Reporter Gene Detection Overview

Reporter gene detection is an important means of functional genomics research, and has been widely used in eukaryotic gene expression regulation research and related drug or protein screening work. A reporter gene detection system that links a cis-regulatory element (ie, a DNA non-coding sequence) to a coding sequence of a very easily identified protein (ie, a reporter gene product) by measuring the expression of the reporter gene in the cell. The amount or activity of the product is used to determine the expression regulation of the cis-regulatory element. This indirect measurement technique provides a simple, effective, and sometimes the only feasible detection system. Reporter gene detection technology, involving reporter gene vector plasmid construction, cell transfection, and reporter gene product assays.

An ideal reporter gene should normally have the following characteristics: it has been cloned and the entire sequence has been determined; the expression product is absent in the recipient cell (ie, no background); other gene products in the cell do not interfere with the detection of the reporter product; The content of the product in the cell can reflect the transcriptional activity state of the gene in real time; the detection method of the reporter gene encoding product should be fast, simple, sensitive and reproducible.

At present, the reporter genes commonly used in laboratories are as follows: luciferase (luc), β-galactosidase (β-gal), chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS) ), secreted alkaline phosphatase (SEAP), green fluorescent protein (GFP), and growth hormone (hGH). Among them, the luciferase reporter gene has the advantages of high detection speed, high sensitivity and low cost, and has been widely used. In the reporter gene detection system, the activity of the reporter gene interacts with specific stimuli. Therefore, in addition to the main reporter gene plasmid, another reporter gene plasmid is usually included, and the same cell is co-transfected as an internal reference for transfection efficiency and cell number and status, and the results of the main reporter gene are normalized.