Transgensis and their Application

Introduction
As the worlds population begins to significantly expand, the urge arises to control the problem of limited resources - agriculturally, medicinally and industrially. Scientists have developed a quick-fix solution to address this urgent crisis. They have genetically engineered living organisms in order to benefit society in all spheres of life through a process referred to as transgenesis.

 Transgenesis is the deliberate act of transferring the genetic material from one organism to another. This foreign DNA is responsible for exhibiting a new function or synthesizing a new substance in the recipient host. In the early 1970s, the first genetically modified organism was produced. A gene from Salmonella was isolated and inserted into E.coli.

Transgenes
A gene cannot be simply transferred between animals in reality. It has to go through a number of procedures first before inserting it into the new host. The gene of interest obtained from the donor animal is isolated and purified. Next, the regulatory components are added to the gene.

 These components are genetically engineered to target specific sites in the host as well as to control the expression of the gene i.e. to enhance or depress certain characteristics by over- or underexpressing the gene. This transgene is inserted into a vector and transformed into bacteria to make many clones of this transgene.

METHODS OF CREATING A TRANSGENIC ANIMAL

Microinjection Method
Retroviral Vector Method

Embryonic Stem Cell Method

Sperm-mediated Transfer Gene Gun

Microinjection Method

Virgin mice are treated with hormones to synchronize their cycles so that super ovulation occurs (i.e. produce a higher number of eggs than normal). These eggs are harvested and fertilized in vitro.
Two pronuclei can be detected in the eggs 8 to 12 hours following fertilization, each containing genetic information from either the mother or father.

Microinjection Method
A microtube is used to hold the fertilized egg in place fine glass needle is used to inject a solution containing the transgene (200-300 copies) into the male pronuclei. Very few of the embryos survive this process, and of these only a few successfully incorperate the foreign DNA into their genome

 The pronuclei are then allowed to fuse naturally to form a diploid zygote which divides by mitosis to form a 2-cell embryo. This embryo is then transferred to the oviduct of a pseudopregnant mouse.

Retroviral Vector Method

The genes which code for proteins vital for viral functioning, especially with regards to its replication are removed. The removed genes are replaced with the transgene sequence which should be of similar size to those genes removed.

The new genome is replicated in vitro.

Oocyte or Zygote

Viral Particle containing Transgene

Helper cell lines are used to package the transgenic genome as viral particles.

The viral particles are then transduced into the zygote, or into an unfertilized oocyte.
This depends on the recipient species. In the case of the unfertilized oocyte, the oocyte is then fertilized in vitro The zygotes are then transplanted into the uterus of a pseudopregnant female.
Zygote Psuedopregnant

Embryonic Stem Cell Method

Donor female mouse from which the fertilized oocyst is removed. The embryonic stem cells are then removed from the inner cell wall of the mouse blastocyst (early mouse embryo). These embryonic stem cells are pluripotent (have the ability to become any cell. The embryonic stem cells are then plated onto culture plates, on to which a layer of feeder cells or leukemia inhibitory factor, that prevents the embryonic stem cells from becoming specialized, has been added. A specific gene of interest (i.e. specific DNA sequence) is added into the culture which transforms the embryonic stem cells by homologous recombination.

 The embryonic stem cells are then tested (Southern blotting), to determine if the cells are fully transformed (i.e. stem cells have taken up the DNA). The transformed embryonic cells (10-15) are removed from culture and injected into blastocysts (8-10) These blastocysts are implanted (in vitro) into the uterus of a pseudopregnant female mouse (uterus is receptive to the developing embryo). These embryos are left to develop within the uterus of the
female

Applications Of Transgenic Animals

Agricultural Applications
1. 2. Modification of milk produced by cows -Transgenic cows can be prompted to produce milk that contains the protein, alpha 1-antitrypsin, that is used in the treatment of diseases. Genetically modifying the growth and composition of livestock- By genetically modifying the growth of animals this can result in new alleles introduced into gene populations of these animals, promoting their success and survival. Cloning of animals-Cloning of white tigers. Increasing the reproductive performance-Transgenic animals can be produced, that produce large number of offspring that are healthy and stand a good chance of survival. Genetically modifying the fiber and wool produced by livestockTransgenic sheep, can produce large quantities of thick, full length, fine, black wool that can be removed with ease.

3. 4.

5.

Industrial Applications:
1. Goats that Produce Spider Silk-Spliced spider genes and inserted into the mammary glands of lactating goats (via microinjection techniques), such that, in addition to the production of milk these goats now secrete tiny silk strands. Testing of Products by Use of Transgenic AnimalsProduce transgenic animals on which to test product safety. Testing of cosmetics and shampoo. Transgenic animals and the environment- Production of genetically modified animals (transgenic animals) that are able to clean up chemical and oil spills.

2.

3.

Medical Applications
1. Models for Human Diseases and Treatments-Mice are used as models for Huntingtons disease to identify possible treatments for the disease.Sheep are used to study cystic fibrosis as resemble the human closely in terms of lung anatomy and physiology, and life span. 2. Reduction of Zoonoses from Livestock - Livestock can be made resistant to these diseases by transgenics, which would mean that they cannot transmit these diseases to humans. 3. Xenotransplantation-The demand for human organs for transplantation is far higher than the supply and the direct use of organs from other animals in humans is associated with severe immune responses and organ failure in the recipient. 4. Production of Therapeutic Human Proteins- 1-antitrypsin is produced by sheep to treat emphysema.