Reporter Gene

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?

Related gene 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

Reporter GeneA 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

lacZ of E.coli is a common Sequence

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Reporter Gene

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

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Reporter Gene

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

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.

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Reporter Gene

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

• 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

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Reporter Gene

• 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).

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.

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Reporter 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

• 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.

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