reporter gene

1. Reporter Gene
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Selection and screening methods for recombinant clones
The most critical step in rDNA technique
Screening: Distinguish a clone from surrounding ones by some visible character.
Selection: Select a clone that survives a selection pressure which discourages the growth
of non-recombinants.
Screening methods depend on what type of information you have in hand?
Relatedgene from another species?
Low stringency hybridization
A piece of genomic DNA?
Hybridization
A mutant?
Complementation
Positional cloning
An antibody?
Expression library screening
A partial amino acid sequence?
Oligonucleotide screening
A specific tissue or embryonic stage?
Subtracted or +/- screening
A functional assay?
Expression screening
Screening methods
Direct Indirect
Insertional inactivation Nucleic acid hybridn
Southern, Colony
Subtractive, differential
Visual screening Immunochemical
Plasmid rescue
Reporter genes Hybrid arrested transln
Hybrid released transln

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Reporter Gene
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.
So, it is very useful in for the monitoring and detection of plant transformation, for
studying the activities of regulatory elements such as promoter and enhancer.
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.
1. Insertional Inactivation
• A selectable marker is interrupted by
the insertion of the target DNA
• Results in a mutant phenotype
• Positive clones are then identified from
the original/ master plate
• Apr/Tcr Aps/Tcr
• CI gene of Lambda Phage
2. Visual Screening
 Involves the use of a visual phenotype for
identification

3. Reporter Gene
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 lacZ of E.coli is a common Sequence
 The vector carries a partial complement of the lacZ.
 Cells are plated with a mixture of IPTG and X-Gal.
 Interrupted sequences lead to white and
uninterrupted to blue colonies
3. Southern Hybridization
 Based on the specificity of nucleic acid
hybridization between Complimentary
Sequences
 One of the most sensitive methods for
recombinant Clone identification
 Involves the use of a radioactively / non
radioactively labelled DNA probe to be
hybridized to the target sequence.
Essentials for S.H
• Stringency
Specificity with which the target sequence
is detected by the probe.
• Rate enhancers
Volume excluders (eg Dextran Sulphate)
• Detergents and blocking agents
Depress non – specific probe binding
Prevent secondary structure formation
4. Subtractive Hybridization
 An ideal method for the detection of
inducible Genes
 Relies on the exclusion of common
sequences by hybridization between
complimentary RNA-DNA

4. Reporter Gene
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5. Colony Hybridization
• Bacterial colonies are replicated from
a master plate
• The colonies are blotted onto a nylon/
nitrocellulose membrane
• The colonies are lysed, the DNA
denatured and cross linked to the
membrane
• The membrane is hybridized with a
radio / non radioactively labelled
probe.
• The radioactively tagged membrane is
kept for autoradiography
Advantages:
• Large number of clones can be
screened.
• Ideal for small genomic libraries
Disadvantages:
• High incidence of false positives
• Not suitable for large genomic
libraries
Reporter Gene
Genes, whose products confer a visible selectable phenotype in the recombinant cells, are
called “reporter genes”.
The most commonly used reporter genes are fluorescent proteins such as G F P, Luciferase,
CAT and β-galactosidase
Types of reporter genes
Reporter genes are mainly of two types:
1)Scorable marker
2)Selectable marker

5. Reporter Gene
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1) 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. These reporter genes were assayed at
the level of protein.
1. G F P
• 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
• Can work as a standalone system
• No special biosynthetic pathways; can be synthesized by any cell system
• Has a unique structure
• 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
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

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• 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
Important examples are:
Chloramphenicol acetyl transferase (CAT)
This gene was isolated from the transposon Tn9 of E.coli and codes for CAT enzyme, which
catalyses the transfer of the acetyl groups from acetyl coenzyme A to chloramphenicol.
GUS (I2-glucuronidase)
Predominant reporter used to study gene expression in plants, based on E.coli gene uidA
encoding I2-glucuronidase enzyme, which catalyze hydrolysis of glucuronides. Transformed
cells turn blue in the presence of substrate, X-gluc. The disadvantage associated with the use
of GUS assay is the destruction of plant material.
Fire fly luciferase (LUC)
Luc gene is isolated from Photinus pyralis. Luciferase enzyme confers the organism the
ability to glow in the dark. The firefly luciferase catalyzes the bioluminescent oxidation of
the luciferin in the presence of ATP, magnesium and oxygen:
Luciferin + ATP ? luciferyl adenylate +PPi
Luciferyl adenylate + oxygen PP' oxyluciferin + AMP + yellow-green light
This gene is not destructive to the plant and reflects real-time gene expression status of the
transgenic tissue under investigation owing to its in-vivo short half life.
Green fluorescent protein (GFP)
It is a small protein of 238 amino acids (26.9 KDa), first isolated from the jellyfish Aequorea
victori. This reporter gene is used for the study of dynamic process (sub-cellular localization
of proteins, etc.) inside the cell and to determine the zygosity of transgenic plants. It exhibits
bright green fluorescence when irradiated with blue light. In 2008, Osamu Shimomura,
Martin Chalfie, Roger Y. Tsien got Noble Prize for the discovery of GFP.
Other scorable markers include LacZ (I2-galactosidase).

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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.
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.
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
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.
Measurement of expression of reporter gene
•Enzyme activity assay of the expressed enzyme encoded by the reporter gene using chromo,
fluoro, luminogenic substrate

8. Reporter Gene
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•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.