Animal TransformationGMOs, Biosafety and Bioethics

10th November 2014

Presented by:Bhagea Ritesh - 1210886

Cécile Christabelle - 1214458Buctowar Rouksaar - 1242569Ghoorbin Keshavi - 1216886

Nazeer Huda - 1214039

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

Objectives of our Presentation

● To shed light on the subject of “Animal Transformation”

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.

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.

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.

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.

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

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.

Methods of Transfection2. 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 perform2. High efficiency3. Don’t alter biological structure/ function of cells4. Can be used for wide range of cell types

● Disadvantages:1. Cell mortality (if using sub-optimal conditions)

Methods of Transfection3. 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).

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

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

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).

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

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).

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.

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.

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.

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 Overviewhttp://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.

Thank you!