1) Reporter genes produce a detectable phenotype that allows transformed cells to be easily identified and selected. They are useful tools for studying gene expression. 2) Common reporter genes include GFP and luciferase, which produce fluorescent and luminescent proteins, respectively, that can be quantified. 3) Selectable marker genes, like antibiotic resistance genes, allow transformed cells to survive selective conditions that kill untransformed cells. This enables isolation of transformed cells.

1. Reporter Genes
Submitted to: Submitted by:
Dr. Nidhi Gupta Aparajita Sharma
Msc. Biotech
Sem 2

2. Marker gene
• It is a DNA sequence with a known location on
a chromosome.
• Used to determine if a piece of DNA has been
successfully inserted into the host organism.

3. Classification of Marker Genes
Marker
genes
Selectable
markers
Negative Positive
Screening
markers
Reporter
genes

4. Reporter Genes
• A reporter or marker gene is a gene, which produces a
specific phenotype, in turn enables the differentiation of
the cells possessing this particular gene from those without
this gene. Hence, the transformed cells can be selected
easily among the thousands of non transformed cells.
• Reporter genes form specific protein products, which are
easily detectable and quantifiable, sometimes even without
destroying the tissue.
• Reporter genes are an invaluable tool to track and study
another associated gene in bacterial and mammalian cell
culture, animals and plants. One can easily find out the
expression patterns of a gene within the cell by fusing its
promoter with one of the several reporter genes and
transfecting inside the living cells.

5. Features of an ideal reporter gene
• Easily quantifiable
• 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. Types of reporter genes
Reporter genes are mainly of two types:
• Scorable marker
• Selectable marker
Scorable marker: -
• Expression of this kind of marker gene results in a quantifiable
phenotype i.e., it will make the cells containing it to look different.
• The main principle behind the use of these reporter genes for the
study of molecular processes in living cells means that in natural
genes, synthetic modification have introduced in order to either
simplify the detection of the product or to distinguish it from
similar genes in the genome.
• Example: GFP and Luciferase.

7. 1) Green Fluorescent protein (GFP):
• Obtained from jelly fish Aequoria victoria
• The gene is cloned upstream to the MCS along with a
strong constitutive promoter
• On exposure to UV the protein emits a green
fluorescent light.
• Ideal system for in vivo detection of gene expression
• A 238 residue polypeptide (Mw 26,888)
• Has a λ max of 509 nm
• A partner protein called aequorin which receives the
light and transfers energy to GFP

8. • No special biosynthetic pathways; can be synthesized by
any cell system
• The protein structure contains a barrell of 11 β strands with
a chromophore in the
• centre
• The chromophore is a small α helix with three a.a residues
at 65-67 (ser, tyr, gly)
• cyclized
• This is a post-translational modification
• Mainly used as a reporter as well as a tag system
• Used to locate proteins within cells / tissues
• Also used to measure the levels of expression

9. 2) Luciferase
• Refers to a group of enzymes that catalyse the
oxidation of luciferin.
• Requires ATP and Mg2+
• In presence of excess of substrate, a flash of
light is emitted, detected by a photometer
or photographic film
• 100 times more sensitive than lacZ.

10. • Obtained from firefly Photinus pyralis or a
marine organism Renilla reniformis
• In the presence of ATP and O2 Luciferin is
converted into oxyluciferin, which emits light
at 560 nm
• ATP+Luciferin+O2 AMP+Oxyluciferin+PPi

11. 2) Selectable marker
• The cells that contain this type of marker gene show
the ability to survive under selective conditions. These
selective conditions would otherwise result in the
death of the cells lacking that specific gene.
• Most commonly used selective agents are antibiotics.
Out of the millions and billions of cells, only few of
them get transformed by the foreign DNA. It is
practically impossible to check every individual cell, so
a selective agent is required to eliminate the non-
transformed cells, leaving only the desired ones.

12. Usually, selectable markers are of two types
a) Antibiotic resistance marker (nptll, hptll, etc.)
• nptll: - Most commonly used neomycin phosphotransferasell
(nptll) gene is isolated from transposon Tn 5 (E.coli K12 strain).
It encodes for aminoglycoside 3* phosphotransferase enzyme
which inactivates a range of antibiotics such as kanamycin,
neomycin, puromycin, etc.
• hptll: - Hygromycin phosphotransferase gene was isolated
from E.coli, which codes for enzyme that inactivates the
antibiotics, Hygromycin B; the latter is more toxic than
kanamycin and kill sensitive cells more quickly.

13. b) Herbicide resistance marker (bar gene, als gene etc.)
• Bar gene
It was originally isolated from Streptomyces
hygroscopicus, and confers resistance to the herbicide
bialaphos (bar). This gene encodes phosphinothricin
acetyl transferase (PAT) enzyme which acetylates
phosphinothricin (PPT), a component of bialaphos. In
normal cells, glutamine synthetase (GS) incorporates
ammonia into protein. Thus maintain the level of
ammonia in cells. PPT is a competitive inhibitor of GS, so
its presence blocks the activity of latter. Consequently,
PPT cells.

14. • Acetolactate Synthase gene (als):
This gene was isolated from Arabidopsis thaliana
and encodes for acetolactate synthase enzyme
that provides resistance against sulfonylurea.
When als gene is transferred to crop of interest,
it will become resistant to sulfonylurea.

15. Measurement of expression of
reporter genes
• Enzyme activity assay of the expressed
enzyme encoded by the reporter gene using
chromo, fluoro, luminogenic substrate.
• Immunological assay of the expressed protein
encoded by the reporter gene.
• Histochemical staining of cells or tissue
typically to localize enzymatic activity
expressed from reporter gene construct cells.

16. THANK YOU