This document discusses reporter genes, which are marker genes used to analyze gene expression. It describes the features of ideal reporter genes and several commonly used ones, including opine synthase, chloramphenicol acetyltransferase, β-glucuronidase, bacterial luciferase, firefly luciferase, and green fluorescent protein. Reporter genes allow quantification of gene expression and regulation by fusing a gene of interest to the reporter gene. The document concludes that reporter gene technology is widely used to study various cellular processes and holds promise for applications in gene therapy and drug development.

1. By
KAUSHAL KUMAR SAHU
Assistant Professor (Ad Hoc)
Department of Biotechnology
Govt. Digvijay Autonomous P. G. College
Raj-Nandgaon ( C. G. )

2.  INTRODUCTION
 FEATURES OF AN IDEAL REPORTER GENE
 COMMONLY USED REPORTER GENES
 Opine synthase
 Chloramphenicol acetyl transferase
 β- Glucuronidase (GUS)
 Bacterial luciferase (Lux F2)
 Firefly luciferase (Luc)
 Green fluorescent protein (GFP)
 β- Galactosidas
 CONCLUSION
 APPLICATIONS
 REFERENCES

3. INTRODUCTION
• Monitoring and detection of plant transformation systems in
order to know whether the DNA has been successfully
transferred into recipient cells is done with the help of a set of
genes, known as marker genes.
• Reporter genes (screenable or scoreable genes) are a type of
marker genes which are useful in the analysis of plant gene
expression.
• A reporter gene is a test gene whose expression results in
quantifiable phenotype.
• The basic principle of using reporter genes means that in the
natural gene, a synthetic modification is introduced in order to
detect the gene product or to distinguish it from similar or
identical genes in the genome.
• The use of reporter genes requires the method of gene transfer,
either transient or stable.
• Reporter gene constructs are comprised of a reporter gene
together with active promoter and terminator regions cloned into
a plasmid vector .

4.  The most use of reporter assay is to analyze how a gene is regulated by
fusing the regulatory sequences of gene of interest to the coding
sequences of a reporter gene.
A diagram of a how a reporter gene is used
to study a regulatory sequence

5. Features of an ideal reporter gene:-
• Easily quantifiable
• Relatively rapid degradation of the enzyme
• High signal-to-noise ratio (Low endogenous background)
• Should not be toxic to cells
• Products of the reporter gene should be resistant to the
chemicals used in the processing
• Assay should be sensitive and reliable

6. COMMONLY USED REPORTER GENES
 Opine synthase
 Chloramphenicol acetyl transferase
 β- glucuronidase (GUS)
 Bacterial luciferase (lux)
 Firefly luciferase (luc)
 Green fluorescent protein (GFP)
 β- Galactosidase
1. OPINE SYNTHASE
The octopine or nopaline synthase genes (ocs,nos) are present in the T-
DNA of Ti or Ri plasmids of Agrobacterium.
The presence of opine in any plant material clearly indicates the
transformed status of the plant cells.

7. There are basically two ways to access the presence of opines in a
transformed plant tissues.
The first one aims at detecting the enzyme activities responsible for the
synthesis of opine.
This technique involves a simple protein extract from the plant sample.
The extract is then incubated with the appropriate opines precursors
arginine, pyruvate and NADH for ocs and arginine, ketoglutaric acid and
NADH for nos, and the reaction products are separated by paper
chromatography.
The second technique aims at directly detecting the presence of opines
from the plant tissue.
This involves extraction of the plant metabolites from the tissue, the
separation of these compounds by paper electrophoresis, and the
detection of opines by appropriate staining treatments.

8. 2.CHLORAMPHENICOL ACETYL TRANSFERASE
The gene for chloramphenicol acetyl transferase is one of the most
commonly used reporter genes in eukaryotic organism.
Two different cat genes have been identified, but the most commonly
used reporter was found in the transposable element Tn9.

9. 3. β-GLUCURONIDASE
During the last few years the bacterial gene
uidA, encoding β- glucuronidase (GUS), has
become the most frequently used reporter
gene for the analysis of plant gene
expression.

10. Luciferase is a generic name for an enzyme that catalyzes a light-emitting
reaction.
4. BACTERIAL LUCIFERASE (Lux F2)
Bacterial luciferases have originated from Vibrio harveyi and V. fischerii.
In these organisms, luciferase is expressed as a heterodimeric flavin
monooxygenase that is responsible for catalyzing a light-emitting
reaction.
The V.herveyi luciferase (Lux) is encoded by the luxA and luxB cistrons
that are part of a longer operon.
The lux genes in plant cells can be expressed as separate subunits or as a
fused polypeptide.
The subunits are correctly assembled in plant cells and the fused protein
has been designated as Lux F2.
The activity of luciferase is normally measured as the initial maximum
light intensity upon mixing the enzyme with its aldehyde and reduced
flavin mononucleotide substrates (FMNH2) in the presence of oxygen.
Light is measured as photons or quanta per second (qu/s), and if
calibrated against a known light standard, the absolute number of enzyme
molecules in the reaction can be calculated.
Light emission can be monitored visually, photographically, or
electronically.

11. 5. FIREFLY LUCIFERASE (Luc)
Enzyme isolated from North American firefly
(Photinus pyralis).
Produces flashes of light in the presence of
luciferin and ATP.
Detected in tissue extracts or even in the
intact plant after watering with luciferin .
Allows non-destructive imaging of plants.
Photinus pyralis

12. 6. GREEN FLUORESCENT PROTEIN
The gene for green fluorescent protein (GFP) has been obtained from
jellyfish Aequorea victoria.
GFP is a small protein of 238 amino acids.
This owes its visible absorbance and fluorescence to a β- hydroxy
benzylidine imidazolinone chromophore.
Upon excitation at 395 nm it emits green light with an emission maximum
at 509 nm.
The intensity of light emissions has been improved upon by mutation in
the chromophore to produce several new proteins including enhanced GFP,
yellow GFP and cyan GFP.
The formation of green fluorescence can be detected under fluorescent
microscope.
GFP-based reporter assays provide a distinct advantage over most other
reporter systems, as they do not require cell permeabilization or the
addition of exogenous substrates. However, because they do not take
advantage of an enzymatic amplification step they are generally less
sensitive than other reporter assays.

13. 8. β- Galactosidase (Gal/ LacZ)

14. APPLICATIONS
STUDYING-
 DNA regulatory elements
 Gene expression
 Protein-protein interaction
 Cellular physiology
 Receptor activity
 Transcription factors
 Intracellular signalling
 mRNA processing
 Protein folding

15. CONCLUSION
Reporter gene technology is widely used to monitor the cellular events
associated with signal transduction and gene expression.
The principle advantage of these assays is their high sensitivity,
reliability, convenience, and adaptability to large scale measurements.
With the advances in this technology and in detection methods, it is
likely that luciferase and GFP will become increasingly popular for the
non-invasive monitoring of gene expression in living tissues and cells.
This technique will be important in defining the molecular events
associated with gene transcription, which has implication for our
understanding of the molecular basis of disease and will influence our
approach to gene therapy and drug development.
The future appears to be promising for the continued expansion of the
use of reporter genes in the many evolving biomedically related
arenas.

16. REFERENCES
BOOKS-
H.S.Chawla: Introduction to Plant
Biotechnology
Biotechnology by U.Satyanarayan
Biotechnology- B.D.Singh
INTERNET www.wikipedia.com