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.