Download - Transgenic animals
Transgenic animals and animal cloning
Mohd bin Mahmud @ Mansor, FIAT
Introduction
95% of all transgenic animals are mice.
5% are rats, rabbits, goat, pigs, sheep, cows and fish.
Among other species, why mice?
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
Transgenic Animals
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
Microinjection of DNAFirst reported in 1985
Most transgenic animals were created this way
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
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
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
"Pharming" and human proteins1970s, 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
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?
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
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
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
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
Transgenic in sheep, goats, cows and pigs
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.
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
Transgenic sheep for wool
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
Transgenic fish
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"
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
Xenotransplantation
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?
Animal cloningIn 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
Other stories
Polly - human blood clotting factor IX
Transgenic pigs - rich with omega 3
Creating transgenic animals
Designing the gene construct
Sourcing the transgene
Make a transgenic embryo
Select +ve transgene cells
Transfecting bovine cells
Making the gene construct
PCR
Analyzing of protein expressed
Fluorescence in situ hybridization
qPCR
Confirming the cow is transgenic
Overall function
Align it all logically
Promoter, gene of interest, poly A tail
Cell or tissue specific promoter
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
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)
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
1. Designing a construct
Poly A
Promotes translation by ribosomes
Protects the mRNA from nucleases
1. Designing a construct
Enhancer
Transcription factor recruitment
Release of RNA polymerase II
1. Designing a construct
Provide by external institution
Come up within a vector
Together with restriction site
Or synthetically constructed
Given that you have the....
2. Sourcing the transgene
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
4. Transfecting bovine cells
Introduction of DNA into a cell
Cells growing in the dish
Using some methods - chemically or physiologically
Some example?
5. Select positive bovine cells
How do we do screening?
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
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
Questions?Thank you.