This is made by using internet resources and only for educational purpose.

1. By : Dr. Ashwini J. Patel
M. V. Sc Scholar (Animal Genetics & Breeding )
College of Vet. Sci. & A. H, Anand.

2. 1) INTRODUCTION
2) HISTORY
3) WHY TRANSGENIC ANIMALS ?
4) STRETERGIES TO PRODUCE TRANSGENIC ANIMAL
5) METHODS / TECHNIQUE FOR PRODUCTION OF
TRANSGENIC ANIMALS
6) APPLICATION OF TRANSGENIC ANIMALS

3. 1) INTRODUCTION
 Transgenesis
 The stable, one or more integration of foreign genes /foreign
DNA into a host’s chromosomes.
or
 Transgenesis either means transferring DNA into the animal
or altering DNA of the animal.
 Transgenic animal
 A transgenic animal is one that carries a foreign gene that
has been deliberately inserted into its genome .
or
 Transgenic animal are genetically modified to contain a gene
from a different species following gene transplantation or
resulting from the molecular manipulations of endogenous
genomic DNA

4. 2) History

5. 2) History:
 Prior to the development of molecular genetics, the only way
of studying the regulation and function of mammalian genes was
through the observation of inherited characteristics or
spontaneous mutations.
 The discovery of DNA and genes opened wide avenues for
research and biotechnological applications.
 The introduction of isolated genes into cells became a common
practice in the 1970s, soon after the emergence of the genetic
engineering techniques.
 During the 1970s, the first chimeric mice were produced.
 It represented a great progress for the understanding of gene
function and mechanisms of action.
 The first transgenic animal i.e. mice, were obtained by
microinjecting the genes into one of the nuclei (pronuclei) of
one day old embryos.

6. U Pennsylvania University of Washington
Ralph Brinster Richard Palmiter
1982 , The First transgenic mouse with a phenotype
A team led by Ralph Brinster and Richard Palmiter made
a construct in which the rat growth-hormone gene was placed
under the control of zinc-inducible metallothionin promoter.

7.  This construct was injected into fertilized mouse embryos and
the resulting transgenic offspring, were fed with extra zinc,
which turned on the metallothionin promoter.
 This resulted in the expression of growth hormone gene and the
resulting high levels of circulating rat growth hormone
dramatically changed the phenotype of the transgenic mice by
stimulating them to grow twice as large as normal.

8. PALMITER, R.D., BRINSTER, R.L.,
HAMMER, R.E., TRUMBAUER, M.E.,
ROSENFELD, M.G., BIRNBERG, N.C.
and EVANS, R.M.
“Dramatic growth of mice that develop
from eggs microinjected with
metallothionin-growth hormone fusion
genes.”
Nature (1982) 300: 611-615.
• The giant mice instilled major excitement in the scientific and
public communities, markedly enhancing attention on the
transgenic mouse system.
• Ralph L. Brinster and Richard Palmiter thus were pioneered in
the development of methods to transfer foreign genes into the
germline of animals.

9.  This technique is still widely used, gene transfer into animals
and plants to generate lines of genetically modified
organisms, known as transgenic animals and plants,
respectively.
 This method could be extrapolated (extend the application of to
an unknown situation by assuming that existing trends will
continue successfully) to other mammals in 1985.
 Other transgenic animals include rats, pigs and sheep etc.
 Transgenic technology led to the development of fish that
enabled to grow faster and livestock that enables to fight
diseases (prion-free cows resistant to bovine spongiform
encephalopathy, known as mad cow disease).

10.  Two other main techniques were Subsiquently developed:
those of retrovirus-mediated transgenesis (Jaenisch, 1976)
and embryonic stem (ES) cell-mediated gene transfer
(Gossler et al., 1986).
 The term transgenic was first used by J.W. Gordon and F.H.
Ruddle (1981).
 The transgenic technology also became an excellent tool in
basic research for understanding the functions and
regulations of a number of mammalian genes.

11.  Thanks to the transgenic technology, because today we have
mouse models for several types of cancer and of human
genetic disorders including chronic hepatitis, diabetes,
Alzheimer's disease and many more.
Transgenic mouse

13. 3) Why Transgenic Animal?
 Transgenic mice are often generated to :
1. characterize the ability of a promoter to direct tissue specific
gene expression
e.g. a promoter can be attached to a reporter gene such as
LacZ or GFP
2. examine the effects of over expressing and misexpressing
endogenous or foreign genes at specific times and locations
in the animals
 Interest in transgenic animals originally fall into two broad
categories:
1. To increase production efficiency of farm animals in a short
duration.
2. Molecular farming: Using livestock to produce medicines,
nutraceuticals and tissues for transplant into humans.

14. 4) Strategies for Producing Transgenic Animals
There are two basic strategies for producing transgenic
animals, which include “gain of function” or “loss of
function” transgenics.
 The basic idea behind the gain of function strategy is that by
adding a cloned fragment of DNA into an animal’s genome to
a new gene product is produced that did not previously
existed in that cell or tissue. E.g. expression of rat growth
hormone in mouse and to get over expression of gene product
in the proper tissue (Palmiter et al., 1982).
 The silencing or loss of gene function is accomplished by the
target gene disruption through the process of homologous
recombination between host genome and exogenous DNA.

15. 5) Techniques / Methods
of
Gene Transfer

16. 5) Techniques/ Methods of Gene Transfer
• There are many techniques, all are listed below
1) Pronuclear injection
2) Transposon
3) Viral technique (Retroviral technique, Lenti
viral transfection)
4) ICSI / Sperm Mediated Gene Transfer
5) Embryonic stem cell / chimeras
6) somatic cell nuclear transfer / cloning
7) Chemical technique
8) Electroporation
9) RNA Interference
• Some of important methods have been described in Houdebine
2003, 2005 and they are summarized in figure demonstated on
next page.

17. Houdebine 2003, 2005

18. 1) DNA Microinjection
 About 1,000 copies of the isolated foreign gene contained in
1–2 pl may be injected into one of the pronuclei of one day
old mammalian embryos.
 This method implies a superovulation of the females
followed by a mating with a male.
 The resulting embryos are collected the next day and
microinjected with DNA. The embryos are then transferred
to hormonally prepared recipient females using surgery
operations.
 The yield of this method in mice is of 1–2 of transgenics from
100 microinjected and transferred embryos. It is lower in all
the other mammalian species and very low in ruminants.

19. Superovulation and mating
Isolation of one cell stage zygote
Microinjection of transgene
Oviduct transfer to pseudopregnant females
Identification of founders by Southern blotting/PCR

20.  In non mammalian species, the pronuclei cannot be
visualized and DNA must be injected into the cytoplasm of
the one day old embryos. This relatively simple technique is
efficient in most fish species.
Generation of sufficient number of eggs for
microinjection / superovularion.
 Pregnant mare’s serum (or=FSH) on day 1
 Human Chorionic Gonadotropin (or=LH) on day 3
 Mated with a fertile male on day 3
Fertilized oocytes microinjected on day 4 with foreign DNA
construct.
The microinjected eggs are implanted the same day or are
incubated overnight and implanted into the oviduct of
pseudopregnant female the next day.

21. Mated female

22. Micro inject purified DNA into a male
pronucleus of fertilized mouse egg.
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.

24.  Generation of pseudopregnant females
 Female mice are tricked into thinking they are pregnant
 A female mouse in estrus is mated with a vasectomized male
 Pseudopregnancy
 If eggs (blastocysts) implanted will become truly pregnant and
will give birth to live offspring.

25. Vasectomy of male mice

26. ~30 cells
200-250
cells
2) Transformation of embryonic stem cells

28.  Micropipette ES cells from the inner cell mass of a blastocyst
(i.e. early mouse embryo) in a strain with a physically
recognizable phenotype (e.g., pigmented).
 Introduce transgenic construct/DNA into ES cells by
electroporation or viral vectors

29.  Culture the cells in presence of antibiotic. Cells which are not
transgenic (black dots) will be killed, while those that have
taken up the DNA and are transgenic (red dots) survice.
•Insert the transgenic stem cells into the blatocyst of a mouse
with a different genetic background trait (e.g., an albino if the
original stem cells came from a pigmented mouse).

32.  Implant the new blastocysts into a pseudopregnant
female with a visible phenotype different from the
blastocyst phenotype (e.g., albino if the blastocyst is
pigmented).

33. • Offspring that have pigmented sections are chimeras that
have incorporated the transgenic sequence into their cell lines.
Select them for further breeding.
• Keep breeding the offspring of the chimeras (two
heterozygotes) and their offspring will have all three
genotypes wild type homozygotes, heterozygotes, and
transgenic homozygotes(fully pigmented mice)

35. Black mouse -
no ES cell integration
Chimeric mouse -
high ES cell integration
Chimeric mouse -
low ES cell integration

36. Method : 2
Method : 1

37. 3) DNA transfer via cloning:
 The foreign gene is transferred into a somatic cell, the
nucleus of which is introduced into the cytoplasm of an
enucleated oocyte to generate a transgenic clone.

38. Cloning by nucleus transfer

39.  Dolly“ 1997, first living offspring derived from
a differentiated cell.

40. 5) Sperm Mediated Gene Transfer
 The sperm cells have the capacity to bind naked DNA or
bound to vesicles like liposomes (Lavitrano et al., 1989;
Chang et al., 2002).

 These sperm cells are in turn used for introducing
exogenous DNA into oocytes either through invitro
fertilization or artificial insemination.
 Sperandio et al. (1996) successfully carried out the sperm
mediated gene transfer in cattle.

41. 6) Retro viral technique
 Retrovirus is single stranded RNA virus which upon
transfection gets converted to double strand DNA and
integrates into the host genome (Eglitis et al., 1988).
 The retroviral method was the first method to produce a
transgenic mouse as reported by Jaenisch et al. (1975).
 The most commonly used retroviral vector is Moloney
murine leukemia virus (Laneuville et al.,1988).
7) Lenti viral Transfection
 This method overcomes the limitations of viral mediated
gene transfer like low expression and silencing of gene
locus.
 Stable transgenic lines could be produced by injection the
lentivirus into the perivitelline space of zygotes.
(Hofmann et al., 2003).

42. 8) Electroporation
 This technique was developed by Puchalski and Fahl (1992).
 In this technique, cells are exposed to electric field which
causes the membranes to become polarized and a potential
develops across the membrane thereby breaking at localized
areas .
 The cell becomes permeable to exogenous molecule.
 The method has a greater efficiency either alone or in
combination with other.
9) Chemical technique
 This technique utilizes the chemical mediated uptake of
DNA or gene fragment by the host cell. The transfection is
carried out effectively by using chemicals like calcium
phosphate or diethyl amino ethyl dextran.

43. 10) RNA Interference
 In this method, small interference RNAs (siRNAs), which
are 20-25 nucleotides long, bind to their complementary
sequences on target in mRNAs and shut down the
expression of genes and there by the production of protein
is stopped.
 This RNA could be used for either transient or stable gene
repression or knock down of specific target genes.

44. Limitations of Transgenesis
 The transgenic technology even though has tremendous
applications in livestock improvement programmes, still it has
lots of limitations:
 Insertional mutations resulting in alteration of important
biological processes.
 Unregulated gene expression resulting in improper expression
of gene products.
 Possibility of side effects in transgenic animals like arthritis,
dermatitis and cancer etc.
 Integration of exogenous DNA sequence in Y chromosome
resulting in transmission only to males.

45. Applications of
Transgenic Animals

46. 1) Transpharmers
 Transpharmers' are transgenic animals engineered to produce
therapeutics, such as insulin in their milk, and used to treat
deficiencies such as diabetes
 The proteins are produced in the mammary glands and
harvested through the animals milk, The process can yield
between 1 and 10 grams of protein per liter of milk
A) Transpharmer Cattle-
Although cows are the most difficult animal to make
transgenic.
 Herman the bull was genetically engineered to carry the
human gene for the production of Lactoferin.
 Human Lactoferin is a protein essential for the immune
system of infants, and is present in mother’s milk, but does not
naturally occur in cows milk.

48. B) Transpharmer Goats
 Transgenic dairy goats have been engineered to carry the
transgene for recombinant human antithrombin III (a blood
thinning protein).
 Those produced 5g/L of recombinant anti-thrombin III protein
in their milk.
 This drug marketed as ATryn® by Genzyme Transgenetics
Corp (GTC) became the world’s first FDA approved drug in
2009 (ATryn, 2009).
Three Transgenic Goats Expressing Recombinant Human Antithrombin
Gene.

50. c) Transpharmer sheep [Tracy(1990-1997)]
 The transpharmer sheep (ewe) was produced for production of
large amounts (< or = 65 grams per litre) of enzymatically
active human alpha 1 antitrypsin in the milk of transgenic
sheep which is used as potential treatment of cystic fibrosis.
 It inhibits a wide variety of proteases and it protects tissues
from enzymes of inflammatory cells, especially neutrophil
elastase but the concentration can rise many fold upon acute
inflammation
 In its absence, neutrophil elastase is free to break down
elastin, which contributes to the elasticity of the lungs,
resulting in respiratory complications such as emphysema, or
COPD (chronic obstructive pulmonary disease) in adults and
cirrhosis in adults or children.

53. 2) Xenotransplanter pig
• Pigs produces a sugar alpha-1-galactose that the human body
recognizes as foreign and rejection of organ occur.
•The transgenic work attempted to knockout the genes
encoding the glycosyl-transferase enzymes that add the sugar
to organ surfaces, to counteract the rejection process

55. 3) Transgenic Food Sources
 Transgenic food sources aim to create animals that grow
larger, mature quicker, are more nutritious and more
efficiently utilize the consumed food that sustains the
animal.
A) Super Fish
 Coho salmon possessing the growth hormone (GH)
transgene appear in market as the transgenic animal food
sources.

57. 4) Scientific/Biological Models
 There are marketable applications as for example giving a
household pet a greater communication capacity with their
owners.
 Transgenic biological models have been used to increase
intelligence in mice, increase size in mice, and in one case to
impart the green fluorescent protein of a jellyfish in a monkey.
A) Super Mouse
 This mouse received a rat growth hormone gene, and the
transgene was expressed to create very large mice relative to
their non-transgenic littermates.

59. B) Smart Mouse
 One of the most significant scientific which ever successfully
conceived was the creation of a strain of smart mice named
Doogie.
 These mice were endowed with the gene NR2B which is a
subunit of the glutamate receptor that predominates during
early development when learning and memory are easier.
 This gene greatly increased a mouse's ability to recognize
objects, learn more effectively and increase spatial learning.

61. • Genetically engineered fluorescent
fish.
• zebra fishes were the first GloFish
available in pet stores.
• They are now sell in bright red,
green, orange-yellow, blue, and
purple and many more fluorescent
colors.
• It is one of the first genetically
modified animals to become publicly
available.
Glo fish.

62. 5) Disease Models
 One of the most important applications of transgenic
technology is the modeling of human diseases.
A) Huntington's Mouse
 Huntington's disease (HD) is a neurodegenerative autosomal
disorder that typically onsets between 30 and 50 years old, but
can start at any age it is characterized by chlorea (involuntary
muscle spasms and loss of motor control)
 Huntington's neural degeneration is caused by a mutation in the
huntingtin gene with CAG triplet nucleotide duplication,
which causes a build up of polyglutamine amino acids in
neurons.

63.  A study in 1998 showed that mice expressing the mutated
human Huntington protein and also showed nuclear inclusion
bodies
 This Huntington's disease model is just a minor sampling of
the scientific knowledge gained from transgenic mice. Other
models have also been constructed for Alzheimer’s disease,
cancer, AIDS, and Parkinson’s disease

64. B) Polio virus receptor
• Normal mice can't be infected with polio virus. They lack
the cell surface molecule that, in humans, serves as the
receptor for the virus.
• Transgenic mice expressing the human gene for the receptor
can be infected by polio virus and even develop paralysis
and other pathological changes characteristic of the disease in
humans.
• So, normal mice can serve as an inexpensive, easily
manipulated model for studying the disease.