ANIMAL

TRANSFORMATION

Prepared by: Angelica P. Maderse, MSc

 HISTORY
• 1928: Frederick Griffith demonstrated the first transformation.
– Found that a strain of Streptococcus pneumoniae could
be virulent when exposed to heat-killed virulent strains
– Hypothesized “transforming principle”
• 1944: Avery Oswald and colleagues discovered that this
process is genetic
– Isolation and insertion of DNA from virulent strain of
Streptococcus pneumoniae into harmless strain called
the process transformation
• 1947 & 1953: The results of Avery et al. were accepted
– Development of genetic markers and discovery of other
methods of transformation greatly helped to clear doubts
 HISTORY
• 1970: Morton Mandel and Akiko Higa
acknowledge the use of E. coli to be used to
take up DNA
• 1972: Stanley Cohen et al., showed that CaCl2
may used to transform plasmid DNA
• 1980’s: Electroporation was developed as a
method of transformation and invention of
Biolistic Particle Delivery System (gene gun) by
John Sanford
• 1982: First transgenic mouse created
 What is Animal Transformation?

• Process where a foreign DNA is introduced and

incorporated/inserted into the cell of living organisms
(animal cell) causing genetic alteration of that cell
– A natural form of genetic exchange/ gene transfer
– In bacterial genetics, this process was described the
uptake of naked plasmid or genomic DNA (essentially
any DNA which had the potential to transform the
phenotype of the recipient cell)
– Concerning animal transformation, the term most often
used is “transfection”
• As “transformation” refers to the progression of
animal cells into cancerous state
 Process of Transfection
• was used specifically to describe the uptake of naked
phage DNA (or RNA), i.e. nucleic acid which had the
potential to initiate a phage replication cycle.
• Two types of Transfection:
a. Transient- transfected DNA not integrated to host
chromosome (create temporary expression)
b. Stable- transferred DNA is integrated (inserted) into
host chromosomal DNA and genetics of recipient
cells is permanent changed/ expression
• Different methods of transfection have different approach
in delivering/inserting foreign DNA
– Ideal method must be high transfection efficiency, low
cell toxicity, minimal effects on normal physiology, be
easy to use and reproducible.
 Methods of Transfection
• Divided into 3 categories:
1. Physical methods
– Electroporation 3. Virus-based method
– Micro injection
– Laser injection
– Biolistic particle delivery
2. Chemical methods
– Calcium phosphate
– Cationic lipids
– DEAE-Dextran
– Cationic polymer
 Methods of Transfection
• Chemical Method
– used naked DNA which was mixed with particular
chemicals to form synthetic complexes which either
interact with the cell membrane and promote uptake by
endocytosis, or fuse with the membrane and deliver the
DNA directly into the cytoplasm.
• Physical method
– involve breaching the cell membrane and introducing
the nucleic acid directly into the cell or nucleus.
• Virus-based method
– deliver their nucleic acid cargo to the nucleus as part of
the infection cycle, often after interaction with cell
surface receptors and either internalization within
endosomes or direct fusion with the plasma
membrane.
Physical Method: Electroporation

• Was first used to transfect mouse lyoma

cells by Neumann and colleagues in 1982
• Allows transfection of cells following their
exposure to a pulse electric field
– Cause a number of nanometer-sized
pores to open in the plasma membrane
for up to 30 minutes allowing uptake of
free DNA from surrounding medium
– When field is turned off, pores close
spontaneously, enclosing the DNA
inside
 Electroporation
Advantages Disadvantages
– High efficiency – Requires specialized
– Highly reproducible capacitor discharge
– Don’t alter the biological equipment (controlling
structure of cells pulse length and
voltage)
– Can be used in different
cell lines – High level of cell death
– Copy number of
transgene can be at
least partially controlled
 Microinjection
• provides direct nuclear delivery of DNA avoiding the
endogenous pathway and also ensures that the DNA is
delivered intact.
• Uses: in introduction of DNA into oocytes, eggs,
embryos of animals either for transient expression
analysis or to generate transgenic animals
• This method was used for transforming cells that were
resistant to any other method of transfection
• Stable transfection efficiencies are extremely high, in the
order of 20%, and very small quantities of DNA are
sufficient.
 Biolistic particle delivery
• Known as or micro-projectile transfection
• delivery of nucleic acids into host cells via high-velocity
nucleic acids coated micro particles
• Involves projecting microscopic heavy metal particles
(gold/tungsten) coated with nucleic acids into host
recipient cells at high velocity using a biolistic device (i.e.
“gene gun”)
• Used to transiently transfect dividing/non dividing cells in
culture and in vivo
• Use in transforming of maize and generating transgenic
cereal plants
• Use in vaccination
 Biolistic Particle delivery
Advantages Disadvantages
• Simple and rapid • Generally have lower
technique efficiency
• Use small amount of DNA • Requires preparation of
• Can delivery large micro-particles
amounts of DNA • Instrument cost
fragments
• Requires little
manipulations of cells
• High reproducibility
 Laser injection
• Use laser light to transiently permeabilize a large number
of cells in very short time
• Various substances (ions, small molecules (siRNAs,
plasmid, protein) can be efficiently laser injected into
numerous cell types
• When the laser induces a pore in the membrane, the
osmotic difference between the medium and cytosol
facilitates the uptake of nucleic acid into the cell
 Calcium Phosphate
• Was first chemical transfection method used in animal cells.
• Used in transient and stable transformation
• Technique where DNA in buffered phosphate solution is mixed
gently with calcium chloride that causes the formation of a fine
DNA-calcium phosphate co-precipitate which settles onto
the cells and some are taken up by endocytosis
• The DNA escapes and reaches the nucleus and can be
expressed
• Procedure was developed in 1973 by Graham and van der
Erb for introduction of adenovirus DNA in rat cells
 Calcium Phosphate
Advantages Disadvantages
• Easily available • Low efficiency
• Inexpensive • Not suited for in vivo
• Can be applied to wide gene transfer to whole
range of cell types animals
• Can be used for transient • Size and quality of
and stable transfection precipitate are crucial to
• CP appears to provide success of transfection
protection against
intracellular and serum • Toxicity especially to
nucleases primary cells
 DEAE- DEXTRAN
• Was first non-viral transfection method verified by
Vaheri and Pagano in 1965
• A soluble polycationic carbohydrate that tightly
associates with negatively charges of nucleic acids
(electrostatic interactions) and promote uptake by
endocytosis
• Aggregates formed are small compared to particles
formed during calcium phosphate transfection
 DEAE-DEXTRAN
Advantages Disadvantages
• Inexpensive • High concentration of
• Easy to perform and DEAE-Dextran can be
quick toxic to the cell
• Can be applied to wide • Transfection efficiency
range of cell types varies with cell types
• Can be used only for
transient transfection
• Produced less than 10%
delivery in primary cells
 Cationic Lipid
• Electrostatic interactions between positive charge of
cationic lipid head groups and negatively charged
phosphates of DNA backbone are main forces that allow
DNA to spontaneously associate with cationic lipids
• It is mixed with neutral lipid (L-
dioleoylphosphatidylethanolamine (DOPE) which can
enhance the gene transfer ability of certain synthretic
lipids
• When liposome encounter nucleic acids, they form
complexes called “lipoplexes” that can be actively
taken up by host cell through endocytosis
• Its successful depend on the factors (lipid
formulation, particle size and method of
preparation)
• An overall net positive charge of complex allow
association of complex with negatively charge of
cell membrane
 Cationic Polymer
• Differ from cationic lipid in that they don’t contain a
hydrophobic moeity and are completely soluble in water
• Includes polybrene, polyethyleneimine (PEI) and
dendrimers
• Ability of cationic polymer more efficiently condense with
DNA
• Cationic polymer cant release their DNA load into
cytoplasm
 Virus-mediated Transfection
• Most commonly used method in clinical research
• Known as transduction - transfer of exogenous nucleic acid into
animal cells as part of a recombinant viral particle
– Transgenes may incorporated into viral vectors by addition to
whole genome or by replacing one or more viral genes (done by
ligation or homologous recombination)

• Enveloped virus- deliver nucleic acid by binding to specific

receptors on the cell surface and then by either fusing directly with
the plasma membrane or, following uptake by endocytosis, by fusing
to the endosomal membrane
– Ex. Herpes virus, Retro virus (murine leukemia virus (MLV)

• Non-enveloped viruses- penetrate or disrupt the plasma or

endosomal membranes with specific virion proteins.
– Ex. adenovirus
 Retrovirus-Mediated Gene Transfer
• Earliest method for
successful gene transfer in
mammals
• Virus gene is replicated with
transgene gene
• Method was successfully
used in 1974 when a simian
virus was inserted into mice
embryos resulting in mice
carrying this DNA
 Virus-Mediated Transfection
Advantages Disadvantages
• Strong immune reactions
• Very high gene delivery against viral proteins
efficiency (95-200%) prohibit multiple
administrations
• Simplicity of infection
• Possibility of chromosomal
insertion and proto-
oncogene activation
• Complicated synthesis
process
• Toxicity, contamination of
live virus
 Application of Animal
Transformation
• Gene therapy
• Production of transgenic animals