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Berger et al. reported a novel, recombinant reporter gene, secreted alkaline phosphatase (SEAP) in 1988. Normally, alkaline phosphatases are not secreted, while the recombinant SEAP originated from placental alkaline phosphatase is efficiently secreted from transfected cells. In the SEAP-transfected cells, the levels of SEAP activity detected in culture media are directly proportional to changes in intracellular SEAP mRNA. This property allows SEAP to act as a quantitative reporter for gene expression. As a reporter molecule, SEAP has several crucial advantages. Because preparation of cell lysates is not required, it is likely to assess the activity of certain promoter/enhancer elements repeatedly through identical cell cultures. Assaying SEAP activity using culture medium is faster, easier, and cheaper than assays for other reporter enzymes. With chemiluminescent assays, SEAP activity can be detected very sensitively. Another important advantage is that background signals owing to endogenous alkaline phosphatases are nearly absent. This is because, unlike other endogenous alkaline phosphatases, SEAP is heat stable and resistant to L-homoarginine. The activity of endogenous alkaline phosphatases present in samples can be eliminated through preheating samples at 65°C and assaying in the presence of L-homoarginine without affecting the SEAP activity.
Detect gene expression with SEAP system
Cell-based microarrays have recently been developed for rapid and simple evaluation of various cellular functions with small volume samples. Lots of research is now focused on the elucidation of gene functions within mammalian cells by using genetic screening systems for genome-wide analysis in living cells. Of the various approaches available for monitoring gene expression within cells, the reporter gene system is the most commonly used approach. So far, several types of proteins have been employed as reporter proteins, such as fluorescent protein, â-galactosidase, and SEAP. SEAP is a particularly useful reporter as it possesses a unique feature in that the reporter protein is secreted into the culture medium. The cellular secretion of SEAP is directly proportional to the changes in the intracellular SEAP mRNA. This characteristic allows the continuous quantification of gene expression. SEAP activity can be sensitively detected by a conventional chemiluminescent or fluorescent assay.
A new electrochemical assay for the detection of SEAP from transfectant HeLa cells is proposed using a microarray device and scanning electrochemical microscopy (SECM). The assay includes two steps: the first is the incubation of a transfected cell in a microarray culture device covered with a substrate modified with anti-SEAP under physiological conditions without any additives. The second step is SECM measurement of secreted SEAP at the antibody-immobilized substrate. This assay ensures accuracy and intactness because the undesired influence of endogenous ALP is eliminated and the transfected cells are incubated in a culture device under suitable conditions. The system successfully detected the expression of SEAP from intact cells at the single-cell level using this assay. The system is useful as a cell-based gene expression assay.
SEAP system for MRI
Non-invasive imaging of gene-expression patterns in whole animals could provide information critical to this end, while current methods lack sensitivity and spatiotemporal precision. Enzymatic reporter systems detectable by magnetic resonance imaging (MRI) address these limitations by combining the relatively high spatial and temporal resolution of MRI with the ability of each genetically expressed enzyme to generate a number of MRI-detectable product molecules. A challenge with the imaging-based detection of some of the most popular reporter enzymes is the need to deliver MRI probes to their sites of action within cells. Lately, researchers describe a new reporter-gene system for MRI that relieves this problem by harnessing an extracellular enzyme, the mammalian EAP.
SEAP is widely used as a stable and heterologously expressible reporter enzyme in conjunction with optically absorbent, fluorescent, or luminescent substrates. For the optimal detection of SEAP activity by MRI, researchers modified an existing sensor for adenosine (Ado), which is produced by SEAP’s hydrolysis of phosphorylated adenosine derivatives. In this system, the reporter enzyme is therefore detected by its generation of product molecules, as opposed to its direct action on an MRI contrast agent. The process is reversed upon the removal or degradation of Ado, is nondestructive to the Ado sensor, and is relatively fast, because SEAP substrates can be used at concentrations well above their Km values without affecting the background MRI signal (Figure 1). It indicated that the reversible detection of an established secreted reporter enzyme, SEAP, is possible by using an MRI contrast agent that selectively monitors products of the SEAP-mediated hydrolysis of phosphorylated purines. MRI detection of SEAP reporter activity may be useful in the opaque cell or tissue culture environments, where optical assays are unreliable. MRI-based assays may be particularly beneficial for screening applications in which data from three-dimensional sample arrays may be acquired in parallel.
Figure 1. SEAP-based reporter-gene system for MRI.