Dual Luminescence-Based Reporter Gene Assay for Luciferase andβ-Galactosidase

Martin, Chris; Wight, P.A.L.; Dobretsova, Anna; Bronstein, Irena

doi:10.2144/96213pf01

Cited by 67 publications

(34 citation statements)

References 8 publications

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“…Cells that received the MF or TS treatments and their controls were processed according to the luciferase assay system (Promega, Madison, WI) as described by Martin et al (1996). Briefly, cellular lysates were assayed for luciferase activity by the addition of 20 μl lysate to 100 μl luciferin substrate.…”

Section: Methodsmentioning

confidence: 99%

Effect of 60 Hz magnetic fields on the activation of hsp70 promoter in cultured INER-37 and RMA E7 cells

Heredia-Rojas¹,

Fuente²,

González

et al. 2010

In Vitro Cell.Dev.Biol.-Animal

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It has been reported that 50-60 Hz magnetic fields (MF) with flux densities ranging from microtesla to millitesla are able to induce heat shock factor or heat shock proteins in various cells. In this study, we investigated the effect of 60 Hz sinusoidal MF at 8 and 80 μT on the expression of the luciferase gene contained in a plasmid labeled as electromagnetic field-plasmid (pEMF). This gene construct contains the specific sequences previously described for the induction of hsp70 expression by MF, as well as the reporter for the luciferase gene. The pEMF vector was transfected into INER-37 and RMA E7 cell lines that were later exposed to either MF or thermal shock (TS). Cells that received the MF or TS treatments and their controls were processed according to the luciferase assay system for evaluate luciferase activity. An increased luciferase gene expression was observed in INER-37 cells exposed to MF and TS compared with controls (p < 0.05), but MF exposure had no effect on the RMA E7 cell line.

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Section: Methodsmentioning

confidence: 99%

Effect of 60 Hz magnetic fields on the activation of hsp70 promoter in cultured INER-37 and RMA E7 cells

Heredia-Rojas¹,

Fuente²,

González

et al. 2010

In Vitro Cell.Dev.Biol.-Animal

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“…This assay uses the chemiluminescent substrate AMPGD (3-(4-methoxyaprio [1,2-dioxethane-3.2′-tricyclo[3.3.1.1 3,7 ] decan-4-yl) phenyl-␤-dgalactopyranonside), which allows for the detection of as little as 2 fg (0.015 amol) of ␤-gal. 30,31 Injected hippocampus was isolated from animals 48 h after infection and homogenized in lysis solution (100 mm potassium phosphate pH 7.8, 0.2% Triton X-100, 1 mm DTT, 0.2 mm phenylmethylsulfonyl fluoride (Sigma) and 5 mg/ml leupeptin (Sigma)). The samples were then centrifuged at 12 500 g for 10 min at 4°C and the supernatant was removed and heated at 48°C for 60 min.…”

Section: Plasmid and Vector Constructionmentioning

confidence: 99%

Drug inducible transgene expression in brain using a herpes simplex virus vector

Oligino

Wang

et al. 1998

Gene Ther

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The ability to regulate transgene expression is likely to be ture half maximal expression was achieved with 10important in the use of gene transfer to treat diseases of RU486, and maximal expression was achieved by 24 h. the central nervous system (CNS). In order to achieve Following stereotactic inoculation of the vector into rat regulatable gene expression we created a replicationhippocampus, expression was increased 150-fold by i.p. incompetent genomic herpes simplex vector containing a administration of RU486. This demonstrates that the RU486-inducible transactivator and a lacZ reporter gene RU486 system functions as a tight on/off switch for regunder transcriptional control of a minimal promoter.ulating expression of a transgene delivered to the brain Reporter gene expression from the vector was regulated via an HSV vector. by administration of RU486 in vitro and in vivo. In cell cul-

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“…This detection system has a large linear range for luciferase concentrations (between 10 715 to 10 78 g) and for bgalactosidase concentrations (between 10 713 to 10 79 g) (Martin et al, 1996). Nevertheless, titration experiments were performed to ensure that linearity standards were maintained in our system.…”

Section: Cell Culture and Transfectionsmentioning

confidence: 99%

Multiple negative elements contribute to repression of the HOX11 proto-oncogene

1998

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The HOX11 proto-oncogene is normally expressed in embryogenesis where it directs the synthesis of the spleen. In adult tissues, HOX11 expression is silenced by an unknown mechanism. Aberrant expression of HOX11 occurs in T-cell acute lymphoblastic leukaemia (T-ALL), where it is thought to be involved in T-cell immortalization. The deregulated expression of HOX11 is frequently associated with chromosomal translocations which juxtapose a T-cell receptor (TCR) gene upstream of the HOX11 gene. In these cases, it is presumed that the activation of HOX11 expression results from bringing the gene under the control of TCR enhancer elements. However, activation of HOX11 also occurs in the absence of an associated translocation in both T-ALL and erythroleukaemia cells, implying that an alternative activation mechanism may exist. We hypothesized that HOX11 may be repressed in normal T-cells and erythroid cells by the action of negative elements which may be deleted or mutated in leukaemia. We therefore conducted a search for negative elements in the human HOX11 promoter which may function to silence its expression in normal cells of the haematopoietic lineages. Since little sequence of the HOX11 promoter was available, we began our investigation by sequencing over 4.5 kilobases of untranslated DNA from upstream of HOX11. The human sequence that overlaps with the 2.1 kb of murine Hox11 is highly conserved, suggesting that a large region of DNA upstream of HOX11 may have a regulatory function. We then used transfection assays to test the ability of portions of the promoter to drive transcription of a reporter gene. These studies identi®ed four negative elements. Two of them (NRE2 and NRE4) function in all cell lines tested, while the remaining two (NRE1 and NRE3) appear to be cell-type speci®c. The DNA sequences of three elements are conserved between the human and mouse HOX11/Hox11 promoters. We propose a model in which the combined action of these negative elements contributes to the overall repression of HOX11 expression in normal blood cells.

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Dual Luminescence-Based Reporter Gene Assay for Luciferase andβ-Galactosidase

Cited by 67 publications

References 8 publications

Effect of 60 Hz magnetic fields on the activation of hsp70 promoter in cultured INER-37 and RMA E7 cells

Effect of 60 Hz magnetic fields on the activation of hsp70 promoter in cultured INER-37 and RMA E7 cells

Drug inducible transgene expression in brain using a herpes simplex virus vector

Multiple negative elements contribute to repression of the HOX11 proto-oncogene

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