Introduction to transgenic animals, their methods of production, maintenance, commercial applications. With examples, Advantages and Disadvantages.

1. TRANSGENIC
ANIMALS.

2. CONTENTS
 INTRODUCTION. Pg. No. 2-3
 PRODUCTION OF TRANSGENIC ANIMALS BY MICRO-INJECTION. Pg. No. 4-7
 PRODUCTION BY EMBRYO CELL TECHNOLOGY. Pg. No. 8-9
 MAINTENANCE OF TRANSGENIC ANIMALS. Pg. No. 9
 COMMERCIAL APPLICATIONS OF TRANSGENIC ANIMALS. Pg. No. 10-15
 ADVANTAGES AND DISADVANTAGES Pg. No. 16-17
 REFERENCES Pg. No. 18
AYESHA NAZEER I MPHARM (PHARMACOLOGY), SCP. 2

3. INTRODUCTION
Genetic manipulation by the addition of genes is a very powerful tool with tremendous
applications in basic biology, drug discovery, gene therapy and in agricultural research. Animals
subjected to such genetic manipulations are known as Transgenic animals.
The added genes are called Transgenes.
Transgenic mouse is becoming very popular animal for studying the disease processes and
testing of newer drugs. Screening of bioactive molecules for drug research is one of the
important areas where transgenic mice have tremendous potential.
During recent years however, transgenic techniques have been extended to other species
including the rabbit, the rat, and also a range of commercially important animals, notably the
cow, the pig, the goat and the sheep.
AYESHA NAZEER I MPHARM (PHARMACOLOGY), SCP. 3

4. AYESHA NAZEER I MPHARM (PHARMACOLOGY), SCP. 4

5. PRODUCTION OF TRANSGENIC ANIMALS BY MICROINJECTION
1. The first stage is to isolate sufficiently large numbers of fertilised eggs for micro-injection.
Achieved by the super-ovulation of young virgin females(appx. 4-5 weeks of age).
 First inject with a source of follicle-stimulating hormone (pregnant mare’s serum)
 48 hrs later inject with human chorionic gonadotrophin to cause artificial leutinizing hormone
surge. This brings about super-ovulation.
2. Isolation of fertilized eggs.
 Paired with males.
 Mated females are identified by presence of vaginal plug. Sacrificed and fertilized eggs removed
from swollen ampullae of the fallopian tubes by dissection.
 Upto 30 zygotes can be isolated per female.
 The zygotes are freed from attached cumulus cells by brief incubation in presence of
hyaluronidase, transferred to appropriate medium and are stored in a CO2 incubator at 37 degree
C.
AYESHA NAZEER I MPHARM (PHARMACOLOGY), SCP. 5

6. 3. Micro-Injection.
 Fertilised eggs are picked up by the gentle suction onto a holding pipette and injected with
micro-needle injection.
 The micro-injection needle, which has an internal tip diameter of approximately 1µm and
contains DNA at a typical concentration of 1- 2 µm/ml.
 Gently and firmly until both the zona pellucida and nuclear membrane of one of the pronuclei
(no cell- division has occurred ) have been pierced.
 Care taken to ensure puncturing of the elastic membrane without touching the nucleoli.(
prevent blockage and damage to egg).
 DNA is injected into the pro nucleus using micro-syringe. Swelling of pro nucleus prior to
removal of the tip indicates the successful injection.
AYESHA NAZEER I MPHARM (PHARMACOLOGY), SCP. 6

7. 4. Reimplantation of micro-injected egg cells.
 Successfully injected eggs are incubated and left to develop to two-cell stage over-night. This
allows to check viability of eggs.
 The developed embryos are reimplanted into the oviduct of anaesthetized pseudopregnant
females. ( experienced mothers mated previous night with vasectomized or infertile males)
 Recovery from anaesthesia and pregnancy is continued to term.
5. Testing for Transgene.
 Polymerase chain reaction(PCR) or Southern blot hybridization Analysis of genomic DNA
isolated from the tail biopsies.
 PCR analysis of whole blood.
 Provided that the foreign gene was incorporated into the genome prior to first cell division,
the transgene should be present every cell of the resultant pup.
AYESHA NAZEER I MPHARM (PHARMACOLOGY), SCP. 7

8. AYESHA NAZEER I MPHARM (PHARMACOLOGY), SCP. 8

9.  Involves the introduction of foreign DNA into embryonic
stem(ES).
 To establish an ES-line, cells are removed from the inner
cell mass of developing blastocyst and are passaged either
on feeder layers, or in the presence of differentiation-
inhibiting activity, to maintain their undifferentiated state.
 Foreign DNA can be introduced by:
 Electroporation
 Transfection
 Micro-injection
 Selected cells are then reintroduced into the blastocyst and
reimplanted into a pseudopregnant female.
 Important distinction between ES-line and the micro-
injection method is that the progeny will be a chimera in
this method.
PRODUCTION OF TRANSGENIC ANIMALS BY EMBRYO CELL
TECHNOLOGY
AYESHA NAZEER I MPHARM (PHARMACOLOGY), SCP. 9

10. AYESHA NAZEER I MPHARM (PHARMACOLOGY), SCP.
MAINTENANCE OF TRANSGENIC
ANIMALS:
 Housing.
 Feeding
 Ventilation.
 Lighting.
 Sanitation
 Routine management practices.
 Special care has to be taken with
transgenic animals as they can be
susceptible to diseases due to altered
metabolic activities.
 The transgenic animals should be
maintained in clean room environment
or in animal isolations.
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11. COMMERCIAL APPLICATIONS
1. Biopharmaceuticals in Transgenic Animals.
 Transgenic sheep for good quality wool production.
 Transgenic livestock produce large quantities of therapeutic proteins.
 Expression of transgene to the mammary gland, an exocrine gland.
 Transgene is fused to the regulatory sequence of a milk protein.
 The future development of human milk substitutes.
A number of biological products such as medicines and nutritional supplements are obtained from transgenic animals.
Research for the manufacture of medicines to treat diseases such as phenylketonuria (PKU) and hereditary
going on. The first transgenic cow, Rosie (1997), produced milk containing human protein (2.4 grams per litre). This
contains the human gene alpha-lactalbumin and could be given to babies as an alternative to natural cow milk.
AYESHA NAZEER I MPHARM (PHARMACOLOGY), SCP. 11

12. AYESHA NAZEER I MPHARM (PHARMACOLOGY), SCP.
PROTEIN ANIMAL USE
Antithrombin
III
Goat Reduce the amount of blood
needed in some surgeries
Factor VIII,
Factor IX
Goat,
Pig,
Sheep
Treatment of Hemophilia
CFTR Sheep Treatment of Cystic Fibrosis
Lactoferrin Cow An anti-bacterial compound
that prevents mastitis in cows.
In humans as anti-infective in
stomach/intestine ulcers,
diarrhoea and hepatitis C
Spider Silk
protein
Goat Production of ultra-strong
weight media and industrial
materials.
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13. 2. Xenografts:
 transplantation has prompted developments in use of animal organs.
 Major barrier here is the Complement-mediated hyperacute rejection(HAR).
 This issue is combatted in porcine-primate xenotransplantation by producing transgenic pigs
expressing human complement inhibitors.
 After transplantation pig’s heart survived in baboons for prolonged periods.
3. Toxicological applications:
 Transgenic animals have been designed which allow the researcher to screen for genotoxic or
carcinogenic compounds.
 Commercially available transgenic mice, including Mutamouse and Big Blue.
 They contain E coli LacZ and lacI genes, respectively.
 They are cloned into the bacteriophage vectors that are integrated into the genome.
 Mice is treated with test chemical. And Genome is extracted. Introduced o Bacteriophage. The
bacteriophage containing the mutant are recognised by their ability to grow in susceptible E coli
host and by resulting Blue color.
AYESHA NAZEER I MPHARM (PHARMACOLOGY), SCP. 13

14. AYESHA NAZEER I MPHARM (PHARMACOLOGY), SCP. 14

15.  Dolly Sheep
 Dolly the sheep was the first
mammal to be cloned from
adult cell. In this, the udder
cells from a 6-year-old Finn
Dorset white sheep were
injected into an unfertilized
egg from a Scottish Blackface
ewe, which had its nucleus
removed. The cell was made
to fuse by electrical pulses.
After the fusion of the
of the cell with the egg, the
resultant embryo was
for six to seven days. It was
then implanted into another
Scottish Blackface ewe which
gave birth to the transgenic
sheep, Dolly.
AYESHA NAZEER I MPHARM (PHARMACOLOGY), SCP.
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16. AYESHA NAZEER I MPHARM (PHARMACOLOGY), SCP. 16

17. ADVANTAGES OF TRANSGENIC ANIMALS:
 (a) Gene requires certain cellular mechanism to help for the production of protein.
The animals used for transgenic purpose naturally carry the mechanism needed to
produce complex protein. These mechanism is absent in cell culture.
 (b) Expression through cell culture or bacterial culture requires constant monitoring
and sampling.
 (c) The isolation and purification of expressed protein in conventional method is
more difficult than purifying proteins from an animal’s milk or body fluid.
 (e) It has been estimated that transgenic animal can produce in its lifetime $100 to
$200 million worth of pharmaceuticals.
AYESHA NAZEER I MPHARM (PHARMACOLOGY), SCP. 17

18. DISADVANTAGES OF TRANSGENIC ANIMALS:
 (a) Transgenic animal project is extremely expensive.
 (b) Generation of transgenic animals are also expensive, because of long gestation period,
litter size and higher maintenance cost of the recipient animals.
 (c) There may be high mortality rate and other deleterious effects on animals used by
researchers to create transgenic breeds. It has been observed that transgenic pigs having
enhanced growth rate and efficient feed conversion exhibit reduced reproductive performance
and may suffer from arthritis and dermatitis etc.
 (d) Transgenic foods have been produced and offer better productivity in terms of both yield
and quantity. However, there are some apprehension about the safety of transgenic foods.
AYESHA NAZEER I MPHARM (PHARMACOLOGY), SCP. 18

19. REFERENCES
 S.K. Kulkarni, HANDBOOK OF EXPERIMENTAL PHARMACOLOGY. (Page No. 29)
 J.M. WALKER, R.RAPLEY, MOLECULAR BIOLOGY and BIOTECHNOLOGY, 4th Edition, PANIMA.
(Page No. 358-361, 363, 375-377)
 https://byjus.com/biology/transgenic-animals/
 https://www.biotechnologynotes.com/animals/transgenic-animals/transgenic-animals-advantages-and-
disadvantages/678
For Images:
AYESHA NAZEER I MPHARM (PHARMACOLOGY), SCP.
https://www.reuters.com/news/picture/genetically-
modified-animals-idUSRTXTZ7A
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