to day 21 post pDNA injection (Takehara et al., 1996). Histological analyses revealed that primarily smooth muscle cells and mesenchymal cells were transfected while epithelial cells had no apparent reporter gene expression. 

In a similar study by Yang and Huang (1996), melanoma tumors in mice were in vivo transfected by the direct intratumoral administration of naked pDNA, resulting in reporter gene expression for up to ten days. In another study, Walker 256 carcinoma tumors in rats were found to express CAT after intratumoral injection of naked pDNA encoding CAT (Nomura et al., 1997). More recently electroporation has been used to deliver naked pDNA to tumors (described further in the following section). 

Ross, G.F., et al. 1998. Enhanced reporter gene expression in cells transfected in the presence of DMI-2, an acid nuclease inhibitor. Gene Ther 5 1244. 

Das A, Niven R (2001) Use of perfluorocarbon (Fluorinert) to enhance reporter gene expression following intratracheal instillation into the lungs of Balb/c mice implications for nebulized delivery of plasmids. J Pharm Sci 90 1336-1344... 

Welsh S, Kay SA (1997), Reporter gene expression for monitoring gene transfer, Curr. Opin. Biotechnol. 8 617-622. 

Measurement of the regulation of cAMP-dependent reporter gene expression, inhibition of constitutive mobilization of intracellular Ca2+, and enhancement of neurotransmitter release have also been used to detect inverse agonism in other Gi/o coupled receptor systems [see 8 for review] ... 

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