Reporter systems are also often used as a standard to compare the transfection efficiency of different transfection experiments. In this case, the control reporter system usually contains a constitutive promoter and a reporter gene, which is different to the reporter gene used by the element under examination. Such a reporter system can also be used to optimize electroporation parameters.
Chloramphenicol Acetyltransferase (CAT)
Chloramphenicol Acetyltransferase (CAT) This enzyme comes from microorganisms and catalyzes the transfer of acetyl groups from acetyl coenzyme A to chloramphenicol. With the CAT assay, the CAT-containing lysates of transfected cells are incubated with 14C-chloramphenicol, which is then acetylated. Acetylated and non-acetylated 14C-chloramphenicol is then separate d using thin-layer chromatography and visualized by autoradiography. If necessary, the distribution of radioactivity can be quantified by a scanning system.
As an increasingly common, non-radioactive alternative, CAT expression is quantified by an ELISA via immunological detection of the CAT enzyme which has been expressed.
The prokaryotic -galactosidase naturally catalyzes the hydrolysis of -galactosides (e.g. lactose). However, the use of non-physiological substrates also enables the quantification of -galactosidase activity in lysates of transfected cells via spectrophotometry (e.g. with 0-nitrophenyl--D-galactoside = ONPG), fluorometry (e.g. with a 4-methyl-umbelliferyl--galactopyranoside compound = MUG) or via chemiluminescence. Detection by chemiluminescence (e.g. with 1.2 dioxetan-galactopyranoside derivatives) is 1001,000 times more sensitive than the other two detection methods, and thus even more sensitive than the luciferase assay (see below). A major advantage of this system is the fact that -galactosidase activity can also be measured in situ.
The -galactosidase reporter gene is often co-transfected together with other reporter systems as an internal control. The -galactosidase activities can be used to compare and standardize different transfection experiments (e.g. luciferase assays).
Human Growth Hormone (hGH)
With the human Growth Hormone (hGH) reporter system in contrast to most other systems the reporter protein is secreted into the culture medium by the transfected cells. This means that cell lysis is not necessary for quantifying the reporter protein. Detection of the secreted hGH is usu ally carried out using 125I-labeled antibodies against the growth hormone. Several manufacturers supply a non-radioactive alternative in the form of ELISA kits, with anti-hGH antibodies bound to the surface of a microtiter plate. First, the hGH from the supernatant of the culture medium binds to the antibody on the plate. Subsequently, the bound hGH is detected in two steps via a digoxigenin- coupled anti-hGH antibody and a peroxidase-coupled anti-digoxigenin antibody. Bound peroxidase is quantified by incubation with a substrate.
Another secreted reporter protein is the SEAP (secreted alkaline phosphatase). This protein is quantified directly by measuring the enzyme activity in the supernatant of the culture medium. Fluorescence and chemiluminescence assays are available for this purpose.
The enzyme from the North American firefly (Photinus pyralis) catalyzes a bioluminescence reaction. In the luciferase assay, the lysates of transfected cells are incubated with luciferin, molecular oxygen, ATP and Mg2+. In the following reaction, the luciferase catalyzes the oxidation of luciferin in oxyluciferin and CO2. In this reaction, light with a wavelength of 562 nm is emitted, which then fades rapidly. The light emitted can be measured in a luminometer or in a liquid-scintillation counter. Light emission is proportional to the amount of luciferase in the lysate, thus enabling the indirect quantification of the transfection rate of the reporter gene. The sensitivity of the luciferase assay is further increased by adding co-enzyme A to the reaction preparation, rendering it 1020 times more sensitive than the CAT assay.
In a fur ther development of the luciferase assay, cells are co-transfected with a control plasmid of a different luciferase from renilla (Renilla reniformis). The activities of firefly and renilla luciferase can be measured separately in one sample. The activity of the renilla luciferase can therefore be used as an internal control for comparing different transfection experiments. Both luciferases are also used in co-transfection experiments for the parallel examination of two cis elements.
Green Fluorescent Protein (GFP)
Unlike other bioluminescent reporters, the green fluorescent protein from the Aequorea victoria jellyfish requires no additional proteins, substrates or co-factors to emit light. When irradiated with UV light or blue light, it emits green light, which enables the examination of gene expression and protein localization in situ and in vivo. In addition, the gene expression can be observed in real time. However, the system is less suitable for quantifying the gene expression.
Variations of GFP with different absorption and emission maxima also
enable double-labeling experiments, such as the simultaneous examination
of gene expression of two promoters in one cell, or the location of two
different fusion proteins in one cell. Other variations of GFP are particularly
designed for expression in mammalian cells or have up to a 35-fold higher
fluorescence. With the aid of a GFP system with a drastically reduced
half-life, dynamic processes can also be examined in vivo in the cell.