报告基因是一个gene with a distinct characteristic when expressed. Because of this distinct characteristic that is easily identified and measured, this reporter gene can be used to monitor the expression of a certain gene.
Green Fluorescent Protein
In this case, the green fluorescent protein (GFP) from jelly fish(Aequorea victoria)was used as a reporter gene because it emits bright green light (λ = 509 nm) when excited with UV or blue light (λ max = 395 nm, minor 475 = nm). Violet/blue light causes a reaction of decarboxylation of Glu22, which helps the formation of anionic fluorescent form of the chromophore. When the gene of interest (GOI) is expressed, GFP will also be expressed resulting in a bright green cell/colony. But if GOI is not expressed, the cells and colonies will not emit the bright green light.
InAequorea victoria, the protein responsible for bioluminescence capability is the Aequorin protein. This protein will emit blue light upon binding with Ca2+ions. The blue light is then absorbed by GFP, which converts it into the green light.
Tet-On and Off System
Tet-off system. Activator: tTA, Operator: TRE, Promoter: Pcmv, Dox: Doxycycline. Gene of interest transcription is blocked in the presence of Doxycycline
Gene expression can be controlled using a regulator gene. A regulator gene can regulate the gene expression by expressing certain protein or miRNA that blocks or actives the gene transcription process. One of the commonly used regulator gene is Tet-off and Tet-on system that utilizes tetracycline (Tc) or doxycycline (Dox,a Tc derivative) as the switch control. In theTet-offsystem, the gene expression is turnedoffwhen Tc or Dox is present in the medium, whereas in theTet-onsystem, the gene expression is turnedonwhen Dox is present. The Tet-on system is responsive only to Dox, and not to Tc. In this case, expression of GOI is being controlled by Tet-off system.
Important components of Tet-off system are:
- Regulatory protein
Regulatory protein that is involved in Tet-off system is a tetracycline-controlled transactivator (tTA) that acts as activator. In this case, the yeast used are stable cell line that contain a Tet-off regulator plasmid which encodes for protein tTA.
- Response plasmid
Response plasmid expresses a gene of interest (In this case, RAD52-eGFP) under the control of the tetracycline-response element, or TRE. Important elements in the response plasmid are TRE, promoter and GOI. TRE acts as an operator that controls the transcription of GOI. GOI is only expressed when tTA binds TRE and activates GOI transcription under condition where Tc or Dox is not present. Tc or Dox can bind to tTA making it unable to bind to TRE, causing GOI not to be expressed.
透照器
透照器is commonly used for fluorescent marker visualization. Transilluminator parts are UV, blue light source and supporting electronics that are placed in a box with appropriate optical filters. A transilluminator must be used carefully because it emits ultraviolet radiation (commonly between 302 nm and 365 nm) that can cause damage to human tissue in the eyes and skin.