Reporter genes & transfection

lecture 3

22 October 2012

pCMV/GFP

cDNA Polyadenylation

site

Polyadenylation site pUC

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

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

use product species

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

β-galactosidase E.coli

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

luciferase photinus 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)

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

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.

Luciferases – reactions

Renilla luciferase – coelenterazine is a substrate

IVIS® Lumina from Caliper Life Sciences

In vivo luminescence measurements

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 Martin Chalfie Roger Y. Tsien

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

took the cDNA of GFP and first expressed it in bacteria and worms. He demonstrated that GFP could be used as a molecular tag.

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.

HPRT-/- cells

HAT medium

Prof.Wacław Szybalski McArdle Laboratory for Cancer Research, Wisconsin, Madison,

USA

HAT medium – the first selective conditions

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 Blocks translation in

eucaryotic cells, in terfering with 80S ribosome sub unit

50 – 1000 µ g/ml

Hygromycin B Hph Interferes with translation 50 - 1000 µ g/ml Puromycin Pac Inhibits translation 1 - 10 µ g/ml Zeocin Sh ble Binds to DNA 0,5 - 1000 µ g/ml Fleomycin Sh ble Binds to DNA 0,1 - 50 µ g/ml Blasticydin S Bcs, BCD Inhibits translation 3 - 50 µ g/ml

- 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 short-term.

Transient and stable transfection Transient 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.

Stable transfection

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 30