1. Transgenic Animals and Plants
Genetic Engineering of plant -> Transgenic plants
Genetic Engineering of animals -> Transgenic animals

2. Definition of Transgenic
Transgenic -> stable introduction of a gene into another organism
-> For Unicellular organisms (such as bacteria or yeast)
all transformed cells are -> transgenic
-> For multicellular organisms (such as animals, plants,..)
difference between:
- manipulation of single cells -> cell line
(expression in insect cells or mammalian cells)
- manipulation of a whole plant or animal -> transgenic
(can have a transgenic offspring!!!)
-> more difficult and expensive to create whole modified organism (transgenic) than just cell line!!!

3. Transgenic versus Cloning
Transgenic -> creation of transgenic animal or plant (introduction of foreign gene into organism)
-> transgenic organisms produced by introduction of foreign gene into germ line
(-> transgenic offspring!!!)
-> introduction of gene into somatic cells -> gene therapy
Cloning -> obtaining an organism that is genetically identical to the original organism
-> such as Dolly the sheep
-> asexual propagation of plants (taking cuttings)

4. Transgenic Plants Why do we need transgenic plants ?
improvement of agricultural value of plant (resistance to herbicides, resistance to insect attack -> Bacillus thuringiensis toxin)
living bioreactor -> produce specific proteins
studying action of genes during development or other biological processes (knock-out plants, expression down-regulated)

5. Transgenic Plants Advantages:
Plant cells are totipotent -> whole plant can be regenerated from a single cell (engineered cells -> engineered plants)
Plants have many offspring -> rare combinations and mutations can be found
Transposons used as vectors
Disadvantages:
Large genomes (polypoid -> presence of many genomes in one cell)
plants regenerating from single cells are not genetically homogenous (genetically instable)

7. Gene – transfer methods

8. Agrobacterium tumefaciens mediated transfer

9. Ti Plasmid

10. Integration of T-DNA into the plant chromosome
-> Tumor formation

11. Gene transfer by cointegration
Recombinant Ti plasmid by recombination

12. Microprojectile bombardment – “Shotgun”

13. Viral Vectors

14. Transfer into protoplasts
Vector + polyethylene glycol
Gene transfer across a protoplast membrane is promoted by some chemicals such as polyethylene glycol

15. Electroporation

16. Control elements on vector
Frequently used promoter: -> 35S promoter from cauliflower mosaic virus

17. Alterations in plant RNA – downregulation of specific genes
PG (polygalacturonase) -> Sensitivity of tomatoes to bruising
Reduced level-> should give harder fruit during shipping
Result: lower level -> did not give harder fruit (more factors responsible for process)
Expression of Antisense RNA of transcript of PG -> reduces level of protein produced

18. Selection marker free transgenic plant -> Transposons

19. Applications for engineering plants
Development of Insect-, pathogen-, herbicide- resistant plants
Flower pigmentation
Modification of nutritional content
Modification of taste and appearance
Bioreactor
Vaccines (Cholera toxin-like protein in potatoes)
Plant yield (alteration of lignin content -> paper industry)

20. Development of Insect-, pathogen-, herbicide- resistant plants
Toxin from Bacillus thuringiensis

21. Development of Insect-, pathogen-, herbicide- resistant plants

22. Development of Insect-, pathogen-, herbicide- resistant plants
Manipulations that make a plant herbicide resistance
Inhibit the uptake of the herbicide
overproduce the herbicide-sensitive target protein (Glyphosate)
reduce ability of target protein to bind herbicide (cyclohexanediones)
plant can degrade herbicide (Bromoxynil, Glufosinate, Cyanamide,..)

23. Development of Insect-, pathogen-, herbicide- resistant plants
Fungus- and Bacterium- resistant plants
Engineering of plants -> express antimicrobial peptides

24. Flower pigmentation
CHS -> Chalone synthetase -> enzyme in biosynthetic pathway of a purple pigment

25. Changed nutrition content
Amino acids (to increase lysine content in the future in animal food)
Lipids (possible to change degree of unsaturation, chain length)
Vitamins (Vitamin E, increase Vitamin A in rice)

26. Modification of taste and appearance
Engineer potatoes -> produce more glucose and fructose at higher temperatures

27. Plants as bioreactor
Therapeutic agents
Antibodies
polymers (PHB)

28. Transgenic Animals
Transgene -> Gene coding for a growth hormone

29. Transgenic Animals Why do we need transgenic animals ?
living bioreactor -> produce specific proteins in the milk (cattle, sheep, goats, pigs)
studying action of genes during development or other biological processes (knock-out animals, expression down-regulated) -> models for studying human diseases -> mice
improvement of agricultural value (fish, bird)

30. Gene-transfer methods
Microinjection
Retroviral method
Engineered Embryonic Stem Cells (ES) method
Knock – out methods (Cre-LoxP system) -> studying gene expression + development

31. The first days of an embryo
Used for retroviral infection
Fertilized egg
Embryonic stem cells (ES)

32. Microinjection into the germ line -> transgenic animal
Gene injected into the male pronuclei

33. Efficiency of the transgenesis process after DNA microinjection

34. Retroviral vectors into the germ line (8-cell embryo infected) -> transgenic animal

35. Engineered Embryonic Stem Cells (ES) into the germ line (blastocyst) -> transgenic animal
Engineered ES -> can form any kind of cell in an embryo
Inner cell mass (ICM) of blastocysts can form all cells of the embryo -> Pluripotent
-> Embryonic stem cells

36. Gene Therapy – Viral gene transfer into somatic cells
Gene transfer into somatic stem cells -> gene therapy
Gene transfer via Virus
Target tissues: Bone marrow, liver, brain,....

37. Gene Therapy – Viral gene transfer into somatic cells
Gene transfer into somatic stem cells -> gene therapy
Used for treating -> genetic diseases, such as diabetes, cancer, color blindness…
Different delivery methods

38. Gene Transfer - what happens on DNA level
Integration into chromosome -> Recombinantion
Recombinantion can be -> homologous – non-homologous
non-homologous event -> more frequently
homologous event -> less frequent but desired
Knock-out mutants -> disrupt functional gene by integration of another gene into target gene
Used for:
-> study human diseases by creating model organisms
-> make minus mutant

39. Homologous recombinantion

40. How do check for homologous recombinantion

41. Construction of a disruption construct

45. Cre-LoxP system:
Inactivation of a gene (knock-out) in a specific cell type
Activation of a transgene in specific cell type
Used for:
Study biological consequences of tissue- specific gene inactivation
-> establishing models for human diseases
-> selective removal of kinesin II gene (expressed in retinal receptor cells)
-> leads to accumulation of opsin and arrestin -> cell death
-> result mimics aspects of a disease (inherited retinis pigmentosa)
-> large deletions in chromosome -> deletion in chr. 22 -> DiGeorge syndrome
(cardiovascular dysfunction)

46. Inactivation of gene in specific cell type (tissue)

47. Cloning of Dolly – Cloning Animals by Nuclear Transfer Technology
Critical for success:
Cell cycle of the somatic cells (udder cells) on plates was critical – they were kept in specific growth stage (diploid stage)
Of the 434 fused oocytes created during the experiment -> only Dolly survived to adulthood
Dolly was real clone (genotype identical) and could reproduce
Dolly was euthanized > suffering from progressive lung disease
Since > cloning of sheep, cows, mice, cats, other animals done
-> many of the clones developed severe diseases as they matured.
Until 1997, arrival of Dolly – not possible to produce an adult animal from a nucleus from an adult animal´s differentiated cell

48. Cloning of Mammals – Reproductive Cloning
Genotype identical
Phenotype is not necessarily identical -> variation due to random events and due to environment

49. Why do clones have health problems?
Telomeres are found at the end of each chromosome.
Shrinking of the telomeric ends of our chromosomes are a sign of aging of the cell.
Each cycle of cell division the telomeres are slightly shortened until they are too short for further replication -> cell death
Dolly´s telomeres (at the age of 3) have been as short as ones of the age of 6 -> clones age “faster”.

50. Why do clones have health problems?
Differentiated cells have certain methylation pattern.
Cloned animals have abnormal methylation pattern originating from nucleus from differentiated cells
Some can be “re-set” (epigenetic reprogramming) to their undifferentiated state, some cannot -> faulty gene activation in cloned animal
-> so few cloned embryos survive
-> surviving clones have severe health problems

51. Transgenic Cattle, Sheep, Goat, Pigs
Production of pharmaceutical proteins -> drugs
Problems:
Highly inefficient
Only 20% of the eggs survive and only 5% of them produce product

52. Transgenic Cattle, Sheep, Goat, Pigs
Protein production: in milk, blood, urin
Animals (pigs) with modification of sugars on surface of organs
-> donor for organ transplants

53. Transgenic Cattle, Sheep, Goat, Pigs

54. Transgenic birds and fish
-> improvement of agricultural value
Transgenic chicken:
Resistant to viral, bacterial diseases
better feeding efficiency (fast growth, better meat quality, more meat
less fat meat, less cholesterol in eggs
maybe use of eggs as bioreactors for protein production
Transgenic fish: -> to support aquaculture
Increase growth rate (growth hormone)
resistance to diseases
Generation of model systems to monitor health hazard
(screening chemicals if they cause mutations)