Passkey Providers and Enabling Portability: FIDO Paris Seminar.pptx
Transgenesis
1. Learning Intentions:
to be able to define transgenesis
to be able to give explanations about the biological ideas associated
with how transgenesis works
to be able to discuss the positive and negative implications of
transgenesis
2. What is transgenesis?
Definitions
• Transgenesis is the process of introducing an
exogenous gene – called a transgene – into a
living organism so that the organism will
exhibit a new property and transmit that
property to its offspring.
• A Transgene is the name given to the
introduced DNA
3. Why use transgenesis instead of
selective breeding?
• More specific — scientists can choose with greater
accuracy the trait they want to establish. The number of
additional unwanted traits can be kept to a minimum.
• Faster — establishing the trait takes only one generation
compared with the many generations often needed for
traditional selective breeding, where much is left to
chance.
• More flexible — traits that would otherwise be unavailable
in some animals or plants may be achievable using
transgenic methods.
• Less costly — much of the cost and labour involved in
administering feed supplements and chemical treatments
to animals and crops could be avoided.
4. Uses of transgenesis
• in toxicology: as responsive test animals (detection of toxicants);
• in mammalian developmental genetics;
• to introduce human genes into other organisms (particularly human) for the study
of disease processes;
• in molecular biology, the analysis of the regulation of gene expression;
• in the pharmaceutical industry, the production of human pharmaceuticals in farm
animals ("pharming"); targeted production of pharmaceutical proteins, drug
production and product efficacy testing;
• in biotechnology: as producers of specific proteins;
• genetically engineered hormones to increase milk yield, meat production; genetic
engineering of livestock in agriculture affecting modification of animal physiology
and/or anatomy; cloning procedures to reproduce specific blood lines;
• to speed up the introduction of existing characters into a strain/breed for
improvement and modification;
• developing animals specially created for use in xenografting, ie. modify the
antigenic make-up of animals so that their tissues and organs can be used in
transfusions and transplants.
6. Agriculture Transgenics On the Market (USA)
Insect resistant cotton – Bt toxin kills the
cotton boll worm
• transgene = Bt protein
Source: USDA
Insect resistant corn – Bt toxin kills the
European corn borer
• transgene = Bt protein
Normal Transgenic
7. Biotech chymosin; the enzyme used
to curdle milk products
• transgene = genetically engineered enzyme
Source: Chr. Hansen
bST; bovin somatotropin; used to increase
milk production
• transgene = genetically engineered enzyme
Source: Rent Mother Nature
8. Next Generation of Ag Biotech Products
• Golden Rice – increased
Vitamin A content (but not
without controversy)
• Turfgrass – herbicide
resistance; slower growing
(=reduced mowing)
• Bio Steel – spider silk
expressed in goats; used to
make soft-body bullet proof
vests (Nexia)
9. Products In The Pipeline
• Tomatoes enriched with flavonols • Oranges resistant
• Soybean and canola oils with to citrus canker
higher levels of vitamin E • Disease-resistant
• Vitamin-enriched rice sweet potatoes
• Decaffeinated coffee • Pest- and disease-
• Bananas to deliver a hepatitis resistant cassava
vaccine • Disease-resistant bananas
• Potatoes to protect
against cholera, E. coli and
Norwalk virus
• Apples to protect against
RSV
Benefits of biotechnology – Better food
10. Tracy (1990-1997): Transgenic
Ewe
• Genetically modified so
that her milk produced
a human protein called
alpha antitrypsin, a
potential treatment for
the disease cystic
fibrosis.
11. GTC Biotherapeutics
• Pharmaceutical product
derived from transgenic
goats modified to produce
therapeutic proteins in their
milk.
• The product, ATryn (an
antithrombrin) received
regulatory approval in the
EU in 2006 and in the U.S. in
2008.
14. Biological Processes
• First, the desired gene must be extracted from the
donor organism.
• This is done using restriction enzymes (restriction
endonucleases).
• It is important that the restriction enzymes cut out the
whole gene required.
• This gene can then be inserted into host cells (another
enzyme, DNA ligase, is very important here)
• http://www.abpischools.org.uk/res/coResourceImport/
modules/hormones/en-flash/geneticeng.cfm
17. Number of
Number of ova Number of
Animal species transgenic
injected offspring
offspring
rabbit 1907 218 (11.4%) 28 (1.5%)
sheep 1032 73 (7.1%) 1 (0.1%)
pig 2035 192 (9.4%) 20 (1.0%)
Microinjection is the most common method at
present and is generally more successful with
laboratory animals than farm animals.
The efficiency of microinjection is quite low:
Figures in parentheses are percent efficiency
compared to original number of ova injected.
(after Hammer et al., 1985)
21. Delivering the DNA into host organism:
plants
• Two major delivery methods
Agrobacterium a biological system based on
the plant pathogen
Agrobacterium tumefaciens
Gene Gun
a mechanical method where
the DNA is “shot” into plant
cells using a gene gun.
Tissue culture required to generate transgenic plants
22. Positive Implications of Transgenesis: Pharming
• Many valuable pharmaceutical products can now be made
using transgenic animals, a few examples below:
– factor VIII blood clotting factor
– fibrinogen blood clotting factor
– haemoglobin as a blood substitute
– human protein C anticoagulant
– alpha-1-antitrypsin (AAT) for treatment of AAT deficiency
– cystic fibrosis transmembrane conductance regulator (CFTR) for
treatment of CF
– insulin for diabetes treatment
– growth hormones for treatment of deficiencies
• These are used to treat human diseases and
defects, improving the quality and quantity of life for
afflicted individuals
examples taken from:
http://users.wmin.ac.uk/~redwayk/lectures/transgenic.htm
23. Positive Implications of Transgenesis: Agriculture Plants
• Improving plants
• Transgenic methods have now been developed for a
number of important crop plants such as rice, cotton,
soybean, oilseed rape and a variety of vegetable crops like
tomato, potato, cabbage and lettuce. New plant varieties
have been produced using bacterial or viral genes that
confer tolerance to insect or disease pests and allow plants
to tolerate herbicides, making the herbicide more selective
in its action against weeds and allowing farmers to use less
herbicide.
• Transgenic technologies are now being used to modify
other important characteristics of plants such as the
nutritional value of pasture crops or the oil quality of
oilseed plants like linseed or sunflower.
24. Positive Implications of Transgenesis: Agriculture
Animals
• Improving Animals
• The main aim in using transgenic technology in animal
agriculture is to improve livestock by altering their
biochemistry, their hormonal balance or their important
protein products. Scientists hope to produce animals that
are larger and leaner, grow faster and are more efficient at
using feed, more productive, or more resistant to disease.
Examples of transgenic breeding programs include:
• producing faster-growing and leaner pigs that use food
more efficiently and resist common diseases
• breeding transgenic sheep that grow better wool without
needing dietary supplements of sulphur-containing amino
acids.
25. Negative implications of transgenesis
GE technology carries certain inherent unpredictability
Some facts
Isolation of a gene from its natural environment and
integration into entirely different organism
Possible transgenic instability due to triggering of the
inbuilt defense mechanisms of the host organism
leading to inactivation or silencing of foreign genes.
26. • Possibilities of integration of foreign gene at a site
predisposed to silencing of genes (position effect).
– Variance in the levels of expression of the transgene in different
environmental conditions (heat, humidity, light…..)
– Possibilities of silencing of genes arising in subsequent generations
• Biosafety concerns arise from:
– Horizontal gene transfer
– Genetic contamination
– Transfer of allergens and toxins from one life form to another and
creation of new toxins and allergenic compounds
27. ..Biosafety issues in transgenic crops
- Concerns
Main
Development of aggressive weeds/ wild relatives by
transfer of transgenic traits
Erosion of land races/wild relatives by genetic
pollution in centres of origin/ diversity
Harm to the non-target organisms
Development of pest resistance by prolonged use
Monoculture and limitations to farmers’ choice in
crop management
Hazard to human and animal health by transfer of
toxins and allergens and by creation of new toxins
and allergenic compounds
28. ….GM foods: Allergenicity; Toxicity
Allergy
It is a hypersensitive reaction initiated by immunologic
mechanisms caused by specific substances called
allergens.
Toxicity
New proteins as a result of intended modification
Unintended new proteins as a result of the modification
Natural constituents beyond their level of normal
variation
29. ….GM foods: nutritional aspects;
unintended effects
Intended and unintended changes in nutrient levels
Bioavailability of nutrients, stability and processing
Presence and effect of anti-nutrients
Impact of individual changes on overall nutritional profile
Unintended effects
Random integration of transgenes
Insertional mutagenesis
Disruption of gene functions
Production of new proteins
Changes in
o Phenotype Metabolites
o Enzymes Toxins
o Genotype
32. The Golden Rice Story
• Vitamin A deficiency is a major health problem
• Causes blindness
• Influences severity of diarrhea, measles
• >100 million children suffer from the problem
• For many countries, the infrastructure doesn’t exist
to deliver vitamin pills
• Improved vitamin A content in widely consumed crops
an attractive alternative
Golden rice project: http://www.goldenrice.org/
33. -Carotene Pathway Problem in Plants
IPP
Geranylgeranyl diphosphate
Phytoene synthase
Phytoene
Problem: Phytoene desaturase
Rice lacks
these enzymes
ξ-carotene desaturase
Lycopene
Lycopene-beta-cyclase
Normal
Vitamin A -carotene
“Deficient” (vitamin A precursor)
Rice
34. The Golden Rice Solution
-Carotene Pathway Genes Added
IPP
Geranylgeranyl diphosphate
Daffodil gene Phytoene synthase
Phytoene
Vitamin A
Phytoene desaturase
Pathway Single bacterial gene;
is complete performs both functions
ξ-carotene desaturase
and functional
Lycopene
Daffodil gene Lycopene-beta-cyclase
Golden -carotene
Rice (vitamin A precursor)
35. Biological implications
1. Ecosystems: the implanted gene could spread to other plants in the
environment, this could have unforeseen side effects
2. Genetic biodiversity: increases biodiversity as introduces rice plants with
novel genes
3. Health or survival of individuals: In remote rural areas Golden Rice could
constitute a major contribution towards sustainable vitamin A delivery,
therefore increase the survival chances of individuals. GR may cause
unforeseen health risks, particularly if it is the first GMO to be widely
consumed by children.
4. Survival of populations: and hence increase survival chance of human
populations living in vitamin deficient/poor areas. However, farmers who
wish to sell it in markets may not want to take the risks of adopting a new
variety (e.g., lower yield, susceptibility to pests and diseases) unless they
are compensated with higher prices or yields. However, such higher prices
would work against its incorporation into the diets of the poor, possibly
causing it to wind up as a niche product for rich consumers.
5. Evolution of populations…
36. • Current breeding and field trialling work is
being carried out by the International Rice
Research Institute (IRRI) in the Philippines
together with PhilRice, the Philippine Rice
Research Institute. PhilRice is preparing a
submission to the regulatory authority of the
Philippines in 2013, which could lead to initial
releases to farmers in 2014.
Editor's Notes
This method involves the direct microinjection of a chosen gene construct (a single gene or a combination of genes) from another member of the same species or from a different species, into the pronucleus of a fertilized ovum. It is one of the first methods that proved to be effective in mammals (Gordon and Ruddle, 1981) which are the most difficult of all cells to genetically manipulate. The introduced DNA may lead to the over- or under-expression of certain genes or to the expression of genes entirely new to the animal species. The DNA construct (usually about 100 to 200 copies in 2 pl of buffer) is introduced by microinjection through a fine glass needle into the male pronucleus - the nucleus provided by the sperm before fusion with the nucleus of the egg. The diameter of the egg is 70 µm and that of the glass needle is 0.75 µm; the experimenter performs the manipulations with a binocular microscope at a magnification of 200 x. The insertion of DNA is, however, a random process, and there is a high probability that the introduced gene will not insert itself into a site on the host DNA that will permit its expression. The manipulated fertilized ovum is transferred into the oviduct of a recipient female, or foster mother that has been induced to act as a recipient by mating with a vasectomized male.
2. Embryonic stem cell-mediated gene transferThis method involves prior insertion of the desired DNA sequence by homologous recombination into an in vitro culture of embryonic stem (ES) cells. Stem cells are undifferentiated cells that have the potential to differentiate into any type of cell (somatic and germ cells) and therefore to give rise to a complete organism. These cells are then incorporated into an embryo at the blastocyst stage of development. The result is a chimeric animal. ES cell-mediated gene transfer is the method of choice for gene inactivation, the so-called knock-out method.This technique is of particular importance for the study of the genetic control of developmental processes. This technique works particularly well in mice. It has the advantage of allowing precise targeting of defined mutations in the gene via homologous recombination.
2. Embryonic stem cell-mediated gene transferThis method involves prior insertion of the desired DNA sequence by homologous recombination into an in vitro culture of embryonic stem (ES) cells. Stem cells are undifferentiated cells that have the potential to differentiate into any type of cell (somatic and germ cells) and therefore to give rise to a complete organism. These cells are then incorporated into an embryo at the blastocyst stage of development. The result is a chimeric animal. ES cell-mediated gene transfer is the method of choice for gene inactivation, the so-called knock-out method.This technique is of particular importance for the study of the genetic control of developmental processes. This technique works particularly well in mice. It has the advantage of allowing precise targeting of defined mutations in the gene via homologous recombination.
3. Retrovirus-mediated gene transferTo increase the probability of expression, gene transfer is mediated by means of a carrier or vector, generally a virus or a plasmid. Retroviruses are commonly used as vectors to transfer genetic material into the cell, taking advantage of their ability to infect host cells in this way. Offspring derived from this method are chimeric, i.e., not all cells carry the retrovirus. Transmission of the transgene is possible only if the retrovirus integrates into some of the germ cells.For any of these techniques the success rate in terms of live birth of animals containing the transgene is extremely low. Providing that the genetic manipulation does not lead to abortion, the result is a first generation (F1) of animals that need to be tested for the expression of the transgene. Depending on the technique used, the F1 generation may result in chimeras. When the transgene has integrated into the germ cells, the so-called germ line chimeras are then inbred for 10 to 20 generations until homozygous transgenic animals are obtained and the transgene is present in every cell. At this stage embryos carrying the transgene can be frozen and stored for subsequent implantation.There is also fusion of host cells with membranous vesicles (eg. liposomes) containing DNA.(Plant cells can be modified using, eg. tobacco mosaic virus or the Ti plasmid of Agrobacterium tumefaciens.)
