2/14/2019 Top 13 Methods of Gene Transfer (With Diagram)

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Top 13 Methods of Gene Transfer

(With Diagram)
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The following points highlight the top thirteen methods of gene

transfer. Some of the methods are: 1. Calcium Chloride (CaCl2)
Mediated DNA Transfer 2. Rubidium Chloride Mediated DNA
Transfer 3. Electroporation 4. Liposome Encapsulation
(Lipofection) 5. Microinjection

Gene Transfer:

Method # 1. Calcium Chloride (CaCl2) Mediated DNA Transfer:

This is used for the transformation of prokaryotic host cells.

Principle:

In the process of transformation all bacterial cells cannot uptake the

exogenous DNA molecule. Those who are capable to take are called competent
cells. So our aim in this step is to make bacterial cells more competent so that
the possibility of transferring of the recombinant DNA into the host cell
increases to a higher fold. CaCl2 makes the cell wall of the bacteria more
permeable to the exogenous DNA and thus increases the competence of the
host cell.

Procedure:

Growing E. Coli cells are isolated and suspended in 50 mM CaCl2 at a

concentration of 108-1010 cells/ml. The cells may be incubated for 12- 24 hr.
to increase the frequency of transformation. The recombinant DNA is then
added.
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Efficient transformation takes only a few minutes and the cells are plated on a
suitable medium for the selection of transformed clones. The frequency of
transformed cells is 106-107 per mg of plasmid DNA; this is about one
transformation per 10,000 plasmid molecules.

The transformed cells are suitably diluted and spread thinly on a suitable
medium so that each cell is well separated and produces a separate colony.
Generally, the medium is so designed that it permits only the transformed cells
to divide and produce colonies. This frequency can be further improved by
using special E. Coli strains, e.g., SK1590, SK1592, X1766, etc.

Gene Transfer:

Method # 2. Rubidium Chloride Mediated DNA Transfer:

The rubidium chloride method is a variant of the calcium chloride method that
offers somewhat higher competency. The process followed is same as before
but just the CaCl2 is replaced with RbCl2. This is also used in the transfor-
mation of the prokaryotic host cell.

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2/14/2019 Top 13 Methods of Gene Transfer (With Diagram)

Gene Transfer:

Method # 3. Electroporation:

Electroporation or electro-permeabilization is the process of applying

electrical field to a living cell for a brief duration of time in order to create
microscopic pores in the plasma membrane called electro-pores. This
technique is used for transferring the recombinant DNA molecule into wide
range of hosts starting from bacteria to plant (plant protoplasts) and animal
cells.

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2/14/2019 Top 13 Methods of Gene Transfer (With Diagram)

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2/14/2019 Top 13 Methods of Gene Transfer (With Diagram)

Principle:

The phospholipid molecules of the plasma membrane are not static. When we
apply electric field to them their kinetic energy increases resulting in the
increase in the membrane permeability at certain points. This is exactly where
we see the formation of electro-pores. The recombinant DNA can pass through
these transient pores before they close.

Procedure:

In this process cells are mixed with the recombinant DNA and the mixture is
placed in a small chamber with electrodes connected to a specialized power
supply. Then a brief electric impulse is discharged across the electrodes, which
makes pores (holes) in the plasma membrane.

These pores remain for some time and are again resealed themselves.
Recombinant DNA enters the cell which are removed and plated in fresh
selective medium. The process of selection is then applied to isolate cells
carrying recombinant DNA.

Gene Transfer:

Method # 4. Liposome Encapsulation (Lipofection):

This technique is found very successful in the transfection of plant protoplasts

and animal host cells.

Principle:

Liposomes are microscopic vesicles developed in a laboratory environment.

Each liposome is a spherical ball like structure made up of phospholipid
bilayers with a hollow central space, allowing liposomes to interact directly
with cells.

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2/14/2019 Top 13 Methods of Gene Transfer (With Diagram)

A liposome can fuse with the cell membrane of the taken host cell and can de-
liver its content to it. The recombinant DNA enclosed in the liposome vesicles
penetrates into the protoplast of the host cell.

Procedure:
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2/14/2019 Top 13 Methods of Gene Transfer (With Diagram)

In this technique the recombinant DNA, which is negatively charged at a near

neutral pH because of its phosphodiester backbone, is mixed with the lipid
molecules with positively charged (cationic) head groups. The lipid molecules
form a bilayer around the recombinant DNA molecules.

This results in the formation of liposomes which are further mixed with the
host cells. Most eukaryotic cells are negatively charged at their surface, so the
positively charged liposomes interact with the cells. Cells take up the lipid-
recombinant DNA complexes, and some of the transfected DNA enters the
nucleus.

Gene Transfer:

Method # 5. Microinjection:

This is the direct introduction of the recombinant DNA into the host cell. This
technique has been used successfully with both plant and animal cells. In this
procedure the cell is held on a glass capillary by gentle suction.

The microinjection needle is made by drawing out a heated glass capillary to a

fine point. Using a micromanipulator (a mechanical device for fine control of
the capillary) the needle has been inserted into the nucleus of the host cell.
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One obvious disadvantage is that this technique is labour-intensive and not

suitable for primary cloning procedures where large numbers of recombinants
are required. However, in certain specialised cases it is an excellent method for
targeting DNA delivery once a suitable recombinant has been identified and
developed to the point where microinjection is feasible.

Gene Transfer:
Method # 6. Biolistic Particle Delivery System:

A gene gun or a biolistic particle delivery system is a device which can directly
bombard small particles coated with the recombinant DNA on the nucleus of
the target cell. This technique is often simply referred to as bio-ballistics or
biolistics and has been successfully used in the transfection of both plant and
animal cells.

In this technique the recombinant DNA is coated with microscopic tungsten

particles known as micro-projectiles, which are then accelerated on a macro-
projectile by firing a gunpowder charge or by using compressed gas to drive
the macro-projectile.

At one end of the ‘gun’ there is a small aperture that stops the macro-projectile
but allows the micro-projectiles to pass through. When directed at cells, these
micro-projectiles carry the DNA into the cell and, in some cases, stable
transformation will occur.

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2/14/2019 Top 13 Methods of Gene Transfer (With Diagram)

Gene Transfer:
Method # 7. Calcium Phosphate Co-Precipitation:

This technique is used for the transfection of plant and mostly animal cells.
The recombinant DNA is mixed with calcium chloride in a phosphate buffer at
neutral pH. This results in the formation of recombinant DNA-calcium
phosphate complex which appears as a thin precipitate. This precipitate is then
added to the host cell.

The precipitate is taken up by the cell by the process of phagocytosis. The re-
combinant DNA enters the nucleus and integrates into the host’s genome. The
transfection efficiency can be increased by exposing the host cell to 10-20%
glycerol or Dimethyl sulfoxide (DMSO).

Gene Transfer:

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2/14/2019 Top 13 Methods of Gene Transfer (With Diagram)

Method # 8. Sonoporation:

Sonoporation, or cellular sonication, is the use of sound (typically ultrasonic

frequencies) for the transfer of recombinant DNA into the target host cell. This
process has been successfully used in a wide range of host cells starting from
bacteria to plant and animal cells.

This employs the acoustic waves to increase the permeability of the plasma
membrane. Taking the advantage of this situation the recombinant DNA
enters the host cell.

Gene Transfer:
Method # 9. Optical Transfection:

Optical Transfection is the process of introducing nucleic acids into cells using
light. This has been successful in transfecting animal cells. In this technique
the plasma membrane of the host cell is exposed to the highly focused laser
beam for a small amount of time (typically tens of milliseconds to seconds),
generating a transient pore on the membrane called photo-pore.

Through the photo-pore the recombinant DNA can enter the host cell.

Gene Transfer:

Method # 10. Impalefection:

Impalefection is a method of gene delivery using Nano materials, such as

carbon Nano fibres, carbon nanotubes, nanowires, etc. This technique is used
for the transfection of plant and animal cells. In this technique needle-like
nanostructures are synthesized perpendicularly to the surface of a substrate.

Recombinant DNA is attached to the nanostructure surface. A chip with arrays

of these needles is then pressed against cells or tissue.

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Gene Transfer:
Method # 11. Magnetofection:

Magnetofection, or Magnet assisted transfection is a method, which uses

magnetic force to deliver recombinant DNA into target host cells. Nucleic acids
are first associated with magnetic nanoparticles. Then, application of magnetic
force drives the nucleic acid particle complexes towards and into the target
host cells, where the cargo is released. This has been successfully used to
transfect the plant and animal cells.

Gene Transfer:
Method # 12. Protoplast Fusion:

This technique is used for introducing gene of interest into plant and animal
cells. In this technique first we transfer the recombinant DNA into a bacterial
cell then dissolve its cell wall by treating it with lysozyme. After this we fuse
the host protoplast with the bacterial cell (lacking cell wall) by the help of
polyethylene glycol (PEG). The transfected cells are then selected by suitable
methods.

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Gene Transfer:

Method # 13. Virus Mediated Gene Transfer:

In other way the gene can be packed into a virus and allow it to infect the host
cell without harming it in any way. This method can be used both for the
transformation of prokaryotic host cell as well as transfection of eukaryotic
host cells. In the case of bacterial host cells the recombinant DNA can be
packed into the empty head of a specially designed bacteriophage (e.g., lambda
phage) and allow the virion to infect the host cell.

Similarly, while transfecting the plant host cells we can follow the similar
strategy by using plant viruses like Caulimo virus and Gemini virus. In the case
of animals, retrovirus infection of embryos has been used for the production of
transgenic mice.

This virus has been found to be an efficient vector system for animals. The
virus carrying the gene of interest transfers it into the genome of embryonic
cells leading to its integration and production of transgenic animals.

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