Genetics

1. Transgenic animals and animal
cloning
Mohd bin Mahmud @ Mansor, FIAT

2. Introduction
95% of all transgenic animals are mice.
5% are rats, rabbits, goat, pigs, sheep, cows and
fish.
Among other species, why mice?

3. Introduction
From mice, researchers now trying similar
procedures on livestocks - as bioreactor
Four routes to create transgenic mammals will be
discussed
The creation of therapeutic proteins

4. Transgenic Animals

5. Creation of transgenic
animals
1. Microinjection of DNA
2. Integration of (retro) viral vector into an oocyte
3. Incorporation of genetically pluripotent stem
cells into early embryo
4. Transfer of genetically altered nuclei into
enucleated oocytes

6. Microinjection of DNA
First reported in 1985
Most transgenic animals were created this way

7. Retrovirus vectors
Replication defective retroviral vectors
Replication competent retroviral vectors
Incubating bovine oocytes in the final stage of
maturation increase rate of transgenesis
Cloning capacity is limited (<10kb

8. Pluripotent stem cells
Capability to developed into many type of cells
Can be maintained in tissue culture
Genetically manipulated and selected
Highly successful in mice, but not so much in
other organisms

9. Nuclear transfer technology
Somatic cloning
Transfer of donor nucleus into cytoplasm of an
enucleated zygote or oocyte
Achieved in cattle, goats, pigs, rabbits, mulls,
horses, cats, dogs and some wild life species

10. "Pharming" and human proteins
1970s, DNA manipulation provided a significant alternative source for
many drugs made of protein
• Human growth hormone: human cadaver
• Insulin: slaughtered pigs
Using DNA technology, insulin can be harvested from recombinant
bacteria
Overexpression of human genes in bacteria not always yield a
functionally active protein
The protein need to be post-translational modified - phosphorylated
or glycosylated to become active

11. Transgenic in sheep, goats,
cows and pigs
The idea of expressing recombinant proteins into
milk started in 1989
Since then until 2002, 25 types of human
therapeutic have been produced in transgenic
animals
Biopharm animals
Why we use animals as bioreactors rather than
bacteria?

12. Transgenic in sheep, goats,
cows and pigs
Example:
• Blood clotting factors (VIII and IX)
• Anti thrombin III - intravascular coagulation
• Collagen - burns and bone fractures
• Fibrinogen - burns and after surgery
• Human fertility hormones
• Human serum albumin
• Human hemoglobin
• Lactoferrin (found in mother milk)
• Tissue plasminogen activator

13. Transgenic in sheep, goats,
cows and pigs
Mostly are using sheep, goats, cows or pigs
Regulated by FDA
Enormous cost involved in production of a drug in
livestock
• US$ 800 million
• Around 15 years
• 7 years to generate transgenic livestock only

15. Transgenic in sheep, goats,
cows and pigs
Amount of blood clotting factor IX needed every
year is 2kg/year
Assuming animals produce 1g of protein per liter
and purification efficiency is 30%, then a pig will
produce 100g, sheep 125g, and a cow 3kg.
So we only need one cow per country to have
enough supply of blood clotting factor IX

16. Transgenic in sheep, goats,
cows and pigs
Cost estimating to produce one transgenic cow is
around US$ 300,000 to US$500,00
There are also costs of failed experiments

17. Transgenic Chicken
Laying eggs containing human protein in egg
whites
Basic strategy:
target expression of a therapeutic protein to a
protein secretory tissue (oviduct of a laying
hens) using a regulatory sequences of one of
the native proteins (ovalbumin) synthesized in
that tissues.

18. Transgenic sheep for wool
Other than production of human proteins
Transgenic sheep with better wool
Diet of high sulphur-containing amino acids
Sheep in cysteine - sulphur rich that is an essential
building block for keratin
Mammal cannot produce their own cysteine - from
gut dwelling bacteria that help them in digestion

19. Transgenic fish
Atlantic salmon expressing growth hormone
Approved for consumption
4-6 times the growth rate
10 - 20% improvement in feed conversion
efficiency
Shorter production time, reduced costs, improve
profitability

20. Xenotransplantation
"Procedure that involves the transplantation,
implantation, or infusion into a human recipient
of either (a) live cells, tissues or organs from
nonhuman animal source or (b) human body
fluids, cells, tissues, or organs that have had ex
vivo contact with live nonhuman animal cells,
tissues or organs"

21. Xenotransplantation
Transgenic pigs
• Provide organs (heart, kidneys) suitable for
human transplantation
• Gene that encodes a human cell-surface protein
• Prevent components of human immune system
from attacking and destroying the organs

22. Animal cloning
The first cloned mammal - Dolly
Cloned from a single adult, somatic cells taken from her mother's
udder
Unfertilized egg cell with the nucleus removed
Fused with the cell from udder
The fused cell then made to divide and develop into a normal embryo
Since then, goats, mice, pigs,cats, rabbits, mules and horses.
Will human be next?

23. Animal cloning
In principle it is possible
But actually the cloning efficiency is extremely low
with only less than 4% embryos developing to live
offspring.
Offspring obtained from cloning also reported to
experience early death or severe abnormalities
Dolly was suffering from arthritis and died
because of lung disease

24. Other stories
Polly - human blood clotting factor IX
Transgenic pigs - rich with omega 3

25. Creating transgenic
animals

26. Designing the
gene construct
Sourcing the
transgene
Make a transgenic
embryo
Select +ve
transgene cells
Making the gene
construct
Transfecting
bovine cells
PCR
Confirming the
cow is transgenic
Analyzing of protein
expressed
qPCR
Fluorescence in
situ hybridization

27. 1. Designing a construct
Overall function
Align it all logically
Promoter, gene of interest, poly A tail
Cell or tissue specific promoter

29. 1. Designing a construct
Promoter
Regulating the spatial and temporal expression pattern of a
transgene
Sequences are isolated from upstream regions of endogenous
mammalian genes
Possibility of being tissue specific and developmental stage
specific
Inducible promoter
• Giving inducible expression

30. 1. Designing a construct
Intron
Leads to significantly greater transgene
expression
Effects on mRNA stabilization
Efficient translocation from nucleus to cytoplasm
Examples: rabbit beta-globin intron or simian
virus 40 (SV40)

31. 1. Designing a construct
Protein coding sequence
Full length cDNA derived from RNA of a gene of interest
Contains a translational start codon (ATG) and
translational stop codon
• So ribosome can properly scan and recognize the
proper translation start and stop sites of mRNA
Kozak sequence upstream of a start codon

32. 1. Designing a construct
Poly A
Promotes translation by ribosomes
Protects the mRNA from nucleases

33. 1. Designing a construct
Enhancer
Transcription factor recruitment
Release of RNA polymerase II

34. 2. Sourcing the transgene
Provide by external institution
Come up within a vector
Together with restriction site
Or synthetically constructed
Given that you have the....

35. 3. Making the gene construct
Just sticking the gene into an expression vector
Vector backbone, opened by RE
And gene of interest, cut with same RE
Mix together all mixtures
Incubation period may vary

36. 4. Transfecting bovine cells
Introduction of DNA into a cell
Cells growing in the dish
Using some methods - chemically or
physiologically
Some example?

37. 5. Select positive bovine cells
How do we do screening?

38. 6. Make a transgenic embryo
Isolate egg from donor
Remove the DNA
Fuse the transgenic cells
Incubated in the lab ~ 7 days
Transfer into surrogate mother

39. 7 Confirming the cow is
.
transgenic
Southern blotting
• Low false positive rate
• Many information can be obtained from the blot
• PCR may be used for large sample (screen positive) but must be
followed by SB atleast once
• PCR is best for offspring
qPCR
Fluorescence in situ hybridization
Protein analysis

40. Questions?
Thank you.