This document discusses various methods of gene transfer, including viral and non-viral methods. Viral methods use recombinant viruses like retroviruses and adenoviruses to insert genes into host cells. Non-viral methods include physical techniques like electroporation and gene guns that force DNA into cells, as well as chemical methods using lipids, polymers, or proteins to transport DNA into cells. Both methods have advantages and disadvantages related to efficiency, safety, and target specificity.
ENGLISH 7_Q4_LESSON 2_ Employing a Variety of Strategies for Effective Interp...
lecture 3 - gene transfer.pptx
1. Gene transfer
Deliberate insertion of genes into cells
Way of conferring transgenes to host cells in protein
production
Means of carrying out gene therapy
Cell membrane does not allow DNA to pass through
2. Methods of gene transfer
Vector/viral mediated transfer: involve use of
recombinant viruses to insert genes ito host cells
Non viral/Direct gene transfer :divided into two
broad categories--physical and chemical
Physical methods involve taking plasmids and
forcing them into cells,electroporation, sonoporation,
or particle bombardment.
Chemical methods use lipids, polymers, or proteins
that will complex with DNA, condensing it into
particles and directing it to the cells
3. advantages
Non viral Viral
Allows delivery of large genes
Inexpensive and easy to
manufacture
Rarely cause immmune
responses
Target specific
High transformation
efficiency
Can infect cells that are not
dividing
4. Direct methods
Chemical methods
Particle gun
Electroporation
Microinjection
Lipofection
Magnetofection
6. Viral mediated
deliver their genes to human cells in a pathogenic
manner.
Retroviruses - A class of viruses that can create
double-stranded DNA copies of their RNA genomes.
Adenoviruses - A class of viruses with double-
stranded DNA genomes that cause respiratory,
intestinal, and eye infections in humans.
Adeno-associated viruses
Herpes simplex viruses
7. Viral mediated
DNA of interest is inserted into the viral DNA
Host cell is infected with the constructed virus
These particles are consisting of a DNA or RNA
genome contained within a protein coat.
They infect host cells incorporate viral DNA into the
host genome, replicating as part of the host (lysogenic
cycle), or multiply inside the host cell before releasing
phage particles by budding from the membrane or
actively lysing the cell (lytic cycle)
Target specific
10. Chemical methods
rely on cationic carriers to complex with negatively
charged nucleic acids for their uptake by cells
dilution/preparation of the carrier
formation of DNA/carrier complexes
addition to cells, and subsequent removal of the non-
endocytosed excess complexes.
The first-generation transfection reagents included
calcium phosphate and diethylamino ethanol (DEAE)–
dextran
11. Chemical methods
Prepare solutions. Mix calcium chloride (in phosphate
buffer) and DNA.
Allow precipitation of extremely small, insoluble particles
containing condensed DNA.
Add complexes to cell culture and incubate. Complexes
adhere to cell membranes, and enter into the cytoplasm by
endocytosis.
DEAE-Dextran and polybrene: Positive-charged polymers
complex with negatively charged DNA molecules to enable
binding to the cell surface.
Remove complexes from cells, wash, and add fresh culture
medium.
Assay for gene expression.
12. Disadvantages/advantages
Simple and inexpensive
low cost and reproducible
Low transfection effieciency
Insufficient protection of transgenes
Sometimes toxic to the cells
13. liposome
is an artificially-prepared vesicle composed of a lipid bilayer. The
liposome can be used as a vehicle for administration of nutrients and
drugs
cationic carrier molecules complex with DNA and neutralise its
electrostatic charge promoting cell-membrane–DNA interaction
A liposome encapsulates a region of aqueous solution inside a
hydrophobic membrane
To deliver the molecules to sites of action, the lipid bilayer fuse with
other bilayers, cell membrane, thus delivering the liposome contents.
By making liposomes in a solution of DNA or drugs (which would
normally be unable to diffuse through the membrane) they can be
delivered past the lipid bilayer
Complexes formed by selfassembly of DNA with liposomes are
generically known as lipoplexes
Lipoplexes are internalised by endocytosis although fusion with the cell
membrane has also been proposed
17. lipoplexes
Once inside the cell, fusion with lysosomes can occur
destroying the insert(no gene expression)
If the endosome is not destroyed DNA trafficking to
the nucleus is made possible and expression occurs
lipoplexes often show high levels of transgene
expression
their nonspecific membrane activity usually precludes
cell-selective targeting
poor efficiency at transfecting nonproliferating cells
21. Particle gun
Gene gun, biolistics, particle bombardment
a device for injecting cells with genetic information
original method utilised a gunpowder acceleration system
to propel DNA-coated tungsten particles at recipient cell
Penetration of the cell membrane result in intracellular
expression of genes encoded by the exogenous DN
helium driven have been developed, in which a helium
pulse is used to accelerate DNA-coated gold microparticles
through the cell membrane
Has a higher transformation efficiency than most nonviral
22. Gene Gun
Advantages:
◦ Can be used in any cell type
Disadvantages:
◦ Damage of a significant number of cells
◦ Reproducibility problems
23. microinjection
mechanical process in which a needle roughly 0.5 to 5
micrometers in diameter penetrates the cell
membrane and/or the nuclear envelope.
desired contents are then injected into the desired
sub-cellular compartment and the needle is removed
Consists of a low power disecting microscope and two
micromanipulators
One is a glass micropipette to hold the cell by partial
suction
The other a glass injection tube to inject DNA into the
cell
29. Polyplexes
They are complexes of cationic polymers with nucleic
acids
Ployplex formation is regulated by electrostatic
interaction, which is affected by:
pH of the media
Ionic strength
Cationic density
32. Polyplexes
Advantages:
◦ Easy to formulate
◦ Simple chemical modification
Disadvantages:
◦ Cytotoxicity
◦ Stability problems
33. Retroviral Vectors
RNA viruses can carry out efficient gene transfer into many cell
types and can stably integrate into the host cell genome
Replace viral genes with therapeutic gene
– Limited size (<8 kb)
– Specific cellular receptors
High efficiency
Stable integration into genome
– Potential for insertional mutagenesis
Most are non pathogenic except, human immunodeficiency
viruses (HIV) and human T-cell lymphotropic viruses (HTLV)
murine leukemia virus (MULV)
vectors themselves have all of the viral genes removed, can
accept up to about 8 kilobases (kb) of exogenous DNA.
34.
35. Vectors based on DNA viruses
Adenoviral vectors. have several positive attributes: they
are large and can potentially hold large DNA inserts (up to
35 kb, see below);
they are human viruses and are able to transduce a large
number of different human cell types at very high
efficiency
they can transduce non-dividing cells; and they can
produced at very high titres in culture. They have been the
vector of choice cells for several laboratories trying to treat
the pulmonary complications of cystic fibrosis, as well as
for a variety of protocols attempting to treat cancer.
36. Problems
• Delivery of DNA
• Achieving high level expression
• Maintaining stable expression
• Tissue-specific expression
• in vivo regulation
37.
38. setbacks
Production in humans is very low, success of GT
reported mainly in mice and canines
Viruses are immunogenic
Mainly requires rapidly dividing hepatocytes in the
liver,hence success greater in neonates