REPORTERS IN

MOLECULAR BIOLOGY
Definitions
In molecular biology, a reporter gene (often simply reporter) is a gene
that researchers attach to a regulatory sequence of another gene of
interest in bacteria, cell culture, animals or plants. Certain genes are
chosen as reporters because the characteristics they confer on organisms
expressing them are easily identified and measured, or because they are
selectable markers. Reporter genes are often used as an indication of
whether a certain gene has been taken up by or expressed in the cell or
organism population.
Common reporter genes

Use of reporter gene:
-galactosidase is
derived from the
Lac-Z gene of E. coli
lac operaon.
-gal is a hydrolase enzyme which catalyzes the
hydrolysis of -galactosides into
monosaccharides.

In E.coli, its natural function is to use Lactose as
a carbon source in the absence of glucose
-GALACTOSIDASE as a
reporter system
STRUCTURE

1024 amino acids

464 Kda

Homo-tetramer

-galactoside into monosaccharide
BIOCHEMICAL MODE OF ACTION
It hydrolyzes a
colorless substrate
X-gal to produce a
Blue colored
compound
With ONPG as substrate,
it produces o-nitrophenol
which is yellow in color

-Galactosidase in Reporter Assay
LacZ can tolerate N-
Terminal Fusions
without any change in
its activity.
Therefore it can be
translationally fused
with the gene of
interest to be used as
a reporter gene.
BLUE-WHITE SCREENING
Vector contains Lac-Z
gene

After transformation,
cells are grown in a
media containing X-gal

The transformed cells
produces blue color due
to hydrolysis of X-gal

Un-transformed cells
remain white
Fluorescent proteins

are members of a structurally homologous
class of proteins that share the unique
property of being self-sufficient to form a
visible wavelength chromophore from a
sequence of 3 amino acids within their own
polypeptide sequence.
From Discosoma - DsRed
RED FLUORESCENT PROTEIN
bright fluorescent
Signals in 8-10 hrs
after transfection
From sea anemone
Entacmaea
quadricolor
- TurboRFP
(excitation/emission maxima are 553
and 574 nm)
CYAN FLUORESCENT PROTEIN
A close-up of the adult red-cyan
morph of the stony coral Acropora
millepora. The red fluorescence is
attributable to amilRFP and the cyan
fluorescence is attributable to
amilCFP
ORANGE FLUORESCENT PROTEIN
Use of green fluorescent protein (GFP) as a reporter gene.
AIDS Research
CAIRO: In the latest string of research to combat AIDS, American researchers
have inserted a fluorescent protein known as GFP (Green Fluorescent Protein) in
cats to monitor the activity of altered genes which scientists say are more resilient to
the feline from AIDS.
Vibrio fischeri
Gene: lux
Subs: N-decyl aldehyde
Renilla reniformis
Gene: Rluc
Subs: Coelenterazine
FIREFLY
Gene:Fluc Gene
Subs: Luciferin
IN-VIVO CELL TRAFFICKING(Jonathan Hardy et al)
Collagen Induced Athritis

T-cells as vehicles to deliver
Immuno-regulatory proteins

CD4+ T-cells with GFP-Luc
translational fusion gene

Luciferin injected

Imaging
Bacterial Bioluminescence
Can be used to test for a specific bacterial
species or possible food born pathogen such as
Salmonella typhimurium.
The lux gene, responsible for bacterial
bioluminescence, has been isolated and cloned.
The cloned lux gene can be injected into a host-
specific phage, which does not have the ability to
express the gene.
If the phage infects a host bacterium, light
emission results.

GLO- FISHES
Tobacco Plants