Direct Gene Transfer

Chemical Mediated

Introduction:
DNA is an anionic polymer, larger molecular weight,
hydrophilic and sensitive to nuclease degradation in
biological matrices.
DNA cannot easily cross the physical barrier of
membrane and enter the cells unless assisted
Various charged chemical compounds can be used
to facilitate DNA transfer directly to the cell.
Chemicals introduced near the vicinity of recipient
cells thereby disturbing the cell membranes,
widening the pore size and allowing the passage of
the DNA into the cell.

Ideal chemical for gene

transfer?
An ideal chemical used for DNA transfer
should have the ability to Protect DNA against nuclease degradation.
Transport DNA to the target cells.
Facilitate transport of DNA across the plasma
membrane.
Promote the import of DNA into the nucleus.

Commonly Used Methods Of

Chemical Transfection
Calcium phosphate
DEAE dextran
Cationic Lipid
Other polymers - poly-L-lysine (PLL),
polyphosphoester, chitosan, dendrimers

PEG Mediated Gene Transfer

PEG Mediated Gene Transfer

PEG is inert & least toxic to cells/protoplast
Protoplasts take ups naked DNA with PEG
PEG in complex with divalent cation can disturb
molecular organization of plasma membrane
Ca2+ neutralizes ve charges on protoplast,
PEG increases rate of endocytosis.

PEG Mediated Gene Transfer

Factors affecting:
Linearized ds-plasmid DNA
Mg/Ca ion conc.

Disadvantage and advantages:

Regeneration of fertile plants from protoplasts is a
problematic for some species.
The DNA used for transformation is also susceptible to
degradation and rearrangement.
advantages: protoplast can isolated and transformed in
number of plants species.

Protocol
To protoplast suspension add linearized plasmid
DNA
To this add 40% of PEG4000(w/v) dissolved in
mannitol & calcium nitrate solution. pH - 8
Incubation 5 mins
Heat shock
DNA enters the nucleus and integrates into the
host genome.

Modified Protocol
Adding PEG after DNA
Heat shock treatment of protoplasts at
460C followed by chilling on ice.
CaCl2/PEG-stimulated DNA uptake in
wheat, tobacco maize

Lipofection:
Introduction of DNA in to cell via Liposome.
The DNA is enclosed in a artificial lipid vesicle called
liposome.
Induced to fuse with protoplast using PEG.
DNA + liposome enders the protoplast through endocytosis
Adhesion of liposomes to protoplast surface
Fusion of liposomes at the site of adhesion
Release of plasmids inside the cell.
Use of vely charged unilammellar liposome
(phosphatidylserine)-DNA easily complexes with these
liposome.

Lipofection
Cationic / neutral lipids from stable complex with DNA
lipoplexes
Liposome preparation-dispersion of phospholipids like
Phosphatidyl Choline in water by sonication
Cationic lipid (Dimethyl Diocta Decyl ammonium
bromide, DDAB) or a synthetic cationic cholesterol
derivative, along with a neutral lipid (dioleoyl-Lalpha-phosphatidyl ethanolamine, DOPE).
Cationic liposome
eg : Lipofectin by Gibco BRL CellFectin by Invitogen

Advantages:
1. Simplicity.
2. Long term stability.
3. Protection of nucleic
acid from degradation

4. Applicable to wide verity

of cell types.
5. High degree of
reproducibility.
6. Biocompatibility- reduced
level of cytotoxicity

Calcium phosphate method:

Method involves the formation of a fine DNA/calcium
phosphate co-precipitate which first settles on the
cells and then internalized by endocytosis.
The precipitate must be formed freshly .
The DNA escapes and reaches the nucleus and can
be then expressed.
This method is suitable only for actively dividing cells
This method gives only 1-2% efficiency.
Increasing efficiency by using Buffer and high-quality
plasmid DNA + DMSO shock.

Calcium phosphate co-precipitation

Ca/DNA coprecipitates, when formed at cell
surfaces, can be taken up by cells
This precipitate is engulfed by cells through
endocytosis and the DNA gets integrated into
the cell genome
in plants, protoplasts are amenable to coprecipitation

Protocol
Mixing DNA with calcium chloride, adding this
in a controlled manner to a buffered
saline/phosphate solution (isotonic)and
allowing the mixture to incubate at room
temperature.
This generates a precipitate (DNA-CaPO 4)

that is dispersed onto the cultured

protoplasts.
Precipitate is taken-up by the cells via
endocytosis or phagocytosis.

Co-precipitation

Calcium phosphate co-precipitation

Advantages
Simple and inexpensive
Applicability to generate stably transfected cell lines
Highly efficient (cell type dependent) and can be applied to a wide
range of cell types.
Can be used for stable or transient transfection
Disadvantages
Toxic especially to primary cells
Slight change in pH, buffer salt concentration and temperature can
compromise the efficacy
Relatively poor transfection efficiency compared to other chemical
transfection methods like lipofection.
Random integration into host cell

DEAE-Dextran
Diethyl amino ethyl dextran is
a soluble high molecular
weight polycationic
carbohydrate
It associates with negatively
charged nucleic acids.
It promotes interactions
between DNA and endocytotic
machinery of the cell.

DEAE-Dextran
Method:
The negatively charged DNA and positively charged
DEAE dextran form aggregates through electrostatic
interaction and form a polyplex.
A slight excess of DEAE dextran in mixture results in
net positive charge in the DEAE dextran/ DNA complex
formed.
These complexes, when added to the cells, bind to the
negatively charged plasma membrane and get
internalized through endocytosis.
Complexed DNA delivery with DEAE-dextran can be
improved by osmotic shock using DMSO or glycerol.

DEAE-Dextran
Advantages
Simple and inexpensive
More sensitive
Can be applied to a wide range of cell types
Can be used for transient transfection.
Disadvantages
Toxic to cells at high concentrations
Transfection efficiency varies with cell type
Can only be used for transient transfectionbut not
forstable transfection
Typically produces less than 10% delivery in primary
cells.

DMSO
25-30% of DMSO increases membrane
permeability & enhance the uptake.
Polybene + DMSO
25-30% of DMSO increases membrane
permeability & enhance the uptake.

Reference:
Gene Cloning and DNA Analysis: an introduction
by Brown TA. 2006.
Biotechnology By B.D. Singh
Principles of Gene Manipulation and Genomics by
S.B. primrose and RM Twyman

Gene Transfer
Transformation: introduction of genetic materials into
bacteria
Transfection: introduction of genetic materials into eukaryotic
cells (e.g. fungi, plant, or animal cells)
Transduction: introduction of genetic materials using viruses
Lipofection: introduction of genetic materials using
liposomes

Stable vs. Transient Gene

Transfer
Stable Gene Transfer: achieved by
plasmid integration in the host genome or
episomal replication of the transferred
plasmid.
Transient Gene Transfer: the foreign DNA
is usually not integrated into the nuclear
genome and will be degraded or diluted
through mitosis