What is GFP and how is it used as a reporter gene?
Green fluorescent protein (GFP) has gained widespread use as a tool to visualize spatial and temporal patterns of gene expression in vivo. We report that GFP is a reliable reporter of gene expression in individual eukaryotic cells when fluorescence is measured by flow cytometry.
Is GFP a reporter gene?
GFP from the jellyfish Aequoria victoria is a versatile reporter for monitoring gene expression in vivo, in situ and in real time. GFP fluoresces bright-green upon exposure to UV light, unlike other bioluminescent reporters which require additional proteins, substrates or cofactors to emit light.
How is a GFP reporter gene useful?
Reporter genes were described in the previous chapter as useful markers to localize protein expression. Fusing a fluorescent reporter gene, such as GFP or its variants, to a protein of interest allows a scientist to observe the location and trafficking of the protein in live cells and tissues (Figure 7.7).
What is the GFP gene?
Gfp refers to the gene that produces green fluorescent protein. Using DNA recombinant technology, scientists combine the Gfp gene to a another gene that produces a protein that they want to study, and then they insert the complex into a cell.
Why is GFP a widely used reporter gene?
GFP is great as a reporter protein because it has no effect on the host when introduced to the host’s cellular environment. Due to this ability, no external visualization stain, ATP, or cofactors are needed.
How do GFP reporters work?
Green fluorescent protein (GFP) causes cells that express it to glow green under UV light. A specialized microscope is required to see individual cells. Yellow and red versions are also available, allowing the investigation of multiple genes at once. It is commonly used to measure gene expression.
Is Lac za reporter a gene?
Posted Dec 10, 2019. The E. coli LacZ gene is often used as a reporter gene since it produces a blue product once it is cleaved by the β-galactosidase enzyme. This ‘reports’ whether or not the gene is expressed by the bacteria when grown in a compatible substrate (such as X-gal).
How many BP is GFP gene?
730 bp
The GFP cDNA consists of 730 bp, which encode a 238 amino acid protein with a molecular weight of 27 kD (2). Wild-type GFP emits a vibrant green fluorescence upon exposure to blue light (450-490 nm). The signal is detectable by fluorescence microscopy and fluorescence-activated cell sorting (FACS) (3).
What is a reporter enzyme?
Reporter genes code for proteins that have a unique enzymatic activity and are used to assess the transcriptional properties of DNA elements. The use of these reporter enzymes allows a more rapid and sensitive method of detection than the analysis of specific transgene transcripts within the transgenic animals.
How is GFP expression detected?
The GFP expression may be detected by fluorescence microscopy, fluorescence-activated cell sorting (FACS) analysis, or fluorometer assays 24–72 h posttransfection, depending on the host cell line used. There is one published report of a stable mammalian cell line expressing GFP (48).
What is a lacZ gene?
The lacZ gene encodes beta-galactosidase, which catalyzes the cleavage of lactose to form galactose and glucose. Beta-galactosidase activity can be identified by both in situ and in vitro techniques when incubated with the beta-galactosidase substrate X-gal.
Why is LAC ZA good reporter gene?
The E. coli LacZ gene is often used as a reporter gene since it produces a blue product once it is cleaved by the β-galactosidase enzyme. This ‘reports’ whether or not the gene is expressed by the bacteria when grown in a compatible substrate (such as X-gal).
Where to insert GFP in a translational reporter?
Ideally, a translational reporter includes the entire genomic locus of a gene (5 ′ upstream region, exons, introns, 3 ′ UTR). GFP can be inserted at any point in the open reading frame, preferably at a site that does not disrupt protein function or topology.
How does insertion of GFP affect protein function?
However, insertion of GFP intragenically can sometimes disrupt protein function or even lead to toxicity of the chimeric product. Finally, translational reporters that exhibit subcellular localization can make cell type identification more difficult because the shape of the cell may not be visible (especially for neurons).
How are reporter genes used in gene analysis?
Reporter gene analysis provides an inexpensive, rapid, and sensitive assay that can be used to study gene delivery and gene expression. Use of reporter genes avoids having to develop a specific probe to evaluate the expression of every new gene of interest.
Can You fusion 5 ′ upstream sequences to GFP?
Fusing 5 ′ upstream sequences to GFP can be done in a number of ways and usually presents no technical challenge. Compared to translational and smg-1 -based reporters, however, promoter fusions may not give a complete representation of the real expression pattern of a gene, both spatially and temporally.