3. Transgenic Animals
■ A transgenic animal is one whose genome has been altered by transfer of gene
or genes from another species or breed.
The photo shows two transgenic mice
positioned either side of a plain mouse. The
transgenic mice have been genetically
modified so that they carry a green
fluorescent protein which glows green under
blue light.
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4. Transgenesis
■ Process of introducing foreign or exogenous DNA into
an animals genome is called transgenesis.
■ Transgenesis is the process of introducing an
exogenous gene called a transgene into a living
organism so that the organism will exhibit a new
property and transmit that property to its offspring.
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5. Why we use Transgenic Technology ?
■ Transgenic animals have been instrumental in providing new insights and
mechanisms of:
1. Development and developmental gene regulation
2. Action of oncogenes
3. Complex cell interactions within the immune system
■ The transgenic technology offers exciting possibilities for generating precise
animal models for human genetic diseases and for producing large quantities
of economically important proteins by means of genetically engineered farms
animals.
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6. Why Mice were chosen for Transgenic
studies?
■ Easily housed, maintained and adapt well to new surroundings.
■ Reproduce quickly, have short lifespan of 2-3 years
■ Relatively inexpensive
■ Mild tempered and docile
■ Medical Trials are uniform
■ Mimic human body
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7. Methods for Producing Transgenic Animals:
■ DNA Microinjection
■ Embryonic Stem Cell-Mediated Gene Transfer
■ Retrovirus mediated gene transfer
■ Transfection of Gametes
■ Nuclear Transfer Method
■ Artificial Chromosome Mediated Gene Transfer
■ Testis cell transplantation method
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8. DNA Microinjection
DNA microinjection is the
dominating technique leading to
random integration of a
transgene via the introduction of
DNA into the pronucleus of a
developing zygote.
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9. Cont..
Fertilized eggs are collected from pregnant female mouse
Fertilized eggs are held under suction at the end of a pipette
The transgene is microinjected into one of the pronuclei with fine
needle
The eggs are then implanted into oviduct of pseudopregnant female
The transgene will be incorporated into chromosomal DNA of about 10%to 30% of the
spring
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10. Embryonic Stem Cell-Mediated Gene Transfer
■ In 1981, the term embryonic stem cells (ES cells) were used to denote a cell
line isolated directly from mouse embryos while, the term embryonal
carcinoma cells (EC) were derived from teratocarcinomas.
■ Embryonic stem cells (ES cells) are harvested from the inner cell mass (ICM) of
mouse blastocysts. They can be grown in culture and retain their full potential
to produce all the cells of the mature animal, including its gametes.
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11. Introduction of foreign DNA into embryonic stem cell by electroporation or
microinjection
Desired genes in cells can be identified by selection using marker gene or
PCR
Transfected cells can be cultivated, Introduced into blastocyst by
microinjection
Implanted into foster mother
Transgenic founder mice are produced
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Steps Involved in Embryonic stem cell mediated
gene transfer technique
13. Retroviral gene delivery method
■ Transgenic mice produced by retroviral transduction of male germ line stem cells.
■ A retrovirus is a virus that carries its genetic material in the form of RNA rather
than DNA.
■ Retroviruses used as vectors to transfer genetic material into the host cell, resulting
into a generation of chimera (an organism consisting of tissues or parts of diverse
genetic constitution).
■ Chimeras are inbred for as many as 20 generations until homozygous (carrying the
desired transgene in every cell) transgenic offspring are born.
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15. Nuclear Transfer Method
■ In this method, the transgenic sheep were
produced by nuclear transfer of fetal somatic
cells.
■ Donor karyoplasts were obtained from a
primary foetal somatic cell line derived from
a 40-day transgenic female foetus produced
by artificial insemination of a non-transgenic
adult female with semen from a transgenic
male.
■ Live offspring were produced with two
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16. Artificial Chromosome Mediated Gene
Transfer
■ A group of nuclei injected with transgene DNA, the eggs are transferred in
medium of incubation and visual evaluation within next few hours.
■ An individual animal develops after receiving the transgene DNA is referred as
founder of a new transgenic lineage.
■ Yeast Artificial Chromosomes (YACs) transgenic mice are generated by using
pronuclear microinjection and represents latest generation of vectors which
have the great advantage of large insert size. This method succeeded in mice
and rabbits
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17. Testis cell transplantation method
■ Testis cell transplantation method is as follows:
A. A single-cell suspension is produced from a fertile donor testis.
B. The cells can be cultured
C. Microinjected into the lumen of seminiferous tubules of an infertile recipient mouse.
D. Only a spermatogonia stem cell can generate a colony of spermatogenesis in the recipient testis.
When testis cells carry a reporter transgene that allows the cells to be stained blue, colonies of
donor cell-derived spermatogenesis are identified easily in recipient testes as blue stretches of
tubule.
E. Mating the recipient male to a wild-type female
F. Produces progeny, which carry donor genes
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18. Transgenic Animals Detection
■ Dot blot technique: Dot blot is a technique for detecting, analyzing, and
identifying proteins, similar to the western blot technique, but differing in that
protein samples are not separated electrophoretically but are spotted through
circular templates directly onto the membrane or paper substrate.
■ PCR Technique: The technique for detecting both foreign and host specific DNA
sequences inside nuclei and chromosomes of single cells of transgenic animals
with the help of polymerase chain reaction (PCR).
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19. ■ Analysis of transgene integration using restriction endonucleases: The
integration of transgene into the genome is confirmed by southern
hybridisation of genomic DNA extracted from transgenic individuals, DNA
digested with suitable restriction enzyme prior to electrophoresis.
■ Analysis of mRNA production: A high purity RNA preparation is obtained from
transgenic individuals and subjected to northern hybridisation, which provides
additional information on transcript size as well.
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21. Application of animal transgenesis may be divided
into 3 major categories
■ To obtain information on gene function and regulation as well as on human
diseases.
■ To obtain high value products (recombinant pharmaceutical proteins and xeno-
organs for human) to be used for human therapy.
■ To improve animal products for human consumption.
■ All these applications directly or not are in someway related to human health.
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22. Applications of Transgenic
animals
■ As disease model: Transgenic animals are produced as disease models
(animals genetically manipulated to exhibit disease symptoms so that
effective treatment can be studied) such as Alzheimer's, cancer, AIDS.
■ Blood replacement: Transgenic swine are used to produce human hemoglobin.
The protein obtained from transgenesis could be purified by using porcine
blood which is similar to human hemoglobin.
■ Xenotransplantation: Xenotransplantation, or heterologous transplant, is the
transplantation of living cells, tissues or organs from one species to another.
Such cells, tissues or organs are called xenografts or xenotransplants. e.g. Pig
organs (particularly kidney and heart) function very similarly to human kidneys
and hearts. 22
23. ■ Disease resistance: To produce disease resistant animals, such as influenza
resistant pigs.
■ Industrial application : Toxicity sensitive transgenic animals to test chemicals.
■ Vaccine safety: Transgenic mice are being used in testing the safety of
Vaccines before they are used in humans e.g. Polio vaccine
■ Chemical safety testing : Transgenic animals are made to carry the genes
which make them more sensitive to the toxic substance then the non
transgenic ones. They are then exposed to toxic substance and effect are
studied.
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24. Some examples of Transgenic animals
Transgenic Cow: The name of the first transgenic
cow is Rosie and it was developed in 1997. They
are mainly developed with two objectives-
increased milk production and molecular farming.
Transgenic Rabbit: The first transgenic rabbit
(Alba) was successfully produced in 1985. Used as
a model for cardiovascular disease, AIDS, Cancer.
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25. ■ Transgenic Fish
Genetic transfer has been done in various fishes such as
common carp, Atlantic salmon, etc. Transgenic salmon
was the first genetically modified animal for food
production. Salmon can grow 6 times faster than wild
type as they have extra copies of growth hormone gene.
■ Transgenic monkey
ANDI, the first transgenic monkey born on October 2nd
,2000, has GFP gene. Glow in dark monkey also have GFP(
green fluorescent protein) and glow under UV light.
Model for studying autism, Alzheimer’s etc.
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26. Ethical Issues
■ Religious concerns: For Hindu, Muslim or Sikh it would be forbidden to eat food
containing genetic material from the animals whose flesh is forbidden.
■ Animal welfare: In vitro procedures might lead to increase gestation length , body
weight and perinatal loss and anomalies. Microinjection increase embryonic and fatal
losses.
■ Economical aspects : Actions carried out by the researchers do not have any more like
simple goal research of the truth but well a race with the property of living and its
patentability which may produce colossal benefits.
■ Environmental effects: Release of transgenic animals into environment may
outcompete the natural population which effect the natural biodiversity and
ecosystem. 26
27. References
■ Bernard et al. Molecular Biotechnology, Principles and Applications of
Recombinant DNA, 4th ed. ASM Press Washington DC, 2010;21: 845-861
■ Heiner, Christine, Animal Biotechnology 1, Embryo Transfer Technologies,
Springer,2018;07: 167-178.
■ Manmohan, Neeraj, Transgenic Animals: Production and Application, IJPSR
(2010), Vol.1 (9): 12-22.
■ Ingrid Moen et al., BMC Cancer, 12/21 (2012), 1-10.
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