The document discusses animal transformation, particularly focusing on the historical and contemporary methods of both transfection and the creation of transgenic animals. It outlines various transfection methods, their applications in agriculture, and the significance of transgenic organisms in medical research. A case study on the genetic transformation of HeLa cells using Agrobacterium is also provided, concluding with the potential benefits of transformation for human advancement.

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!