Lecture II

Plasmid vectors & transfection

Mammalian expression plasmid

How to make a plasmid working

in mammalian cells?

Expression of foreign genes in eucaryotic cells requires

the eucaryotic promoter in a plasmid vector

1. Viral promoters : CMV, SV40

2. Eucaryotic promoters

- constitutive:

- non-selective: -globin

- tissue specific:

- inducible

3. Complex

Poly-A is required for expression of genes

in mammalian cells

AAUAAA

Mammalian expression plasmid

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

luciferase – various forms are used

-galactosidase

Green fluorescent protein (GFP)

Secreted alkaline phosphatase (SEAP)

Human growth hormone

-glucuronidase

Detection of the expression of reporter genes

-Autoradiographic tests

-Colorimetric tests

- fluorescence emission

- chemiluminescence emission

-Protein detection: ELISA tests

Chloramphenicol acetyltransferase (CAT)

CAT

transfers acetyl groups from Acetyl-CoA to chloramphenicol

procarytic enyzme

protein is very stable (half-life is 50 hours)

Detection:

radioactive test

Separation of acetylated forms from non-acetylated ones by

means of chromatography

- qualitative rather than quantitative

non-isotopic tests are recently available

It becomes historical...

CAT – detection of activity

Chloramphenicol

Acetylated forms

non-transfected transfected

Wstawienie sygnału poliadenylacji przed MCS

redukuje podstawową transkrypcję

Staining of the cells:

-galactosidase performs hydrolysis of -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

-galactosidase

Expression of b-galactosidase in various cells after

lipotransfection

Cells from kidney Vascular smooth muscle cells

of a monkey (rat)

Luciferases

-fire fly 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

Luciferin is activated by luciferase in an

ATP-dependent step to form a luciferin-

adenylyl intermediate; when oxygen is

present this intermediate is rapidly

converted to a peroxyluciferin product

that decays to oxyluciferin with the

emission of photons.

Firefly light

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

Cardiac

ischemia

Gene

transfer

Stem cell engraftment

Hematopoesis from a single stem cell…

Bladder

cancer

Wstawic nasze wyniki pokazujące wykorzystanie

IVIS – powiedzieć o tym, że taki sprzęt będzie

Zakupiony w ramach BMZ

Biotechnologia Molekularna dla Zdrowia www.wbbib.uj.edu.pl/bmzbmz

2008-2012

Biotechnologia Molekularna dla Zdrowia www.wbbib.uj.edu.pl/bmzbmz

Biotechnologia Molekularna dla Zdrowia www.wbbib.uj.edu.pl/bmzbmz

Strona internetowa projektu

Biotechnologia Molekularna dla Zdrowia www.wbbib.uj.edu.pl/bmzbmz

Web site

Biotechnologia Molekularna dla Zdrowia www.wbbib.uj.edu.pl/bmzbmz

Nowoczesna aparatura

IVIS

(Image Visualisation and Infrared Spectroscopy)

Renilla reniformis luciferase

Sea pansy

Renilla reniformis luciferase

Luciferase Reporter Gene Assay

Luciferases – reactions

Renilla luciferase – substrate is coelenterazine

Double luciferase assay – Promega (Madison, USA)

Green fluorescent protein - from Aequorea victoria (jellyfish): equorin emits blue light after

Ca2+ binding. The light is absorbed by GFP, which emits green ligth.

-Expression of GFP in other organisms causes green fluorescence

after stimulation with blue light or UV.

Aequorea victoria are brightly luminescent jellyfish, with glowing points around the margin of the

umbrella. Light arises from yellow tissue masses that each consist of about 6000-7000 photogenic

cells. The cytoplasm of these cells is densely packed with fine granules that contain the components

necessary for bioluminescence [1:2] . In other bioluminescent coelenterates these have been

characterised as 0.2 micron diameter particles enclosed by a unit membrane, and have been termed

lumisomes [4] . The components required for bioluminescence include a Ca++ activated photoprotein,

aequorin, that emits blue-green light, and an accessory green fluorescent protein (GFP), which

accepts energy from aequorin and re-emits it as green light [5] . GFP is an extremely stable protein of

238 amino acids [6] . The fluorescent properties of the protein are unaffected by prolonged treatment

with 6M guanidine HCl, 8M urea or 1% SDS, and two day treatment with various proteases such as

trypsin, chymotrypsin, papain, subtilisin, thermolysin and pancreatin at concentrations up to 1 mg/ml

fail to alter the intensity of GFP fluorescence [7] . GFP is stable in neutral buffers up to 65oC, and

displays a broad range of pH stability from 5.5 to 12. The protein is intensely fluorescent, with a

quantum efficiency of approximately 80% and molar extinction coefficient of 2.2 x104 cm-1 M-1 [5]

(after correction for the known molecular weight). GFP fluoresces maximally when excited at 400 nm

with a lesser peak at 475 nm, and fluorescence emission peaks at 509nm .

GFP

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,

and demonstrated that these

variants could be used as

genetically-encoded intracellular

sensors for calcium, enzyme action,

and glutamate.

Color variants of GFP

Green fluorescent protein

Human saphenous vein endothelial cells (HSVEC)

Green fluorescent protein

Not

transgenic

GFP ?

green fluorescent pig...

Green fluorescent pig...

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

Transient and stable transfection

Transfekcja plazmidem zawierającym gen selekcyjny, np. gen oporności na

neomycynę. Po transfekcji komórki są hodowane w pożywce zawierającej

neomycynę (lub jej analog, jak G418). Komórki, które nie zostały stransfekowane

plazmidem z genem NeoR, nie produkują enzymu rozkładającego G418 i giną. Przeżywają

tylko komórki stabilnie stransfekowane plazmidem z genem selekcyjnym.

Selection of stably transfected cells

1. Selective genes/resistance to antibiotics

a) aminoglicosides antibiotics – neomycin transferase (G418)

b) hygromycin – hygromycin phosphotransferase B

c) puromycin

d) zeocin (bleomycin).

Antibiotic Resistance gene Mechanism of action Concentration

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

eucaryotic cells,

interfering with 80S

ribosome sub-unit

50 – 1000 g/ml

Hygromycin B Hph Interferes with translation 50-1000 g/ml

Puromycin Pac Inhibits translation 1-10 g/ml

Zeocin Sh ble Binds to DNA 0,5-1000 g/ml

Fleomycin Sh ble Binds to DNA 0,1-50 g/ml

Blasticydin S Bcs, BCD Inhibits translation 3-50 g/ml

Antibiotics used for selection of stable transfectants

Principles of ideal delivery system

1. Simple

2. Safe

3. Provide prolonged expression of transgene at

therapeutic levels

Overview of non-viral gene delivery

Transfections of plasmid DNA

„naked” DNA (physical methods)Chemical vehicles

injection electroporationBiolistic

(gene gun)

microinjection

macroinjection

Large volume

(hydrodynamic methods)

Microinjection of plasmid DNA

First transgenic mouse

Brinster et al., 1982

Plasmid used for establishment of first transgenic mouse

Electroporation

Electropermeabilisation, electrokinetic enhancement

Transfection of cells following their exposure

to 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 (eg. skeletal muscle)

- length of pulses

- shape and type of electrodes

- cell size

Electroporation

ElectroporationGene Pulser II

Nucleofection

Based on the physical method of electroporation, but nucleofection uses

a 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 interest

straight 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 transfection

of primary cells and difficult-to-transfect

cell lines in 96-well format.

Amaxa Nucleofector Systems

Nucleofection

about 50% efficiency

Gene-gun technology

bazooka

Gene-gun technology

Chemicals, proteins, RNA or DNA are first coated on dense metallic particles of

0.25 to 5 m in diameter, and these are accelerated into target cells or tissues to

deliver these substances directly inside cells.

Gene-gun technology

Advantages of gene gun technology

Barry et al.., 2004

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)

Macroinjection

Injection of naked DNA

Naked plasmid, containing lacZ gene has been injected into the

leg muscle of a mouse.

Naked DNA - applications

1. Duchenne muscular dystrophy; patients; mdx 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...