Reporter genes & transfection

Reporter genes & transfection

lecture II18th October 2010

pCMV/GFP

cDNAPolyadenylation

site

PolyadenylationsitepUC

Bacterial origin of replication

For expression in cells

To generate stable cell line

For amplification of the plasmid in

bacteria

Mammalian expression plasmid

To determine transfection efficiency

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 are used to study

• when, • where, and • how much a gene is expressed

Reporter genes

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

lacZ

luc

CAT

GUS

GFP

useproductspecies

Widely used reporter system, The enzyme hydrolyzes the colorless substrate X-gal to a blue precipitate

β-galactosidaseE.coli

Highly sensitive reporter enzyme that oxidizes luciferin and generates a bioluminescent products (photons)

luciferasephotinus pyralis (firefly)

A useful reporter for in vitro assays but proteins gives poor resolution in situ CAT transfers nonradioactive acetyl groups to radioactive chloramphenicol

Chloramaphenicol acetyltransferase (CAT)

E.coli

Generally used reporter in plant systems, hydrolyzes colorless glucuronides (e.g. X-gluc) to yield colored products for localization of gene expression

β-glucuronidase (GUS)E.coli

A very widely used system, reporter that emits fluorescence after irradiation with UV,

Green fluorescent protein (GFP)

Aequorea victoria (jellyfish)

Chloramphenicol acetyltransferase (CAT)


•1st reporter gene used to monitor transcriptional activity in cells

•Bacterial enzyme that transfers acetyl groups from acetyl-CoA to chloramphenicol, detoxifying it

Ways of Detection:

•Reaction quantified using radiolabeled substrates (14C-chloramphenicol)

- qualitative rather than quantitative

•ELISA (non-radioactive) is recently available

It becomes historical...


Advantages: low background in eukaryotic cells, high stability - protein is very stable (half-life is 50 hours)

Disadvantages: use of radioisotopes

CAT assay : TLC

TLC: Thin Layer Chromatography

CAT – detection of activity

Chloramphenicol

Acetylated forms

non-transfected transfected

origin

CAT assay: ELISA

Anti-CAT – antibody to CATAnti-CAT-DIG - digoxigenin-labeled antibody to CATAnti-DIG-POD Fab fragment - antibody to digoxigenin conjugated to peroxidase (anti-DIG-POD) and binding to digoxigeninABTS - peroxidase substrate

β-galactosidase

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, non-radioactive

•Disadvantages: background activity in mammalian cells (reduced at higher pH values),

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

β-galactosidase

Expression of β-galactosidase in various cells after lipotransfection

Cells from monkey kidney Vascular smooth muscle cells(rat)

Histochemical staining for β-galactosidase expression in rat hepatocellular carcinomas

Heller et al, 2000

β-Galactosidase staining of transgenic mice embryos

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

• short lasting emission

• short half-life of luciferase – about 3 hours

• high sensitivity

Luciferases

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.

Renilla reniformis luciferase

Sea Pansy

Renilla reniformis luciferase

Luciferases – reactions

Renilla luciferase – coelenterazine is a substrate

Measurement of bioluminescence

Modified from: www.xenogen.com

Shutter

Lens and Aperture

Heated Sample Stage

Illuminator

Electronics Tray

CCD Chip

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Spread of cancer cells expressing luciferase can be monitored using

non-invasive CCD camera

Luciferase activity can be detected in vivo

In vivo optical bioluminescent imaging

Cardiacischemia

Genetransfer

Stem cell engraftmentHematopoesis from a single stem cell…

Bladdercancer

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Growth of tumors in lungs after intravenous injection with B16(F10) melanoma cells

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Green fluorescent protein

- from Aequorea victoria (jellyfish): equorin emits blue light afterCa2+ 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 Martin Chalfie Roger Y. Tsien

discovered GFP during the study of the bioluminescent protein aequorin, themechanism by which certain jellyfish glow

took the cDNA of GFP andfirst expressed it in bacteriaand worms. He demonstrated that GFPcould be used as a moleculartag.

reported the S65T point mutationthat greatly improved GFPfluorescent characteristics.His lab also evolved GFPinto many other color variants,

Color variants of GFP

Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.top63

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 timesbrighter 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. victoriavariants 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; InvitrogenmBanana, 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.


Human saphenous vein endothelial cells (HSVEC)

Bright-field photograph of four agouti coat-colored mouse pups.

Dark-field photograph taken under epifluorescence optics revealing the

transgenic color of the mice.

EGFP - green FP variantECFP - cyan FP (ECFP) variant giving a bluecyan fluorescenceEYFP - yellow FP variant

Histochem Cell Biol (2001) 115:49–58

The color of mice: in the light of GFP-variant reporters

Hadjantonakis, A-K., et al., BMC Biotechnology 2002

In this study embryonic stem (ES) cell-mediated transgenesis have been used to test the enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein

(EYFP), two mutant and spectrally distinct color variants of wild type (wt) GFP

Dual non-invasive reporter visualization in embryos and adult mice


Transgenic salamander developed by: Lidia Sobkow, Werner Straube, Heino Andreas, Elly Tanaka Max-Planck Institute for Molecular Cell Biology and Genetics, Dresden

Double transgenic mouse embryo, 18.5 days

Gloria Kwon, (GFP and RFP)

GFP ? green fluorescent pig...

Green fluorescent pigs...

When lit up in the dark, the pigs glow green

In daylight, their eyes and skinare green-tinged

http://news.bbc.co.uk/2/hi/asia-pacific/4605202.stm

Fluorescent zebrafish

daylight UV light

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.

Transient and stable transfection

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 ConcentrationGeneticin (G418) APH(3’) I , APH(3’)II Blocks translation in

eucaryotic cells, interfering with 80S ribosome subunit

50 – 1000 µg/ml

Hygromycin B Hph Interferes with translation 50-1000 µg/mlPuromycin Pac Inhibits translation 1-10 µg/mlZeocin Sh ble Binds to DNA 0,5-1000 µg/mlFleomycin Sh ble Binds to DNA 0,1-50 µg/mlBlasticydin S Bcs, BCD Inhibits translation 3-50 µg/ml

Principles of ideal delivery system

1. Simple

2. Safe

3. Provide prolonged expression oftransgene at therapeutic levels

Overview of non-viral gene delivery

The major approaches of DNA delivery

Chemical delivery

Physical delivery

Viral vectors

Bactofection (bacterial vectors)

Transfections of plasmid DNA

„naked” DNA (physical methods)

chemical vehicles

injection electroporation biolistic (gene gun)

microinjection

macroinjection

large volume(hydrodynamic methods)

Microinjection of plasmid DNA

Macroinjection - Injection of naked DNA

Naked plasmid, containing lacZ gene has been injected into the leg muscle of a mouse

Electroporation

Electropermeabilisation, electrokinetic enhancement

Transfection of cells following their exposureto pulsed electric field

first used with animal cells by Neumann et al. - 1982

Electroporation

1. Strength of electric field

- usually in mammalian cells the best transfection efficiency is obtained when the voltage is between 250-750 V/cm.

- time: 20-100 msec - about 20-50 % of cells survive such a treatment

2. The degree of permeabilisation of the cells depends on:

- electric field intensity: the larger the „functional” size of the cell, the lower is the field strength necessary (e.g. skeletal muscle)

- length of pulses- shape and type of electrodes - cell size

Electroporation

Gene Pulser II

Nucleofection

• Based on the physical method of electroporation, but nucleofection usesa combination of optimized electrical parameters, generated by a special device called Nucleofector, with cell-type specific reagents.

• Provides ability to transfect non-dividing cells like neurons or blood cells.

• Optimal nucleofection conditions depend upon the individual cell type,not on the nucleic acid being transfected (DNA, RNA, siRNA, shRNA,…).•This combination permits the crucial step: delivery of the DNA of intereststraight into the nucleus. As the DNA quickly reaches the nucleus, expression can begin without delay.•Transfection efficiencies of over 50% can be obtained in most cell lines as well as in primary cells. For some primary cells, such as human dermal fibroblasts, efficiencies exceed 90%

Nucleofector 96-well Shuttle System

system for the highly efficient transfectionof primary cells and difficult-to-transfectcell lines in 96-well format

Amaxa Nucleofector Systems

Nucleofection vs Lipofection

Gene-gun technology

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Gene-gun technology

Chemicals, proteins, RNA or DNA are first coated on dense metallic particles of0.25 to 5 µm in diameter, and these are accelerated into target cells or tissues to deliver these substances directly inside cells.

Gene-gun technology

The Helios Gene Gun



Prof. Halina Gabryś,Department of Plant Physiology and Biochemistry

Gene-gun technology

Gene-gun technology

• DNA- or RNA-coated gold particles are loaded into the gun

• a low pressure helium pulse delivers the coated gold particles into virtually any target cell or tissue.

Gene gun technology – properties

1. Efficiency Typically 5-10% of cells within 10-20 cell layers of the target site.

2. Unsuited to gene therapy requiring high number of transfected cells

3. Unsuitable in tissues difficult to expose (brain, lumen of the lung)

4. Suitable for addressing targets in culture- targets at the surface of the body (skin, eye) or internal organs that can be exposed surgically (eg. Liver, muscles, spleen)

Naked DNA - applications

1. Duchenne muscular dystrophy; patients; mdx mice (dystrophin-deficient mice)

2. Transfer of genes encoding secretory proteins, eg. VEGF

3. Animal models of autoimmune diseases

4. Genetic vaccines

Low transfection efficiency in large animals...

Yla-Herttuala S, Alitalo K, Nature Med. June 2003

Problems with transfection of naked DNA

1. Cell type dependent – only few cell types can be effectively transfected

2. Maximal expression – after 14 days in skeletal muscles

3. Long-term expression in skeletal muscle – episomal, even up to 2 years

4. Muscle regeneration enables higher expression

5. Promoters – better viral than cell specific ?

6. Efficiency is reversely dependent on the animal size...

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Reporter genes & transfection