Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
1995-12-28
2003-08-05
Horlick, Kenneth R. (Department: 1637)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S006120
Reexamination Certificate
active
06602657
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to methods of detecting transcriptional activity in a cell using two or more reporter genes.
BACKGROUND OF THE INVENTION
Reporter gene assays are useful in the study of gene regulatory elements because reporter gene activity, i.e., production of the reporter protein, is directly proportional to transcriptional activity of the regulatory elements of the gene. A reporter gene construct for use in these assays contains one or more gene regulatory elements which are of interest, the minimal sequence requirements for transcription of a functional mRNA and the coding sequence for a reporter protein. Alam, J., et al,
Anal. Bioch
., 188: 245-254 (1990). Analysis of constructs containing various deletions within the regulatory region enables mapping of regulatory sequences necessary for transcription and cell specific expression.
The reporter protein typically has a unique enzymatic activity or structure which enables it to be distinguished from other proteins present. The activity of the transcribed reporter protein, or quantification of the expressed protein, provides an indirect measurement of gene expression. Reporter gene assays enable the identification of sequences and factors that control gene expression at the transcriptional level. Bronstein, I., et al,
BioTechniques
, Vol. 17, No. 1, p. 172 (1994). Other uses for reporter gene assays include: identification of sequences and factors that control genes at the translational level, study of mechanisms and factors that influence and alter gene expression levels and drug screening in cell-based assays.
In single reporter gene assays with poor sensitivity it is difficult to distinguish negative results caused by the lack of expression or low level assay sensitivity. This problem can be overcome with assays of greater sensitivity. Multiple gene assays are commonly used to provide controls for efficiency of transfection. In such assays, cells are transfected with a mixture of two separate plasmids, each having a different reporter gene. The expression of one reporter gene is controlled by different regulatory regions being studied while the other reporter gene, acting as a control, is generally constitutively expressed by a standard promoter or enhancer. The activity of the experimental reporter enzyme is normalized to the activity of the control reporter enzyme.
In the known examples of assaying multiple reporter gene expression, a separate aliquot of the sample must be used in a separate assay to test for the activity of each enzyme. Alam, J., et al,
Anal. Bioch
., 188: 245-254 (1990). The necessity of testing a separate portion for each enzyme decreases the precision of the assay and may introduce experimental errors into the results. Therefore, a multiple reporter gene assay which is sequentially performed on the same aliquot of cell extract would simplify the assay procedure and minimize experimental errors. The use of multiple reporter genes can improve the efficiency of high throughput screening for drug discovery.
To provide relevant experimental information, reporter assays must be sensitive, thus enabling the detection of low levels of reporter protein in cell lines that transfect poorly. The sensitivity of a reporter gene assay is a function of the detection method as well as reporter mRNA and protein turnover, and endogenous (background) levels of the reporter activity.
Commonly used detection techniques use isotopic, calorimetric, fluorometric or luminescent enzyme substrates and immunoassay-based procedures with isotopic or color endpoints. Many of these systems, however, have disadvantages that limit their usefulness in these assays. For example, isotopic substrates and immunoassays are limited by the cost, sensitivity and inconvenience of using radioisotopes. Fluorometric systems require external light sources which must be filtered to discriminate fluorescent signal, thereby limiting the sensitivity and increasing complexity of the detection system. Furthermore, fluorescence from endogenous source can interfere with fluorometric measurements. Colorimetric systems lack the sensitivity desired for sensitive reporter gene assays. Chemiluminescent and bioluminescent assays, on the other hand, have been found to be more rapid and sensitive than colorimetric assays and fluorometric assays. Jain, V. K. and Magrath, I. T.,
Anal. Biochem
., 199: 119-124 (1991). It would be, therefore, desirable to have a multiple reporter gene assay as aforesaid, which uses a luminescent detection system.
A number of genes are currently used as reporter genes including chloramphenicol acetyltransferase (CAT), secreted alkaline phosphatase, luciferase, &bgr;-galactosidase, &bgr;-glucuronidase, and human growth hormone, among others. Bronstein, I., et al,
Anal. Biochem
., 219: 169-181 (1994). &bgr;-galactosidase and CAT are two of the most widely used reporter genes. See Alam, et al., 1990). &bgr;-galactosidase detection is commonly performed with calorimetric substrates which lack sensitivity. Fluorescent substrates are also used to detect &bgr;-galactosidase, however, those assays also lacks sensitivity and are limited by background autofluorescence and signal quenching. The most widely used assay for CAT is radioisotopic, exhibits only moderate sensitivity, and suffers from a narrow dynamic range. &bgr;-Glucuronidase (GUS) is a very widely used reporter gene in plant genetic research and to a lesser extent in mammalian cells. A common assay for GUS uses a fluorescent substrate, but is limited by background autofluorescence and signal quenching. Luciferase has become a more widely used reporter gene as it is quantitated using a very sensitive bioluminescent assay utilizing the substrate, luciferin.
Sensitive chemiluminescent assays, not limited to reporter gene-assays, have been described using dioxetane substrates. Bronstein, U.S. Pat. No. 4,978,614, incorporated herein by reference. These dioxetane substrates emit visible light following enzyme induced degradation. Enhancement of the chemiluminescent degradation of 1,2-dioxetanes by enhancer substances comprised of certain water soluble substances, such as globular proteins that have hydrophobic regions, has been described. Voyta et al., U.S. Pat. No. 5,145,772, incorporated herein by reference; These dioxetane substrates are also used in reporter gene assays for alkaline phosphatase, &bgr;-galactosidase, and &bgr;-glucuronidase for example. See e.g., Bronstein, I., et al,
Anal. Biochem
., 219: 169-181 (1994) and citations therein. However, no reporter gene assay using dioxetane substrates has been described in which the products of multiple reporter genes are sequentially quantitated in the same aliquot of cell extract.
Simple, rapid and highly sensitive, combined multiple reporter gene assays to detect commonly used reporter genes which do not use radioisotopes or require external light sources are highly desirable.
It is desirable to have a multiple reporter gene assay in which the reagents enhance the light signal produced by the reporter enzymes. It is also important that the signal from one reporter enzyme in a multiple reporter gene assay does not significantly interfere with the signal from the other reporter enzymes during measurement of their maximum light signal. It would be useful to have an assay which produces enhanced levels of light and therefore increases assay dynamic range and sensitivity and enables the use of a wide variety of instruments.
SUMMARY OF THE INVENTION
The method of the present invention provides a rapid, highly sensitive, non-isotopic method for sequentially detecting multiple reporter gene products in a single aliquot of cell extract. The method of the present invention comprises quantifying the activity of a first reporter enzyme by measuring the light signal produced by degradation of a substrate by the first reporter enzyme, and quantifying the activity of a second reporter enzyme by measuring the light signal produced by degradation of a second substrate by the second reporter enzyme, wherein both qu
Bronstein Irena Y.
Fortin John J.
Martin Chris S.
Voyta John C.
Horlick Kenneth R.
Kelber Steven B.
Piper Rudnick LLP
Tropix, Inc.
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