Animal Biotechnology

Animal Biotech Animals provide a number of products we use in every day

life: Milk Leather Wool Eggs Meat

Animal Biotechnology Transgenics

Characterizing genetic variability

Reproductive technologies

Artificial insemination (AI) and preservation of semen

Improving health through developing vaccines

Diagnostics and epidemiology

Nutrition and feed utilization

Transgenics

Transgenics are genetically modified organisms with DNA from another source inserted into their genome

A large number of transgenic animals have been created

Mice Cows Pigs Sheep Goats Fish Frogs Insects

Currently, no transgenic animal or animal product is approved by the FDA or USDA for human consumption

Goals of transgenic animal creation

Improve livestock animals

Genetic modification of animals may be aimed at improving their milk, meet,

wool etc production.

Use of animals as bioreactors

Genes are transferred into the animals to obtain a large scale production of

proteins encoded by these genes in milk, urine or blood of such animals; such

animals are called bioreactors and this method is called molecular farming or

gene farming.

Research into animal and human disease

A special case of gene transfer is alleviating or eliminating the symptoms

and consequent miseries of genetic diseases.

Specific transgenic animals strains or lines are created to fulfill specialized

experimental and/or biomedical needs.

Eg. Konck out mice strains in which specific genes have been replaced

or knocked out by their disrupted counterparts through a process of

homologous recombination.

Transgenic Animal Creation

Making a transgene1. Structural gene which selected as per requirement

2. Vectors; are carriers used to transfer the gene of interest from one organism to another. Eg. SV40 vectors, bovine papilloma vectors, retrovirus vectors, bacculovirus vectors

3. Requirements of promoter/ enhancer seq in the transgene so that it can be expressed in the host cells.

- A transgene must be integrated into the host genome for obtaining transgenic cells/animals.

- Transgene must be present in proper orientation in relation to and in association with various sequences required for its efficient transcription and translation in host cell.

- A transgene must be integrated into the host genome for obtaining transgenic

cells/animals.

- Transgene must be present in proper orientation in relation to and in association

with various sequences required for its efficient transcription ans translation in host

cell.

The sequences may be listed as follow;

An efficient promotors/ enhancer

The translation initiation codon (AUG in mRNA, and ATG in DNA)

The chain termination codon

Transcription termination sequence

Polyadenylation cleavage/ addition site

Gene Constructs

Gene Construct

Schematic representation of a gene construct for expression in animal/ plant cell.

P/E ATG TAG Poly- (A)

Gene

Construction of Transgenic Animal

Isolation of transgene

Integration of gene into the genome

Insertion into vector

Development of organism

Mosaic organisms Non transgenic organisms

Fully transgenic organisms

Introduction into cell

Creation of Transgenic Animals by Nuclear Injection

In vitro fertilization is used to start a transgenic animal.

Harvested eggs and sperm are fertilized, and before the pronuclei fuse, the transgene is injected into the male pronucleus.

The embryo continues to divide in culture and is then implanted into a mouse.

The “foster mother” mouse has been treated with hormones so that she accepts the embryo and carries on with the pregnancy.

The offspring are screened for stable integration of the transgene.

Founder mice have one copy of the transgene.

Gene injected into the male pronuclei

Detection of Transgenics and Transgene Function

Dot Blot technique

PCR amplification using genomic DNAs extracted from mice

Detection of mRNA Expression

Assays for protein Expression

Large Transgenic Mice Express the rat somatotropin gene

Under the control of the promoter from an unrelated mouse gene, metallothionein , which is normally expressed in the liver

Instead of being made in the pituitary gland, the normal site for growth hormone, the rat somatotropin is manufactured in liver

Human somatotropin has also been expressed in mice and also gives bigger mice.

Dwarfism

Recombinant human somatotropin (rHST) is now used to treat the hormone-deficient type of dwarf.

Large Transgenic Mice

Express the rat somatotropin gene

Under the control of the promoter from an unrelated mouse gene, metallothionein , which is normally expressed in the liver

Instead of being made in the pituitary gland, the normal site for growth hormone, the rat somatotropin is manufactured in liver

Human somatotropin has also been expressed in mice and also gives bigger mice.

Trendy Transgenic Mice

Marathon Mouse

Can run about 1800 meters—more than a mile—before exhaustion. This is twice as far as a normal mouse can last.

Marathon mouse has enhanced PPAR-delta—a regulator of several genes involved in burning fat and in muscle development.

Mighty Mouse

Lack myostatin, a protein that slows muscle growth. The result is colossal muscle development.

There are one-two known cases of a human with a genetic defect leading to lack of myostatin. A German boy, born in Berlin in 2000, has muscles twice the size of other children his age.

Recombinant protein production The somatotropin gene from Cow

Can be expressed in bacteria - the production of large amounts of the hormone, rBST.

Used in the dairy industry to increase milk production.

Used to boost adult cow’s somatotropin, resulting in increased milk production.

If cloned in Cow?

Production of heterologous proteins

Recombinant proteins can be produced in the milk from transgenic cows or other farm animals.

The cloned genes are placed under the control of a regulatory region that will allow gene expression only in the mammary gland.

For small-scale production, transgenic goats are often used.

E.g. transgenic goats producing rTPA

Knockout Mice The target gene is cloned and

disrupted by inserting a DNA cassette.

This work is usually done in bacteria.

Once the construct is made, it is put back into a mouse by injection into the male pronucleus during fertilization.

After the transgenic offspring are born, two heterozygotes are crossed to create a homozygous knockout mouse.

These are then screened for defects due to inactivation of the target gene.