Production of transgenic animals.

1. TOPIC
PRODUCTION OF TRANSGENIC ANIMALS
SUBMITTED BY: MOANUKSHI KICHU
M.PHARM 1ST YEAR (PHARMACOLOGY)
SUBMITTED TO: Dr.SAUMYA DAS
Professor & H.O.D of Pharmacology

2. INTRODUCTION
Transgenic animals are those animals in which a foreign gene has been deliberately inserted
into its genome or genetic materials .The foreign gene is constructed using recombinant DNA
technology.
Foreign genes are inserted into the germ line of the animals, so it can be transmitted to the
progeny.
These transgenic animals are used to study biological functions of specific genes to develop
animals models for diseases of humans or animals to produce therapeutic products vaccines
and for biological screening.
1st transgenic animal(mice)- created by Rudolf Jaenisch , in 1974.
In 1982, Ralph Brinster and Richard Palmiter created a transgenic animal (supermouse) – by
inserting a human growth hormone gene in a mouse genome . offspring was larger than the
parents.

3. Animal used for Transgenics Are:
 Chickens
 Pigs
 Rabbits
 Mice( most commonly)
 Cows
 Sheep
 Goats etc

4. Step 1- Construction of a transgene
Using Recombinant DNA technology
Parts-
o Gene to be expressed
o Vector DNA
o Promoter & Termination sequence
Production of Transgenic animals

5. (I).DNA Microinjection
 It was first discovered by Dr. Marshall A. Barber in early 19th century.
 In this method , the fertilized eggs are harvested from a female
mouse. Then the foreign gene are microinjected into the eggs and
the eggs are re-emplanted into a pseudopregnant female mouse.
Step 2- Introduction of foreign gene into the animal

7. Steps:
A female mice is superovulated by giving hormonal therapy
The eggs are collected and fertilized in invitro condition
Now foreign gene is inserted into the male pronucleus by using a micropipette
Eggs with transgene are kept for overnight in incubator to develop to a 2 cell stage
Eggs are then implanted into the uterus of a pseudo-pregnant female mice.
The offspring are check to determine whether the foreign gene are incorporated into
its genome or not by using PCR test

8. (II).Embryonic Stem cell-mediated Gene Transfer
The ES Cells are obtained from the inner cell
mass of a blastocyst.
Transgene is incorporated into the ES Cell by
oMicroinjection
oBy a retro virus
oBy electroporation
Transgenic stem cells are grown in vitro.
Then they are inserted into a blastocyst and
reimplanted in a pseudo pregnant foster
mother .

9. (III).Retrovirus Mediated Gene Transfer
 In this method, gene transfer is mediated
by a carrier of vector.
 Retroviruses are commonly used as
vector.(which carries genetic material in
the form of RNA rather than DNA).
• 8 celled embryo are taken from donor
female mice.
• The retroviral vector which contain the
transgene infects the embryo.
• It is then implanted in the uterus of
pseudopregnant mouse.
• Offspring of transgenic mice produced.

10. (IV).Sperm- mediated gene transfer
• In 1989 Lavitrano et al. described SMGT as a simple and
efficient technique to produce transgenic mice.
• It is a transgenic technique, uses the sperm as a natural vector
to transport the internalize fertilization to produce genetically
modified organism.
• Use of linker protein to attach DNA to sperm which transfer
the new DNA during fertilization .
• In this technique, DNA was mixed with sperm cell before in
vitro.30% of offspring mouse were integrated foreign DNA.

11. Step 3- Screening for transgenic positives
 Transgenic progenies are screened by PCR to examine the site of incorporation of
the gene.
 Some transgenes may not be expressed if integrated into a transcriptionally
inactive site.

12. Step 4 – Further animal breeding is done to obtain maximal expression
 Heterozygous offsprings are mated to form homozygous strains.
PROBLEMS
 Multiple insertion - too much proteins
 Insertion into an essential gene – lethality
 Insertion into a gene leading to gene silencing
 Insertion into a different area can affect the gene regulation

13. SOME EXAMPLES OF TRANSGENIC ANIMALS:
TRANSGENIC FISH
 Superfish
 Increased growth and size.
 Growth hormone gene inserted into fertilized egg.
 Transgenic salmon grows about 10 - 11 times faster than normal fish
 Glo fish
 GM freshwater zebra fish(Danio rerio)
 Insertion into a different area can affect the gene.
.
TRANSGENIC MOUSE
 Alzheimer's mouse
 In the brain of Alzheimer's patients, dead nerve cells are entangled in a
protein called amyloid.
 Mouse made by introducing amyloid precursor gene into fertilized egg of
mice.
 Oncomouse
 Mouse model to study cancer.
 Made by inserting activated oncogenes.

14.  Smart mouse
 Biological model engineered to over express NR2B receptor in the synaptic pathway.
 This makes the mice learn faster like juveniles throughout their lives.
TRANGENIC PIG
 Enviro pig
 Pigs have trouble fully digesting a compound known as phytate found in many cereal grains used to feed them.
 Transgenic pigs are created by introducing phytase gene of E.coli
 Phytase enzyme is thus produced in the salvary gland of pig
 It degrades indigestible phytate with the release of phosphate that is readily digested by pigs.
 Pig for organ transplant
 pigs with human genes, in order to decrease the chance of organ rejection by human body.
TRANSGENIC LIVESTOCK
 Bioreactors whose cells have been engineered to synthesis marketable proteins.
 More economical than producing desired protein in cell culture.
Transgenic cattle
 Transgenic cows are made to produce proteins lactoferrin and interferons in their milk.
 Prion free cows resistant to mad cow disease.
Transgenic sheep
 For good quality wool production.
Transgenic goat
Goats that could express tissue plasminogen activator, anti thrombin III, spider silk etc in milk.

15. IMPORTANCE OF TRANSGENIC ANIMALS
 Medical importance
 Disease model
 Bioreactors for pharmaceuticals
 Xenotransplantation
 Agricultural importance
 Disease resistant animals
 For improving quality and quantity of milk, meat, eggs and wool production
 Industrial importance
 Toxicity sensitive transgenic animals to test chemicals.
 Spider silk in milk of goat.

16. ISSUES RELATED TO TRANSGENIC TECHNOLOGY:
 Blurring the lines between species by creating transgenic combinations.
 There may be health risks associated with transgenics.
 There may be long term effects on the environment when transgenic animals
are released into the field.
 Various bioethicist argue that it is wrong to create animals that would suffer as a
result of genetic alteration.

17. RECENT TRENDS:
 Transgenic goats engineered to produce human breast milk (Journal Transgenic Research, August
2012)
 University of California scientists created the transgenic goats by transferring human genes for
breast milk enzymes and proteins into goat embryos.
 Produce 60 percent of the lysozyme and lactoferrin found in human mother's milk.
 For babies of mothers who aren't present, or can't nurse them, milk from these transgenic goats
could provide the next-best alternative.
 Bioluminescent transgenic mouse model for study mammary gland tumour development. (PLOS
ONE, aug 2012)
 Research done by Cancer Biology and Therapeutics Group, Ireland

18.  Transgenic technology is a field that is under constant evolution.
 Many transgenic animals have been successfully created for a variety of purposes, and
the prospects are enormous.
 It holds great potential in many fields including agriculture, medicine and industry.
 With proper research and careful use the transgenic animals can go a long way in solving
several problems for which science doesn't have a solution till now.
CONCLUSION:

19. REFERENCE:
► Satyanarayana U, Biotechnology (2010), 1st edition, Books and allied (P) Ltd, Kolkata.
► Channarayappa, Molecular Biotechnology - Principles and Practices (2006), 1st edition,
University Press Pvt Ltd, Hyderabad.
► Smith E John, Biotechnology (1996), 3rd edition, Cambridge University Press►
http://dsc.discovery.com/technology/tech- 10/genetic-engineering/10-transgenic- animals-
03.html
► www.biomedcentral.com/1471-2407/12/209
►http://news.e-healthsource.com/index.php? p=news1&id=534197

20. Thankyou