Download Reporter genes

Reporter genes & transfection
lecture 3
22 October 2012
Mammalian expression plasmid
For expression
in cells
cDNA
To determine transfection
efficiency
pCMV/GFP
For amplification
of the plasmid in
bacteria
pUC
Bacterial origin of replication
Polyadenylation
site
To generate
stable cell line
Polyadenylation
site
Reporter genes
Genes used for determination of the transfection efficiency.
Expression of a reporter has to be easily detectable. It has to
be a gene which is not naturally present in transfected cells.
Reporter genes
chloramphenicol acetyltransferase - CAT
β-galactosidase
secreted alkaline phosphatase (SEAP)
luciferase – various forms are used
green fluorescent protein (GFP)
human growth hormone
β-glucuronidase
Detection of the expression of reporter genes
- autoradiographic tests
- colorimetric tests
- fluorescence emission
- chemiluminescence emission
- protein detection: ELISA tests
A comparison of some commonly used reporter genes
Reporter
gene
species
product
E.coli
β-galactosidase
Widely used reporter system,
The enzyme hydrolyzes the colorless
substrate X-gal to a blue precipitate
photinus pyralis
(firefly)
luciferase
Highly sensitive reporter enzyme that
oxidizes luciferin and generates a
bioluminescent products (photons)
E.coli
Chloramaphenicol
acetyltransferase (CAT)
A useful reporter for in vitro assays but
proteins gives poor resolution in situ CAT
transfers nonradioactive acetyl groups to
radioactive chloramphenicol
E.coli
β-glucuronidase (GUS)
Generally used reporter in plant systems,
hydrolyzes colorless glucuronides (e.g. Xgluc) to yield colored products for
localization of gene expression
Aequorea victoria
(jellyfish)
Green fluorescent
protein (GFP)
A very widely used system,
reporter that emits fluorescence after
irradiation with UV,
lacZ
luc
CAT
GUS
GFP
use
β-gal (β-galactosidase)
• E. coli enzyme (encoded by lacZ) that hydrolyzes
galactosidase sugars such as lactose
• Widely used to monitor transfection efficiency
• Many assay formats:
colorimetric, fluorescent, chemiluminescent
Transfected cells
Non-transfected cells
β-galactosidase
In bacteria
After transfection
β-gal (β-galactosidase)
•Advantages: simple colorimetric assays, nonradioactive
•Disadvantages: background activity in
mammalian cells (reduced at higher pH values),
β-galactosidase
Staining of the cells:
β-galactosidase performs hydrolysis of b-galactosidates, usually X-gal
(5-bromo-4-chloro-3indolyl-B-D-galactoside)
Various quantitative tests:
 colorimetric: ONPG (o-nitrofenyl-B-D-galaktopiranozyd
 Fluorimetric: MUG – 4-methylumbelliferyl-b-D-galaktozyd
 Chemiluminescent: 1,2-dioeksyetan
SEAP (secreted alkaline phosphatase)
• Mutated form of human placental alkaline phosphatase
• Secreted outside the cell (can assay sample repeatedly
and non-destructively by sampling culture medium)
• Heat-stable (eliminate endogenouse AP activity by heating
samples in 65°C)
• Colorimetric assay, read on plate reader
Luciferases
• firefly Photinus pyralis & sea anemone Renilla reniformis
•Bioluminescent reactions requires luciferin (substrate), ATP,
Mg2+ions and oxygen
Luciferases
• short lasting emission
• short half-life of luciferase – about 3 hours
• high sensitivity
Firefly light
1. Luciferin is activated by luciferase in an ATP-dependent step
2. Luciferin-adenylyl intermediate is formed
3. When oxygen is present this intermediate is rapidly converted to a
peroxyluciferin product
4. Peroxyluciferin decays to oxyluciferin with the emission of photons.
Luciferases – reactions
Renilla luciferase – coelenterazine is a substrate
In vivo luminescence measurements
IVIS® Lumina from Caliper Life Sciences
Green fluorescent protein
- from Aequorea victoria (jellyfish): equorin emits blue light after
Ca2+ binding. The light is absorbed by GFP, which emits green light.
- Expression of GFP in other organisms causes green fluorescence after
stimulation with blue light or UV.
The Nobel Prize in Chemistry 2008
Osamu Shimomura
discovered GFP during the
study of the bioluminescent
protein aequorin, the
mechanism by which certain
jellyfish glow
Martin Chalfie
took the cDNA of GFP and
first expressed it in bacteria
and worms.
He demonstrated that GFP
could be used as a molecular
tag.
Roger Y. Tsien
reported the S65T point mutation
that greatly improved GFP
fluorescent characteristics.
His lab also evolved GFP
into many other color variants,
Blue fluorescent proteins
• blue variants of GFP result from the substitution of histidine for tyrosine
at position 66 in the chromophore
•the original blue variant and an enhanced blue fluorescent protein (EBFP)
version containing secondary mutations to increase folding efficiency and
brightness still lack the necessary photostability for use in live cell imaging
• some mutations resulted in improved FPs named Azurite, SBFP2, and
EBFP2 which are brighter and more photostable than EBFP but remain less
than half as bright as EGFP.
• the most promising BFP is derived from a sea
anemone and is named TagBFP
• compared to EBFP2 TagBFP is more then 1.8 times
brighter and is much more pH-stable.
Cyan fluorescent proteins
• replacing Tyr66 in the chromophore of GFP with tryptophan produces a
cyan-emitting variant CFP
• several CFP variants have been constructed to improve photostability
• the most notable is Cerulean which is twice as bright as CFP
• a recently described monomeric teal-colored FP (mTFP1) derived from
coral exhibits even higher brightness, acid insensitivity, and photostability
than any of the cyan A. victoria variants
•Studies to optimize the folding of monomeric A. victoria
variants have resulted in supercyan derivatives
that are significantly brighter than the parent proteins
• a commercially available derivative
AmCyan1; Clontech
Yellow fluorescent proteins
• the first YFPs were created after the crystal structure of GFP revealed that
Thr203 was positioned very close to the chromophore.
• further development led to the generation of EYFP,
which is one of the brightest and most popular FPs
• however, EYFP is far from perfect. The FP is very
sensitive to acidic pH and becomes only ~50% fluorescent at pH 6.5. In
addition, EYFP is also sensitive to chloride ions and photobleaches much
more quickly than the GFPs.
• many of the problems with EYFP have been solved through mutagenesis.
The Citrine variant is brighter and more resistant to
photobleaching, acidic pH, and other environmental effects
• a commercially available derivative
Topaz; Invitrogen
mBanana, ZsYellow1; Clontech
Orange fluorescent proteins
• FPs exhibiting emission in the orange wavelengths (~560-585 nm) have all been
derived from the Anthozoa species
•although many of these probes are referred to as red FPs, popular variants such as
DsRed and TagRFP actually have emission profiles that are clearly more orange than
red
• currently, the most useful orange FPs are mKusabira Orange (mKO) and its faster
folding derivative mKO2, TagRFP, mOrange2, and tdTomato (a tandem dimer)
• mOrange2 and tdTomato are members of the mFruit family pioneered by Tsien and
coworkers. tdTomato is the brightest FP of any color, but it contains two copies of the
dimeric Tomato FP joined with a 12-residue linker.
Aequora victoria is the
bioluminescent jellyfish
from which GFP was cloned
Discosoma sp. is the
non-bioluminescent reef coral
from which DsRed was cloned
Red fluorescent proteins
• the Tsien laboratory successfully created mRFP1
• continued mutagenesis efforts on mRFP1, resulted in a series of monomeric
FPs leading to first true far-red genetically engineered FP, mPlum
•these FPs were named after common fruits that bear colors similar to their
emission profiles and are thus referred to as the mFruits
• several members of mFruit series, like mApple, mCherry, and mPlum –
they differ e.g. in their brightness: mApple is one of the brightest-red FPs,
whereas mCherry is only half as bright as mApple
•a wide variety of other red FPs has been isolated
from the Anthozoa species, among these are AsRed2,
HcRed1 (and its tandem-dimer variant), and JRed
which are commercially available from Clontech
and Evrogen.
Transfection
• the process of introducing nucleic acids into cells
• the term is used for non-viral methods of NA delivery in eukaryotic cells
• two types of transfection are possible: transient and stable
• for most applications, it is sufficient if the transfected genetic material is only
transiently expressed. Since the DNA introduced in the transfection process is
usually not integrated into the nuclear genome, the foreign DNA will be
diluted through mitosis or degraded.
• for long expression, stable transfection protocol have to be applied: a marker
gene is co-transfected, which gives the cell some selectable advantage, such as
resistance towards a certain toxin.
• if the toxin is then added to the cell culture, only those few cells with the
marker gene integrated into their genomes will be able to proliferate, while
other cells will die.
HAT medium – the first selective conditions
HPRT-/- cells
HAT medium
Prof.Wacław Szybalski
McArdle Laboratory
for Cancer Research,
Wisconsin, Madison,
USA
HPRT+/+ cells
Selection of stably transfected cells
1. Selective genes/resistance to antibiotics
•
•
•
•
aminoglicosides antibiotics – neomycin transferase (G418)
hygromycin – hygromycin phosphotransferase B
puromycin
zeocin (bleomycin).
Antibiotics used for selection of stable transfectants
Antibiotic
Resistance gene
Mechanism of action Concentration
Geneticin (G418)
APH(3’) I , APH(3’)II
Hygromycin B
Puromycin
Zeocin
Fleomycin
Blasticydin S
Hph
Pac
Sh ble
Sh ble
Bcs, BCD
Blocks translation in
eucaryotic cells,
interfering with 80S
ribosome subunit
Interferes with translation
Inhibits translation
Binds to DNA
Binds to DNA
Inhibits translation
50 – 1000 µg/ml
50-1000 µg/ml
1-10 µg/ml
0,5-1000 µg/ml
0,1-50 µg/ml
3-50 µg/ml
Transient and stable transfection
Transient transfection
- In most cases plasmid DNA exists as an episomal element and is gradually
degraded. Additionally it is lost during the replication of cells, thus the percentage of
transduced cells gradually decreases. Therefore the effects of transfection are shortterm.
Stable transfection
- Some vectors - especially retroviral vectors - introduce the transgene to the host
genome. Such DNA replicates together with the rest of chromosome and is traded to
the all daughter cells. Therefore, the effects of transfection are long-term.
- Integration can occur also for other vectors e.g. plasmids or adenoviral ones, but
this process is very uneffective ranging from 1 per 100 to 1 to 1000 successfuly
transduced cells.
- Transgene usually builds into more-less random site of genome.
- Site-specific integration using a homologous recombination mechanism is
possible, but more diffucul and rarerly used.
History of development of gene transfer techniques
(1958 – Alexander et al. – purified polio virus RNA infects cells)
1962 – Szybalski &Szybalska – transfer of cellular DNA – calcium ions
and spermin
1965 – Vaheri & Pagano – DEAE dextran
1973 – Graham & van der Eb – calcium phosphate (human adenovirus 5 DNA)
1979 – Mulligan – transfection of plasmid with rabbit β-globin gene to monkey
kidney cells
1982 – Souther & Berg – selection of cells resistant to neomycin
1987 – Felgner – cationic liposomes
1987 – Wu & Wu – receptor-mediated transfection (polylysine)
1988 – Johnson – gene gun
1995 – Boussif et al.- poliethylenoimine
1996 – Tang i wsp. - dendrimers
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