ansfection methods

Calcium phosphate precipitation

DEAE-dextran (dimethylaminoethyl-dextran)
Lipid mediated lipofection
Electroporation
Retroviral Infection
Microinjection

Transfection of eukaryotic cells

Transfection
What is Transfection?
Transfection is a method of transporting DNA, R
NA and/or various macromolecules into an eukaryo
tic cell by using chemical, lipid or physical based me
thods.
Methods: (just a few examples)
Method
CaPO4, DEAE
Liposome Based
Polyamine Based

Application
DNA Transfection
DNA Transfection
DNA Transfection

Early Years
Transfection = transform
ation + infection
The infection of cells by t
he isolated nucleic acid fr
om a virus, resulting in the
production of a complete v
irus

1928: Frederick
Griffith transforms
Streptococcus
pneumoniae
1944: Avery, Macleod
and McCarty discover
DNA is the transforming
factor
1964: Foldes and
Trautner use the term
transfection

1965: Vaheri and Pagano

describe the DEAE-Dextran
transfection technique

Transfection vs. Transformation

Transformation: genetically altering ce
lls by transporting in foreign genetic
material.
Transfection: the process of transporti
ng genetic material and/or macromol
ecules into eukaryotic cells through ty
pically non-viral methods.

Purpose/uses of Transfection
Study gene function
Study protein expression
Transfer DNA into embryonic stem cell
s

How it works
Utilize Chemical, lipid or physical meth
ods (direct microinjection, electropora
tion, biolistic particle delivery) for tran
sportation of genetic materials or mac
romolecules.

Transfection method 1: DEAE-De

xtran
Facilitates DNA binding to cell membranes and
entry via endocytosis
DEAE-D associates with DNAcomplex binds t
o cell surface
Add chloroquine to transfection medium
Add plasmid DNA to DEAE-Dextran medium
Incubate cells with mixtureefficient
transfection results in 25-75% death
DMSO shock

Transfection method 1: DEAE-De

xtran
Major variants: number of cells, concentration o
f DNA and concentration of DEAE-Dextran
Only method that cannot be used for stable tran
sfections

Transfection method 3: liposomemediated

Use of cationic lipids
chemical and physical similarities to biological
lipids
spontaneous formation of complexes with DN
A, called lipoplexes

High efficiency for in vitro transfections

Can carry larger DNA than viruses
Safer than viruses
Low in vivo efficiency
http://homepage.usask.ca/~sdw132/images/lipid_nanoparticle.gif

How it works Chemical and

lipid methods
Neutralize negative ch
arges on DNA
Chemical method: Calc
ium phosphate; create
s precipitates that settl
e on cells and are take
n in.
Lipid or Polymer metho
ds: interact with DNA,
promotes binding to ce
lls and uptake via endo
cytosis.

Experimental method/proces
s
(chemical methods)

HBS = HEPES-Buffered Saline

Transfection method 2: electropo

ration
Use of high-voltage electric shocks to introduce DNA into cells
Cell membranes: electrical capacitors unable to pass current
Voltage results in temporary breakdown and formation of pore
s
Harvest cells and resuspend in electroporation buffer
Add DNA to cell suspensionfor stable transfection DNA should be
linearized, for transient the DNA may be supercoiled
electroporate
Selection process for transfectant

Transfection method 2: electropo

ration
Variants: amplitude and length of electric pulse
Less affected by DNA concentration-linear correl
ation between amount of DNA present and amou
nt taken up
Can be used for stable transfections

How it works Physical metho

ds
Electroporation: use of high
voltage to deliver nucleic aci
ds; pores are formed on cell
membrane.
Direct Microinjection: Use o
f a fine needle. Has been us
ed for transfer of DNA into
embryonic stem cells.
Biolistic Particle delivery: Us
es high-velocity for delivery
of nucleic acids and penetra
tion of cell wall.

Advantages/Disadvantages
Advantages:
Provides the ability to transf
er in negatively charged mo
lecules into cells with a neg
atively charged membrane.
Liposome-mediated transp
ort of DNA has high efficie
ncy. Good for long-term st
udies requiring incorporatio
n of genetic material into t
he chromosome.

Disadvantages:
Chemical Reagents: not use
ful for long-term studies.
Transfection efficiency is de
pendent on cell health, DN
A quality, DNA quantity, co
nfluency (40-80%)
Direct Microinjection and Bi
olistic Particle delivery is an
expensive and at times a dif
ficult method.

Exploring protein function

1) Where is it localized in the cell?
Approaches:
a) Make antibodies - immunofluorescence
b) Express the protein in cells with a tag
Fuse to GFP
2) What is it doing in the cell?
Approaches:
a) Reduce protein levels - RNA interference
b) Increase protein levels over-express
c) Express mutant versions

Transfection!!!!

Transfection =
Introduction of DNA into mammalian cells

Gene is transcribed and translated into protein

= expressed

Direct introduction of the DNA

Electroporation - electric field

temporarily disrupts plasma
membrane
Biolistics (gene gun)- fire
DNA coated particles into cell
Microinjection

Virally-mediated introduction of the DNA

Infection:
Use recombinant viruses to deliver DNA
Retroviruses
Adenoviruses

Carrier-mediated introduction of the DNA

Positively charged carrier molecules
are mixed with the DNA and added to
cell culture media:
Calcium Phosphate
DEAE Dextran
liposomes
micelles
Carrier-DNA complexes bind to plasma
membrane and are taken up

Types of Transfection

Transient:
Expression assayed 24-48 hours
post transfection
Stable:
Integration of the transfected DNA
into the cell genome - selectable
marker like neomycin resistance
required
stably transfected cell line

DNA expression vector transfected:

Insert gene
in here

For
expression
in cells

pCMV/GFP

pUC

Ne
o
m
ycin
r
e
s
i
s
tanc
e

n
icilli
Amp ance
st
resi

For
amplification
of the
plasmid in
bacteria

Polyadenylation
site

40 r
SV ote
om
Pr

V r
CM ote
om
r
P

GFP

To
generate
stable cell
line

Polyadenylation
site

Bacterial origin of replication

Three ways to make Green fluorescent

protein GFP fusion constructs:
PROTEIN X

GFP
PROTEIN

GFP

PROTEIN Y

GFP

EXPERIMENT:
Transfect unknown GFP fusion protein
Protein X, Y or Z

Visualize GFP protein fluorescence by fluorescence

microscopy in living cells

Counter-stain with known marker to compare

localization patterns in living cells
= vital stain

Some Cellular Organelles

Compartments/organelles examined
Protein sequences sufficient for localization
Vital stains
Nuclei
Mitochondria
Secretory Pathway:
Endoplasmic Reticulum
Golgi Complex
Endocytotic Pathway:
Endosomes

Nucleus
Transport through nuclear
pore
signal = basic amino acid
stretches
example: P-P-K-K-K-R-K-V

Import of proteins into nucleus through nuclear pore

Nuclear Stain:
Hoechst 33258
binds DNA

Mitochondria
Transmembrane transport signal
Example: H2N-M-L-S-L-R-Q-S-I-R-F-F-KP-A-A-T-R-T-L-C-S-S-R-Y-L-L

Protein being transported across mitochondrial membranes

Mitochondrial dye =
MitoTracker Red
Diffuses through
membranes
Non-fluorescent until
oxidized
Accumulates in
mitochondria and oxidized
Mitotracker
DNA

Cellular components of the secretory and endocytic pathways

lysosome

plasma
membrane

late
endosome
nuclear envelope
endoplasmic reticulum

early
endosome

CYTOSOL

cis
Golgi
network

Golgi
stack

trans
Golgi
network

Golgi apparatus

Endoplasmic Reticulum
Entry into E.R.:
Transmembrane transport signal
= hydrophobic amino acid stretches
at amino terminus

Example: H2N-M-M-S-F-V-S-L-L-V-G-I-LF-W-A-T-E-A-E-Q-L-T-K-C-E-V-F-Q

Retention in E.R. lumen:

Signal = K-D-E-L-COOH
at carboxy terminus

Endoplasmic Reticulum marker

ER-Tracker Blue-White

Live bovine pulmonary artery

endothelial cells

Mitotracker Red and ERblue/white

nucleus

Golgi

From the ER, secreted and membrane proteins move to the Golgi, a
series of membrane-bound compartments found near the nucleus

Golgi marker
BODIPY-TR ceramide

Ceramide = lipid
When metabolized,
concentrates in the
Golgi
Red fluorophore

Cultured Epithelial Cells

DNA (Hoechst)
Golgi (ceramide)

MDCK Cells

Madin-Darby Canine
Kidney
Polarized Epithelial
Cells

DNA (Hoechst)
Golgi (ceramide)
Lysosomes (LysoTracker)

Molecular Probes, Inc.

Rhodamine transferrin

Does the fluorescent green protein co-localize?

Immunofluorescence / confocal microscopy

Immunofluorescence / confocal microscopy

Immunofluorescence / confocal microscopy

B or T cells in suspension, adherent cells on chambered coverglass or chamberslides, cryostat sections
of unfixed, OCT embedded tissue:
1. wash cells 1x cold RPMI (no wash for cryostat sections).
2. Fix 20 min 4% paraformaldehyde in 0.1M phosphate buffer pH 7.4, 0.03M sucrose on ice. **
3. wash 2x PBS/1%BSA. From now on everything can be at room temp. or on ice.
4. Permeabilize 5 min RT in 0.2% saponin, PBS, 0.03M sucrose, 1% BSA.
5. wash 1x PBS/BSA.
6. Block 15 min 5% normal goat serum (NGS) in PBS/BSA.
7. wash 1x PBS/BSA.
8. 1 diluted in PBS/BSA 60 min RT; 100 l per tube or section.
9. wash 3x PBS/BSA.
10. Block 15 min 5% NGS in PBS/BSA.
11. wash 1x PBS/BSA.
12. 2 diluted in PBS/BSA 30 min RT; 100 l per tube or section.
13. wash 3x PBS/BSA; (wash 1x in PBS/BSA then 2 x 10 min in Molecular Probes SlowFade Light buffer
if using Slow Fade Light S-7461 to coversip); pellet cells and put up in two drops of Molecular Probes
Slowfade Light antifade medium. Pipet about 15 m l on slide and coverslip. For chambered coverglass
just put two-three drops in each chamber after wash.

High-throughput loss-of-function screens using RNAi cell microarrays.

An example of a high-density, high-content RNAi cell-microarray screen in

cultured D. melanogaster Kc167 cells.

Experimental Animals

Model systems

E. coli
- easy and inexpensive to maintain
- ,
-
Pathogenic sickness, death
- Metabolism

Eukaryote

 Yeast
- Eukaryote prokaryote

- Genetic study easy
- haploid, single-celled
- Grow very rapidly, inexpensive
- 2
Saccaromyces cerevisiae; budding
Schizosaccaromyces pombe; fission

Cloning the yeast origin of replication

- yeast genome cut restriction enzyme
- fragments clone into plasmid vector
- recombinant plasmids growth and
select
- yeast survive in media lacking leucine
- selected yeast cells contain leu2+ gene

Nematoda worm
- Small and easy to keep
- Three days to develop
Multicellular organism
Caenorhabditis elegans
Fruit fly
Identification of mutant
mutation
-
- 12
- Drosophila melanogaster

Zebrafish
- Reproduction rapidly and high number
- vertebrate
-
-
Danio rerio
Amphibians
-
- 1920
- Xenopus leavis

Chicken

;
- ,
- Gallus gallus

Mouse
- vertebrate, mammals
Human
; ,
- Expensive to maintain, Reproduce
slowly
Knock out mouse
gene

Human cell culture

Human blood tissue
Primary culture
; derived directly from living tissue
- Grow for a short period time and stop
-

Plants
- Grow slowly, long generation time
-
- Large genome
- Transposon ; corn
- Arabidopsis thaliana