This presentation consists of introduction of animal transformation as well as the methods and applications.

1. Animal Transformation
GMOs, Biosafety and Bioethics
10th
November 2014
Presented by:
Bhagea Ritesh - 1210886
Cécile Christabelle - 1214458
Buctowar Rouksaar - 1242569
Ghoorbin Keshavi - 1216886
Nazeer Huda - 1214039

2. Contents
➔ Objectives of this presentation
➔ Browsing through history
➔ An introduction to transformation
➔ Methods of transfection
➔ Transgenic animals
➔ Transgenic animals: DNA microinjection
➔ The application of transgenic organisms in
agriculture
➔ Case study: The genetic transformation of
HeLa cells by Agrobacterium
➔ Conclusion
➔ References

3. Objectives of our Presentation
● To shed light on the subject of “Animal Transformation”

4. Browsing through History
+ 1928: First demonstration of transformation by Frederick Griffith.
○ He found that a strain of Streptococcus pneumoniae could be made
virulent when exposed to heat-killed virulent strains.
○ He then hypothesized the “transforming principle”.
+ 1944: Oswald Avery and colleagues discovered that this principle was
genetic.
○ After isolation of DNA from a virulent strain of S. pneumoniae and
inserting it into a harmless strain to successfully make it virulent, they
called the process “transformation”.
+ 1947 & 1953: After much dispute about the experiment of Avery et al., the
results were finally accepted.
○ The development of genetic markers and discovery of other methods
of transformation greatly helped to clear the doubts.

5. Browsing through History
+ 1970: Acknowledgement by Morton Mandel and Akiko Higa that
Escherichia coli may be used to take up DNA.
+ 1972: Stanley Cohen et al., showed that CaCl2
may also be used to
transform plasmid DNA.
+ Late 1980s: Development of the electroporation method for
transformation and invention of the Biolistic Particle Delivery
System (gene gun) by John Sanford.
+ 1982: First transgenic mouse created.
○ A gene for a rat growth hormone was inserted into a mouse
embryo.

6. An Introduction to Transformation
● Process whereby an exogenous DNA is directly taken up and
incorporated into a cell causing genetic alteration of that cell.
○ Exogenous DNA comes from the surrounding and
○ Is taken up through the cell membrane.
● Can occur naturally in some species of bacteria.
○ But can also be induced artificially in other cells.
○ Other means to introduce exogenous genetic material into
bacteria: conjugation and transduction.
● Concerning animal transformation, the term most often used is
“transfection”.
○ As “transformation” refers to the progression of animal cells
into a cancerous state.

7. Methods of Transfection
There are different methods of transfection. Each method has a
different approach to be considered, depending on cell type and
purpose.
The ideal method must have:
❏ high transfection efficiency,
❏ low cell toxicity,
❏ minimal effects on normal physiology,
❏ be easy to use,
❏ reproducible.

8. The methods are divided into 3 categories:
1. Chemical methods
- Calcium Phosphate
- Lipids
- Cationic polymer
2. Physical methods
- Electroporation
- Microinjection
- Laserfection
- Sonoporation
- Biolistic particle delivery
Methods of Transfection
3. Biological method
- Virus-based

9. Methods of Transfection
● Advantages:
1. Deliver nucleic acids to cells in a
culture dish with high efficiency
2. Easy to use, minimal steps required;
adaptable to high-throughput systems
3. Using a highly active lipid will reduce
the cost of lipid and nucleic acid, and
achieve effective results
● Disadvantage:
1. Not applicable to all cell types
1. Lipid-Mediated Gene Delivery
● Also referred as lipofection or liposome-based gene transfection.
● Mode: Uses lipids to cause a cell to absorb exogenous DNA.

10. Methods of Transfection
2. Electroporation
● Cell exposed to a high-intensity electric field
(destabilizes the mbr)
● Mbr is highly permeable to exogenous molecules
present in the surrounding media
● DNA moves into the cell through these holes
● When the field is turned off, the pores in the
membrane reseal, enclosing the DNA inside.
● Advantages:
1. Easy to perform
2. High efficiency
3. Don’t alter biological structure/ function of cells
4. Can be used for wide range of cell types
● Disadvantages:
1. Cell mortality (if using sub-optimal conditions)

11. Methods of Transfection
3. Viral-Based method
● Most commonly used method in clinical research
● Also known as transduction
● Example: Retrovirus murine leukemia virus (MLV)
● Mode:
● Establish sustainable transgene expression in humans.
● Integrates its DNA into the host genome which is expressed in
the host.
● The integrated MLV DNA replicates as the host genome does.
● Segregates into daughter cells, which enables sustainable
transgene expression (Kim and Eberwine, 2010).

12. Methods of Transfection
Viral-Based method (ctd)
● Advantages:
1. Very high gene delivery efficiency, 95–100%
2. Simplicity of infection
● Disadvantages:
1. Strong immune reactions against viral proteins
prohibit multiple administrations
2. Possibility of chromosomal insertion and proto-
oncogene Activation
3. Complicated synthesis process
4. Limitation on gene size
5. Toxicity, contamination of live virus

13. Transgenic Animals
● “Transgenic” is a genetically modified
organism with DNA from other source
inserted into its genome.
● To date, there are three methods for
producing transgenic animals:
1. DNA microinjection
2. Retrovirus-mediated gene
transfer
3. Embryonic stem-cell mediated
gene transfer

14. Transgenic Animals: DNA Microinjection
● Desired gene construct (single genes
or combination of genes recombined
and cloned) from another member of
the same/different species into the
pronucleus of the reproductive cell.
● Manipulated cell (first cultured in vitro)
to develop embryonic phase, is then
transferred to female recipient.
● First transgenic mammal Herman, the
bull (Lelystad, 16 Dec 1990).

15. The Application of Transgenic Organisms in
Agriculture
● Possibility of producing new strains or breeds of animals that carry new
beneficial, or improved genetic information.
● Examples of strains that have been developed:
○ Swine: leaner, more feed-efficient and faster-growing (have
additional copies of the growth hormone gene)
○ Mice: having the regulatory elements of the human
immunodeficiency virus (HIV) genome which are used as non-
infectious models for the study of AIDS

16. The Application of Transgenic Organisms in
Agriculture
● Knowledge gained in their studies is important in many fields
including:
○ cancer research; immunology; developmental biology; gene
expression and regulation; and models for human genetic
diseases such as muscular dystrophy, Lou Gehring's disease,
and sickle cell anemia.
● Potential applications for transgenic animals include:
○ manipulation of milk composition, growth, disease resistance,
reproductive performance, and production of pharmaceutical
products by livestock (known as pharming).

17. Case Study: Genetic transformation of HeLa
cells by Agrobacterium
● Transforming cells by Agrobacterium tumefaciens is a way to
transfer DNA.
● A. tumefaciens transfers oncogenes to host plant causing
tumours.
● A. tumefaciens needs a Ti-plasmid and a virulence region to
function.
● It can transform human cells by integrating its T-DNA into the
cellular genome.
● In this study, human HeLa R19 cells were used as host.
● A. tumefaciens C58C1 with Ti plasmid pGV3850 was used.

18. Case Study: Genetic transformation of HeLa
cells by Agrobacterium
● Pure cultures of HeLa cells were infected with A. tumefaciens to
produce transformants.
● chvA and chvB genes were important for binding to HeLa cells.
● These are normally required to bind bacteria to plant cells.
● Also, important genes: virA, virB, virG, virD and virA were also
very important for transformation.
● These make the plant-transforming protein machinery for
transformation of HeLa cells.
● Thus, it was proven that animal cells could be transformed by A.
tumefaciens.

19. Conclusion
● Transformation or transfection is one way of modifying
organisms for the betterment of humans.
● Many methods are present and some animals have already
been subjected to it.
● A lot of medical research could be based on application of
transfection on alarming diseases such as cancer, HIV, and
diabetes.
On the overall, transfection would be a valuable tool for the
development of humans.

20. References
● Mammalian and Plant Cell Culture http://web.mnstate.
edu/provost/biotech/Transfection%20and%20Infection%20of%
20Mammalian%20Cells%20Handout.pdf [ Date accessed: 5th Nov
2014]
● Transfection Methods Overview
http://www.bio-rad.com/webroot/web/pdf/lsr/literature/10-
0826_transfection_tutorial_interactive.pdf [ Date accessed: 5th Nov
2014]
● Kim, T.K. and Eberwine, J.H. (2010). Mammalian cell transfection: the
present and the future. Analytical and Bioanalytical Chemistry 397(8),
3173-3178.
● Kunik, T., Tzfira, T., Kapulnik, Y., Gafni, Y., Dingwall, C., and Citovsky,
V. (2001). Genetic transformation of HeLa cells by Agrobacterium.
Proceedings of the National Academy of Sciences 98(4), 1871–1876.

21. Thank you!