Presented by :- Neha M.Sc biotech III sem

 What is transgenic animal?  How are they made?  Microinjection method  Blastocyst method  Retrovirus method  Why are they being made?  Some examples of transgenic animals  Risk associated with them  Conclusion  References

 A transgenic animal is animal whose chromosomes have been changed to carry the genes of other organisms.

 There are three main ways to produce transgenic animals: microinjection, blastocyst injection, and the help of a retrovirus.

 This involves injecting a trangene, the gene used to create a transgenic animal, into a fertilized egg.  There is little ability to determine where the transgenic is located and whether it will survive.  Because of this, most eggs do not survive or do not have the transgene, but between 1% and 30% of the eggs injected can produce a live transgenic animal.

 This involves placing the transgene into an embryonic stem cell or ES cell.  ES cells are cells that are able to develop into all types of tissues.  The cells are injected into blastocysts, which is an early stage of development. The injected ES cells become part of the animal and the animal becomes a mixture of transgenic and original cells.

Institute of Laboratory Animal Science University of Zurich Production of transgenic mice by ES cell gene transfer

 A retrovirus is a virus that can insert itself into a host’s chromosomes.  This virus is inserted into the cells.  The animals that contain the virus must breed to produce a transgenic animal.

Pronuclear microinjection Lentivral infection ES based transgenesis pros Relatively simple and efficient Long transgenes possible Potentially all species Very efficient Single copy insertions No technical equipment Works in many species Long transgenes possible Gene targeting possible Single copy insertions cons Random integration Multicopy insertions ( Strain limitations) High embryo mortality 9.5 kb packaging limit Safety issues (?) Only random integration Technically difficult Time consuming Species / Strain limitations

 Transgenic animals are being made to help economic traits, be used as disease models, and help in medical research.

 Scientists created cattle to produce milk containing certain human proteins which may help in the treatment of emphysema.  It also helps in breeding. Farmers can produce transgenic animals that have the needed traits for doing a specific job.

 Scientists are creating animals with specific human diseases and cancers, so that they can study the affects it has on the body. They hope this will help them find a cure for the different diseases and cancers.

HIV normally will only cause disease in humans and chimpanzees. By knocking out a mouse’s immune system and using human stem cells to create a human immune system, HIV mice are susceptible to AIDS.

 Tracy has been genetically engineered by PPL Therapeutics to produce alpha1-antitrypsin (AAT), useful for treating emphysema.  Human lacking ATT enzyme suffer progressive lung degeneration (emphysema) unless treated with 200g ATT (derived from human blood) per year.  Tracy, has secreted 1.5 kg ATT per lactation.

 Every year people die because of lack of a replacement heart, liver, or kidney.  Transgenic pigs may be able to supply these organs.  Research is trying to find a way to remove a pig protein and replace it with a human protein.

 In 1984, Baby Fae received the heart of a baboon and survived for three weeks.  Genetic modifications can make the donor more compatible with the recipient, thus reducing the chance of rejection by the recipient’s immune system.

 The Beltsville hogs had a human gene for growth hormone inserted into their genome.  The gene resulted in an increased pace of growth, arthritis, blindness, and general suffering. The experiment was stopped early and the hogs were euthanized.

 Bioreactors whose cells have been engineered to synthesize marketable proteins  DNA constructs contain desired gene and appropriate regulatory sequences (tissue- specific promoters)  More economical than producing desired proteins in cell culture

YACs contained many of these heavy and light chain segments Knock out Mouse Segments, replace wuth Human segment genes Fully human antibody made

This mouse had a gene from a jellyfish which expresses green fluorescent protein. This technique is widely used to demonstrate the presence of engineered genetic changes.

An ad for a company which makes transgenic mice for research. Note the coat color on the back indicating cells from two different sources.

Applications Increasing casein content of milk increase cheese production Lactose free milk (transgene lactase) Resistance to bacterial infections In vivo immunization transgene is specific Heavy and Light chain genes which create Ab against a specific antigen

Easy to purify - few other proteins in milk Doesn’t harm transgenic animal- no change to physiology rProtein is authentically modified post-translationally Mammary Gland-specific Promoters promoter and transcription termination sequences are present

 Pig organs are rejected acutely due to the presence of human antibodies to pig antigens.  Once human antibodies are bound to pig organs, human complement is activated and triggers the complement cascade and organ destruction.  Transgenic pigs with complement inhibitors have been produced and are gaining feasibility as a source of xenogeneic organs for transplantation.

PIGS PST porcine somatotropin (growth hormone) adverse effects- kidney, stomach, heart, sterility human Hemoglobin to replace whole blood transfusions SHEEP Increase wool production keratin promoter growth factor

 Naked human Hb from pigs  Human lactoferrin in cows’ milk  HGH in mouse urine (uroplakin promoters)  Human antibodies in mice (H and L chain tgenics  hybridomas)  CfTCR in goats  Tissue plasminogen activator (TPA) in goats  Human antithrombin III in goats  Malaria antigens in goats (vaccine)  Alpha-glucosidase in rabbits (Pompe’s disease

Ethical concern is ever increasing as the technology grows, including the issue of lab animal welfare  These ethical issues include questions such as: Should there be universal protocols for transgenesis? Should such protocols demand that only the most promising research be permitted?

 Is human welfare the only consideration?  What about the welfare of other life forms?  Should scientists focus on in vitro (cultured in a lab) transgenic methods rather than, or before, using live animals to alleviate animal suffering?

Negative impact on other species  Risks are similar to exotic species which can become invasive  Africanized honey bee  Gypsy moth  Feral pigs  Risk is increased if the transgene enhances ecological adaptation  Salt water tolerance in fish  Cold tolerance  Increased growth rate or final size  Disease resistance  Nutrient utilization

Genetic Modification: or ?

 Gupta, p.k.; “biotechnology & bioinformatics” ; “transgenic animals” ; 2009 ; ed III ; pg ; kalyani publication.  Singh, b.d.; “biotechnology” ; “transgenic organisms” ; 2010 ; ed I ; pg ; kalyani publication   