Lecture 4.2 (reporter gene assay) Flashcards
Give an example of a promoter that contains a single transcription binding site.
cAMP response element (CRE)
What kind of promoters reduce the potential interference seen with less selective promoters?
Promoters that contain a single transcription binding site
What is the advantage of using promoters that contain a single transcription binding site?
reduce the potential interference seen with the less selective promoters
How to overcome the problem of ‘promiscuity’ of endogenous promoters?
to choose a promoter that does not bind transcription factors native to the cellular system being studied
e.g. yeast Gal4 system
Give an example of a promoter gene that does not bind to transcription factors native to cellular systems being studied
yeast Gal4 system
The utilisation of yeast Gal4 system as a reporter gene assay requires the transfection of:
- Gal4 transcription factor (DNA binding domain coupled to regulatory or binding domain)
-reporter gene whose expression is regulated by Gal4 response elements
Gal4 is a Zn2Cys6 binuclear cluster containing transcription factor that
binds DNA as a _________ .
homodimer
What should be considered when choosing a reporter gene?
the product has unique
enzymatic activity or easily identified from the mixture of intra- or extracellular proteins.
What is vital to the success of a reporter gene assay system?
the sensitivity of the detection procedure
For reporter gene assay system, what may the detection method be based on?
photometry, colorimetry, radiometry, fluorescence or immunoassay
bioluminescence and chemiluminescence in recent years
What are the upsides of using bioluminescence and chemiluminescence as detection systems for reporter gene assay system?
higher sensitivity and ease of detection
Two types of reporter gene are commercially available:
- Intracellular reporter genes
- Extracellular reporter genes
What is the full form of CAT?
Chloramphenicol O-acetyltransferase
What is CAT?
a bacterial enzyme
What is the reaction catalysed by CAT?
Acetyl-CoA + chloramphenicol = CoA + chloramphenicol 3-acetate
The CAT gene protein product is easily assayed and detected even at __________ levels
attomole (10-18)
What are the detection options using CAT?
- Radiolabelled [14C]chloramphenicol and acetyl-CoA
–> separate using TLC –> autoradiography or perform liquid scintillation counting - use [3H]acetate or [3H]acetyl-CoA
- using fluorescent BODIPY 1-deoxychloramphenicol substrates
Explain the principle of autoradiography
exposing photographic film or a photosensitive imaging plate to sample containing radioactive material
–> emitted radiation create image on film/plate
Explain the principle of liquid scintillation
detection scintillation light produced when beta particles emitted by radioactive isotopes interact with a liquid scintillator
What is a scintillator?
scintillator is a cocktail containing molecules that emit light when excited by the beta particles
What is the downside of TLC?
development of the signal can take several hours
using TLC for CAT assay, compare the distance travelled by unacetylated chloromphenicol, mono-acetylated chlorophenicol and di-acetylated chloramphenicol?
di-acetylated
chloramphenicol»_space; mono-acetylated chlorophenicol»_space; unacetylated chloromphenicol
Why is florescent substrate a popular choice for CAT assay as opposed to using radiolabelled C or H?
simplified handling procedures and higher speed of sample processing
What is the main disadvantage of CAT assays when scaling up to high throughput formats?
lack of simple separation and detection methods
What gene is beta-galactosidase a product of?
bacterial lacZ gene
What is the drawback of using beta-galactosidiase for assay development?
it is an enzyme endogenous to most mammalian cell lines
–> experiments to distinguish between exogenous and endogenous enzyme
Deficiencies in beta-ga;actosidiase can lead to which disease?
galactosialidosis or Morquio B syndrome
What is Morquio B syndrome?
inherited autosomal recessive mucopolysaccharide
storage disease
–> leads to dwarfism
What is the substrate for beta-galactosidase?
- ONPG (o-nitrophenyl-beta-D-galactopyranoside) for colorimetric assay
- phenylgalactose-substituted dioxetane or AMPGD for chemiluminescence detection
- firefly luciferin beta-D-galactopyranoside
substrate for bioluminescence
What is ONPG converted to by the action of beta-galactosidase?
ONP anion
–> can be quantified colorimetrically
What substrate for beta-galatosidase shall be used if the assay is to be adapted to chemiluminescence detection?
What advantage does it serve over ONPG colorimetric assay?
phenylgalactose-substituted dioxetane
–> 20-fold improvement in sensitivity over the ONPG colorimetric assay.
For detection using beta-galactosidase assay, what is the advantage of using AMPGD over ONPG or phenylgalactose-substituted dioxetane?
50,000-fold more sensitive than the ONPG
–> able to detect as little as 2 fg of beta-galactosidase
What is luciferin beta-D-galactopyranoside
substrate converted into under the action of beta-galactosidase?
firefly luciferin
–> measured using firefly luciferase assay
What is Aequorin?
photoprotein isolated from jellyfish Aequorea Victoria
What are the 3 components of Aequorin?
- apoaequorin
- coelenterazine chromophore
- oxygen
What is the detection method of using Aequorin based reporter gene based on?
In presence of calcium ions, the photoprotein complex emits a flash of blue
luminescence (max wavelength 469nm)
What are advantages to using an aequorin based reporter gene assay?
high sensitivity and low backgrounds
What is the downside of using an aequorin based reporter gene assay?
- requires the exogenous application of coelenterazine
- bioluminescent flash triggered by Ca2+ lasts less than 5 seconds
–> use luminometer with ability to initiate and rapidly detect the reaction
Aequorin stably expressed in the cells detects the
increased [Ca2+]i and emits
luminescence.
True or False
true
