Gene transfer, methods of gene transfer, Viral and non Viral methods and application

1. GENE TRANSFER TECHNOLOGY
IN ANIMALS
VIRALAND NON-VIRAL METHOD
PRESENTED BY,
RAHUL KUMAR GOSWAMI

2. INTRODUCTION
Gene transfer is to transfer a gene and DNA segments
from one organism to another.
This area of genetic manipulation makes
important contributions to domesticated animals
in relation to immunology, vaccines, aging, and
cancer.
Gene transfer represents a relatively new possibility
for the treatment of rare genetic disorders and
common multifactorial diseases by changing the
expression of a person's genes.

3. Gene transfer method in animals
There are four major strategies for gene transfer to
animal cells, two of which are considered :-
biological mechanisms using virus (transduction)
Virus (transduction); the transferred gene
represents part of the viral genome.
or bacterial that invade animal cells (bactofection)
Bacteria (bactofection); the gene will be
transferred as a plasmid.
Biochemical and physical methods which do not
involve infection thus termed transfection.

4. The gene transfer results can be transient and
stable transfection.
Transient transfection
In transient transfection, the transfected DNA is not
integrated into host chromosome. DNA is transferred
into a recipient cell in order to obtain a temporary but
high level of expression of the target gene.
Stable transfection
Stable transfection is also called permanent
transfection. By the stable transfection, the transferred
DNA is integrated (inserted) into chromosomal DNA
and the genetics of recipient cells is permanent
changed.

5. Non viral methods (Bio chemical and Physical methods)
Biochemical methods
Calcium phosphate 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 at the time of
transfection.
The DNA escapes and reaches the nucleus and can be then
expressed.
Since the cells must be coated by the calcium complex,
monolayers of cells must be used for maximum efficiency.
However, this method gives only 1-2% transfection
efficiency.

6. DEAE-dextran mediated (diethylaminoethyloethyl-dextran)
There are three points that DEAE-dextran mediated transfection
differs from calcium phosphate coprecipitation.
(1) It is used for transient transfection.
(2) (2) It works more efficiently with cell lines of BSC-1, CV-1 and
COS, etc.
(3) (3) It is more sensitive.
protocols :-
(1) Harvest exponentially growing cells by trypsinization and transfer
then into 60-mm tissue culture dished at a density of 105 cells/dish.
(2) Add 5 ml complete growth medium.
(3) Incubate 24 hours at 37oC with 5% CO2.
(4) Prepare DNA/DEAE-dextran/TBS-D solution by mixing 2 mg of
superoiled plasmid DNA into 1 mg/ml DEAE-dextran in TBS-D.
(5) Remove medium and wash tree times with PBS and twice with
TBS-D.

7. DEAE-dextran mediated cont.
(6) Add DNA/DEAE-dextran/TBS-D solution 250 ml.
(7) Incubate 60 min at 37oC with 5% CO2.
(8) Remove DNA/DEAE-dextran/TBS-D solution.
(9) Wash with TBS-D three time and PBS twice.
(10) Add 5 ml medium supplemented with serum and chloroquine (0.1
mM).
(11) Incubate 4 hours at 37oC with 5% CO2.
(12) Remove medium.
(13)Wash with serum-free medium three times.
(14) Add to cells 5 ml of medium supplement with serum, and
incubate 48 hours at 37oC with 5% CO2.
(15) Harvest the cells after the 48 hours transfer-
ction.
(16) Analyze RNA or DNA by hybridization, or analyze expressed
protein by radiommunoassay, immunoblotting, immuniprecipitation,
or by enxzy moatic activity in cell extract.

8. Lipid mediated method
This method can be used for both transient and stable
transfection, and it can be used for adherent cells,
primary cell lines, and suspension cultures.
For the following protocol, the Lipofectamine
Reagent from Invitrogen Corporation will be used.
Lipofectamine Reagent is a 3:1 (w/w)
Liposome formulation of the plycationic lipid 2,3-
dioleyloxy-N-[2(sperminecardox-ido)ethyl]-N,N-
dimethyl-1-propanaminium trifluoro-acetate
(DOSPA) and the neutral lipid dioleoyl
phosphatidylethanolamine (DOPE) in membrane-
filtered water.

9. Lipid mediated method protocol :-
1) Put about 40,000 cells per well of a 24-well plate in 0.5 ml of the
appropriate complete growth medium (add 10% serum if it needs).
(2) Incubate the cells at 37oC in a CO2 incubator until the cells are 50-
80% confluent (about 20 hours, depending on the cells).
(3) Dilute 3 mg DNA into 25 ml medium without serum for each well
and mix.
(4) Dilute 3 ml Lipofectamine Reagent into 25 ml medium without
serum for each well and mix.
(5) Combine diluted DNA (Step 3) and Lipofect-amine Reagent (Step
4) and incubate at room temperature for 30 min. In this step the DNA-
liposome complexes are formed.
(6) Replace the medium in the cells with 0.2 ml transfection medium
without serum.

10. Lipid mediated method cont.
(7) Add 0.15 ml medium without serum to the tube containing the
complexes for each well.
(8) Incubate the cells with the complexes for about 10 hours at 37oC in a
CO2 incubator. The incubating time will be flexible by the cell type.
(9) Add 0.4 ml growth medium containing double the 2× normal
concentration of the serum without removing the transfection mixture.
(10) Replace the medium with fresh, complete medium at 20 hours
following the start of transfection if continued cell growth is required.
(11) Assay cell extracts for transient gene expression at 24-72 hours after
transfection, depending on the cell type and promoter activity.
(12) To obtain stable transfectants, passage the cells 1:10 into the selective
medium after 72 hours of transfection for the reporter gene transfected.

11. Physical method of gene transfer or transfection in
animal (Non viral methods)
In this method, naked DNA is deposited directly
into the cell by exploiting a physical force.
This includes
Microinjection
particle bombardment
ultrasound
electroporation
Which ever method used, the result is called
transformation which is a change in the
recipient cell’s genome caused by the acquired
transgene.

12. Microinjection
Direct transfer of DNA into the cell without a carrier is called DNA
microinjectin. This can only be done for only few cells at a time.
This technique is used mainly for large cells such as oocytes, eggs and the
cells of early embryos. The DNA can be directly injected into tissues, such
as skin, muscle or internal organs or it can be injected into the blood.

13. Particle bombardment or Gene gun (Biolistics)
Some cells, tissues and intracellular organelles are impermeable to foreign
DNA
To resolve this problem in gene transfer, the gene gun was made by Klein
at Cornell University in 1987.
The gene gun is part of the gene transfer method called the biolistic (also
known as biobalistic or particle bombardment) method.
In this method, DNA or RNA adhere to biological inert particles (such as
gold or tungsten).
By this method, DNA-particle complex is put on the top location of target
tissue in a vacuum condition and accelerated by powerful shot to the
tissue, then DNA will be effectively introduce into the target cells.
Uncoated metal particles could also be shot through a solution containing
DNA surrounding the cell thus picking up the genetic material and
proceeding into the living cells.
The efficiency of the gene gun transfer could be depended on the
following factors: cell type, cell growth condition, culture medium, gene
gun ammunition type, gene gun settings and the experimental experiences,
etc.

14. Particle bombardment or Gene gun (Biolistics) cont.

15. Ultrasound
Involves the exposure of cells to a rapidly
oscillating probe such as the tip of sonicator.
In this method the application of ultrasound
waves to a dish or cells or a particular tissue
results in the formation and collapse of bubbles in
the liquid, including the cell membrane, a process
known as cavitation.
The transient appearance of such cavities allows
the DNA to cross the membrane into the
cytoplasm. This method can be used both for in
vivo or in vitro as the plasmid DNA is left
structurally intact.

16. Electroporation
is a physical transfection technique involves creating transient
nano-meter size pores in the cell membrane by exposing cells to
a brief pulse of electricity. The most critical parameter is the
intensity and duration of electrical pulse.
Steps of the electroporation transfection:
(1) Harvest cells in the mid- to late-logarithmic phase of
growth.
(2) Centrifuge at 500 g (2000 rpm) for 5 min at 4oC.
(3) Resuspend cells in growth medium at concen-
tration of 1 X 107 cells/ml.
(4) Add 20 mg plasmid DNA in 40 ml cells.
(5) Electric transfect by 2.1 kV / 25uF for 2.6 milliseconds.
(6) Transfer the electroporated cells to culture dish and culture
the cells.
(7) Assay DNA, RNA or protein and continuously culture the
cells to get positive cell lines.

17. Electroporation cont.

18. Biological mechanisms virus mediated gene transfer in
animals
Virus particles have a natural ability to adsorb to the surface of
the cells and gain entry. This can be exploited to deliver
recombinant DNA into animal cells.
Several classes of viruses has been used for gene transfer and
at least 8 has been used in clinical trials.
Transgenes may be incorporated into viral vectors either by
addition to the whole genome or by replacing one or more viral
genes. This can be done by ligation or by homologous
recombination.
If the transgene replaces a none essential gene the vector is
described as helper-independent
If it replaces an indispensable gene, then this vector will be
helper dependent.
It is generally recommended to use vectors from which all viral
coding sequences has been deleted such vectors are described
as fully deleted or gutted or gutless vectors.
These vectors contain just the cis-acting elements required for
packaging and for genome replication.
High capacity for foreign DNAbecause no viral gene products
are made, the vector has no intrinsic cytotoxic effects.

19. virus mediated gene transfer in animals cont.
Adeno virus
These are viruses with a linear, double stranded genome, of
approximately 36 kb. These are used frequently due to certain
advantages;
Stability
high capacity for foreign DNA
wide host range including none-dividing cells
Ability to produce high titer stocks up to 1011 pfu/ml

20. Virus mediated gene transfer cont.

21. Adeno-associate virus (AAV)
These viruses are not genetically related to adenovirus but is so-
called because it was first discovered as a contaminant in an
adenovirus isolate.
The AAV is a single stranded DNA and is a member of the
parvovirus family.
It is naturally replicating deficient, thus it requires the presence
of another virus such as adenovirus to complete its infection
cycle. AAV replicates lytically and produces thousands of
progeny virions.
The dependence of AAV on a heterologous helper virus such as
adeno virus provides an unusual degree of control over vector
replication making AAV one of the safest vectors to use for
gene therapy.
Other advantages of this viral vector is the wide host range that
it exhibits including none dividing cells.

22. Adeno-associate virus (AAV) cont.

23. Method
The AAV genome is small (5 kb) and
comprises a central region containing rep (replicase)
and cap (capsid) genes flanked by 145 kb inverted
terminal repeats.
Foreign DNA replaces the cap region and gets
expressed by indigenous promoter. However rep
proteins might interfere with the expression process
thus responsible for some of the cytotoxic effects of
the virus.
Then virus is bind to host cell and use host cell
machinary and divide their viral genome.
And the viral genome codes the protein.

24. Baculovirus
Baculovirus promote high level of transgene
expression in insect cells but can also infect
mammalian cells.
Have rode shape capsid and large double-stranded
DNA genomes. They productively infect arthropods
particularly insects.
BV vectors are used mainly for high level transient
protein expression in insects and insect cells.

25. Retrovirus-Mediated Gene Transfer
The most useful vectors for the purpose of gene isolation are those that
lend themselves to the production of libraries consisting of
overlapping fragments of genomic DNA, ideally encompassing the
entire genome several times.
This can best be done at 4-16 cell stage embryos.
However, it can be done up to midgestation, but with
incomplete infections i.e low infectivity rate.
Immediately following infection, the retrovirus produces a
DNA copy of its RNA genome using its reverse
transcriptase.
Completion of this process requires that the host cell
undergoes the S phase of the cell cycle. Therefore,
retroviruses effectively transduce only mitotically active
cells.
Modifications to the retrovirus frequently consist of
removal of structural genes, such as gag, pol, and env,
which support viral particle formation.

26. Retrovirus-Mediated Gene Transfer cont.
Additionally, most retroviruses and complementary lines
are ecotropic in that they infect only rodents, such as rats
and mice, and rodent cell lines rather than humans.
The DNA copy of the viral genome, or provirus, integrates
randomly into the host cell genome, usually without
deletions or rearrangements.
Because integration is not by way of homologous
recombination, this method is not used effectively for site-
directed mutagenesis.
Very high rates of gene transfer are achieved with the use
of retroviruses.

27. Retrovirus-Mediated Gene Transfer cont.