1. Fundamentals of Biotechnology Transgenic Animals and their Application By Haji Akbar

2. Introduction As the world’s 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.

3. 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. Using the results from this experiment as the foundation, together with the advancements in biotechnology, scientists are now able to solve the problem of these resource shortages by using transgenic animals.

4. 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.

5. 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.

6. Various M ETHODS OF C REATING A T RANSGENIC A NIMAL Microinjection Method Retroviral Vector Method Embryonic Stem Cell Method Sperm-mediated Transfer Gene Gun etc

7. 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.

8. Continue!!! A microtube is used to hold the fertilized egg in place while an extremely 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

9. 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.

10. Retroviral Vector Method The genes which code for proteins vital for viral functioning, especially with regards to its replication are removed. The cis ends of the genome are left as these are important in replication of the transgenic genome. 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. Viral Particle containing Transgene Oocyte or Zygote

11. Packaging or ‘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 ♀

12. 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 in turn, transforms the embryonic stem cells. by homologous recombination.

13. 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) which were 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

14. Creation of Pseudopregnant Mother For mice: These are produced by mating a female mouse with a sterile (neutered) male. This elicits the hormonal changes needed to make her uterus receptive to the implanted embryos. The chimeric offspring thus produced are tested to determine whether any are transgenic. This is done by removing some tissue, usually from the tail of the offspring, isolating the DNA and analyzing it to determine the presence or absence of the transgene.

15. Continue!!! These are then mated with a non-transgenic mouse to produce heterozygous offspring, which are then mated to produce mice that are homozygous for the transgene. Continuous mating of such homozygous Individuals will ensure the establishment of the desired transgenic trait. X X Pregnant ♀ ChimeraHeterozygous Offspring Homozygous Offspring

16. Applications Of Transgenic Animals

17. Agricultural Applications 1. Modification of milk produced by cows Problems: The amount of milk that is produced by cows is not adequate. In addition, the milk lacks nutrients that are essential for humans and other animals. Solution: Recombinant DNA technology, promotes beneficial changes in the composition of milk. Examples: Transgenic cows can be prompted to produce milk that contains the protein, alpha-1-antitrypsin, that is used in the treatment of lung diseases (e.g. emphysema or cystic fibrosis).

18. 2. Genetically modifying the growth and composition of livestock Problems: The lack of vitamins, minerals and large amounts of fat present in these livestock. Solution: Genetically modify and manipulate the genes that are responsible for growth in livestock. In addition, essential fats such as, omega-3-fatty acids, can be expressed by livestock (example sheep or fish) when genetically modified. Example: By genetically modifying the growth of animals this can result in new alleles introduced into gene populations of these animals, thereby, promoting their success and survival.

19. 3.Cloning of animals Problems: a dramatic decrease in species richness and abundance of many species. And only a fraction of a particular species have a desirable trait that is beneficial (i.e. profit) to the farmer. Solutions: 1) Genetically modify (i.e. produce transgenic animal), such that, that animal expresses the desired trait (e.g. wool or meat). 2) Clone species that are endangered, get these species to propagate and place them in a sanctuary. Example: Cloning of white tigers

20. 4. Increasing the reproductive performance and prolificacy Problems: The mating of animals does not always result in the production of offspring, and the offspring produced can be deformed. Solution: Transgenic animals can be produced, that produce large number of offspring that are healthy and stand a good chance of survival. By genetically modifying the parents, with a specific gene of interest that has a desirable trait, offspring produced is guaranteed to express that trait. Moreover, by genetically modifying the parents this would in turn increase the reproductive performance of the offspring.

21. 5. Genetically modifying the fiber and wool produced by livestock Problems: Livestock produce too little wool or that is of poor quality. Solutions: Produce transgenic animals, whereby, the quality, color, yield and ease in shearing of wool (sheep) and fiber (goat) can be expressed by these animals; which in turn, is beneficial to the farmer and textile industries. In addition, these animals can be further modified to yield a specific length, fineness and crimp of wool and fiber. Examples: Transgenic sheep, can produce large quantities of thick, full length, fine, black wool that can be removed with ease.

23. Industrial Applications: 1. Goats that Produce Spider Silk Problem: Silk is required in textile industries, production of sporting equipment, production of medical micro- sutures and even in the police force for the production of bullet proof jackets. Solution: In 2001, Nexia biotechnologies in Canada 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. These strands are extracted from the milk and weaved into thread. Furthermore, scientists can manipulate the structure, quality and type of thread produced by these goats

25. 2. Testing of Products by Use of Transgenic Animals Problem: Humans and animals alike need hygiene and cosmetic products that are safe to use. Solution: Produce transgenic animals on which to test product safety. Example: Testing of cosmetics and shampoo.

26. 3.Transgenic animals and the environment Problem: Due to human requirements important but toxic materials are transported across countries. However, during transportation accidents occur (i.e. spillages at sea or land) which is detrimental to the animals that occupy those habitats (e.g. spillage at sea endangers marine life) Solution: Production of genetically modified animals (transgenic animals) that are able to clean up chemical and oil spills. Example: Transgenic microorganism produce proteins, that expresses enzymes which are able to clean up chemical spills

27. Medical Applications 1. Models for Human Diseases and Treatments Transgenic animals can be used to model human diseases, especially genetic diseases. Possible treatment regimes can then be tested on these ‘models’ Examples: Mice are used as models for Huntington’s 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.

28. 2.Reduction of Zoonoses from Livestock Problem: Many livestock species have diseases that can be passed on to humans. Solution: Livestock can be made resistant to these diseases by transgenics, which would mean that they cannot transmit these diseases to humans. Example: Creutzfeld-Jacob disease (Mad Cow disease) is caused by bovine spongiform encephalopathy (BSE)being transmitted to humans from cattle. BSE affects proteins coded from Prp gene, so research into whether cattle without a functioning version of this gene are resistant to BSE, is in progress at the moment.

29. 3.Xenotransplantation Problem: 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. Solution: The use of pigs which have similar physiology and anatomy to humans. The α1,3 galactose gene in pigs is knocked out so that α1,3 galactose is not produced as this has been shown to be antigenic.

30. 4.Production of Therapeutic Human Proteins Problem: Some of the proteins used by the human body cannot be produced by bacteria or yeasts. Solution: Production (pharming) of these proteins by transgenic animals such as mammals and chickens, which perform similar post translational modifications to that of humans. The proteins can then be recovered from the milk, eggs, semen or blood from the animal. Examples: α 1-antitrypsin is produced by sheep to treat emphysema. α-glucosidase is produced by rabbits to treat Pompes disease