Protocols and Applications Guide (US Letter Size) - Promega

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|||||||| 8Bioluminescence Reporters CONTENTS I. Introduction 1 A. Luminescence versus Fluorescence 1 B. Bioluminescence Reporters 2 II. Luciferase Genes and Vectors 3 A. Biology and Enzymology 3 B. Gene Optimization 4 III. Luciferase Reporter Assays and Protocols 7 A. Single-Reporter Assays 7 B. Dual-Reporter Assays 9 C. Live Cell Substrates 11 D. Luciferase Reporter Cell Lines 11 E. Selecting a Reporter Gene and Assay: Tables 8.1–8.3 12 IV. References 15 Protocols & Applications Guide www.promega.com rev. 3/09 PROTOCOLS & APPLICATIONS GUIDE
|||||||| 8Bioluminescence Reporters I. Introduction Genetic reporter systems have contributed greatly to the study of eukaryotic gene expression and regulation. Although reporter genes have played a significant role in numerous applications, both in vitro and in vivo, they are most frequently used as indicators of transcriptional activity in cells. Typically, a reporter gene is joined to a promoter sequence in an expression vector that is transferred into cells. Following transfer, the cells are assayed for the presence of the reporter by directly measuring the reporter protein itself or the enzymatic activity of the reporter protein. An ideal reporter gene is one that is not endogenously expressed in the cell of interest and is amenable to assays that are sensitive, quantitative, rapid, easy, reproducible and safe. Analysis of cis-acting transcriptional elements is a frequent application for reporter genes. Reporter vectors allow functional identification and characterization of promoter and enhancer elements because expression of the reporter correlates with transcriptional activity of the reporter gene. For these types of studies, promoter regions are cloned upstream or downstream of the gene. The promoter-gene fusion is introduced into cultured cells by standard transfection methods or into a germ cell to produce transgenic organisms. The use of reporter gene technology has allowed characterization of promoter and enhancer elements that regulate cell, tissue and development-defined gene expression. Trans-acting factors can be assayed by co-transfer of the promoter-reporter gene fusion DNA with another cloned DNA expressing a trans-acting protein or RNA of interest. The protein could be a transcription factor that binds to the promoter region of interest cloned upstream of the reporter gene. For example, when tat protein is expressed from one vector in a transfected cell, the activity of different HIV-1 LTR sequences linked to a reporter gene increases and the activity increase is reflected in the increase of reporter gene protein activity. Reporters can be assayed by detecting endogenous characteristics, such as enzymatic activity or spectrophotometric characteristics, or indirectly with antibody-based assays. In general, enzymatic assays are quite sensitive due to the small amount of reporter enzyme required to generate the products of the reaction. A potential limitation of enzymatic assays is high background if there is endogenous enzymatic activity in the cell (e.g., β-galactosidase). Antibody-based assays are generally less sensitive, but will detect the reporter protein whether it is enzymatically active or not. Fundamentally, an assay is a means for translating a biomolecular effect into an observable parameter. While there are theoretically many strategies by which this can be achieved, in practice the reporter assays capable of delivering the speed, accuracy and sensitivity necessary for effective screening are based on photon production. Protocols & Applications Guide www.promega.com rev. 3/09 A. Luminescence versus Fluorescence Photon production is realized primarily through fluorescence and chemiluminescence. Both processes yield photons as a consequence of energy transitions from excited-state molecular orbitals to lower energy orbitals. However, they differ in how the excited-state orbitals are created. In chemiluminescence, the excited states are the product of exothermic chemical reactions, whereas in fluorescence the excited states are created by absorption of light. This distinction of how the excited states are created greatly affects the character of the photometric assay. For instance, fluorescence-based assays tend to be much brighter, since the photon used to create the excited states can be pumped into a sample at a very high rate. In chemiluminescence assays, the chemical reactions required to generate excited states usually proceed at a much lower rate, so yield a lower rate of photon emission. The greater brightness of fluorescence would appear to correlate with better assay sensitivity, but this is commonly not the case. Assay sensitivity is determined by a statistical analysis of signal relative to background or "noise", where the signal represents a sample measurement minus the background measurement. The limitation of fluorescence is that it tends to have much higher backgrounds, leading to lower relative signals. The reason fluorescence assays have higher backgrounds is primarily because fluorometers must discriminate between the very high influx of photons into the sample and the much smaller emission of photons from the analytical fluorophores. This discrimination is accomplished largely by optical filtration, since emitted photons have longer wavelengths than excitation photons, and by geometry, since the emitted photons typically travel a different path than the excitation photons. But optical filters are not perfect in their ability to differentiate between wavelengths, and photons can also change directions through scattering. Chemiluminescence has the advantage that, since photons are not required to create the excited states, they do not constitute an inherent background when measuring photon efflux from a sample. The resulting low background permits precise measurement of very small changes in light. Fluorescence assays can also be limited by the presence of interfering fluorophores within the samples. This is especially problematic in biological samples, which can be replete with a variety of heterocyclic compounds that fluoresce, typically in concentrations much above the analytical fluorophores of interest. The problem is minimized in simple samples, such as purified proteins. But in drug discovery, living cells are increasingly used for high-throughput screening. Unfortunately, cells are enormously complex in their chemical constitutions, which can exhibit substantial inherent fluorescence. Screening compound libraries is also inherently complex, since, although each assay sample may contain only one or a few compounds, the data set from which the drug leads are PROTOCOLS & APPLICATIONS GUIDE 8-1
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CONTENTS I. Nucleic Acid Amplificat
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|| 2RNA Interference CONTENTS I. RN
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||| 3Apoptosis I. Introduction A. C
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|||| 4Cell Viability CONTENTS I. In
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||||| 5Protein Expression I. Introd
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|||||||||| 10Cell Imaging I. Introd
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