Introduction History Landmarks Events in Transgenic Livestock Research Techniques/ Method for Gene Transfer Examples of transgenesis Importance Application Limitation Issue related to Transgenic Technology Ethical concerns and how to Overcome

1. REPRODUCTIVE BIOTECHNOLOGY
Dr. Dhaval Chaudhary
M.V.Sc Scholar
Dept. of Animal Genetics and Breeding
College of Veterinary Science and Animal Husbandry, AAU, Anand
Transgenesis

2. Content
• Introduction
• History
• Landmarks Events in Transgenic Livestock Research
• Techniques/ Method for Gene Transfer
• Examples of transgenesis
• Importance
• Application
• Limitation
• Issue related to Transgenic Technology
• Ethical concerns and how to Overcome

3. Introduction
• 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.
• Transgenesis can be facilitated by liposomes by liposomes, enzymes,
plasmid vectors, viral vectors, pronuclear injection, protoplast fusion,
and ballistic DNA injection.

5. Definitions
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

6. 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.

7. • 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.

9. • 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.

10. • 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.
• 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.

11. • 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).

12. • 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.

13. • 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.

15. Why Transgenic Animals Produced?
• To help scientists to identify, isolate and characterise genes in order to
understand more about their function and regulation.
• To provide research models of human diseases, to help develop new
drugs and new strategies for repairing defective genes (“gene
therapy”).
• To provide organs and tissues for use in human transplant surgery.
• To produce milk which contains therapeutic proteins; or to alter the
composition of milk to improve its nutritional value for human
infants.
• To enhance livestock improvement programmes.

16. 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.

17. Techniques/ Methods of Gene Transfer
There are many techniques, all are listed below
1) DNA Microinjection
2) Retro virus- Mediated Gene Transfer
3) Embryonic stem cell / chimeras
4) Somatic Cell Nuclear Transfer / Cloning
5) Use of Transposons
6) Sperm Mediated Gene Transfer
7) Electroporation
8) Chemical technique
9) RNA Interference
• Some of important methods have been described in Houdebine 2003, 2005.

18. Houdebine 2003, 2005
Different methods to generate transgenic animals: (1) DNA
transfer via direct microinjection into pronucleus or cytoplasm of
embryo; (2) DNA transfer via a transposon: the foreign gene is
introduced in the transposon which is injected into a pronucleus;
(3) DNA transfer via a lentiviral vector: the gene of interest in a
lentiviral vector is injected between the zona pellucida and
membrane of the oocyte or embryo; (4) DNA transfer via sperm:
sperm is incubated with the foreign gene and injected into the
oocyte cytoplasm for fertilization by ICSI (intracytoplasmic sperm
injection); (5) DNA transfer via pluripotent cells: the foreign gene
is introduced into pluripotent cell lines (ES: embryonic stem cells:
lines established from early embryo, EG: embryonic gonad cells:
lines established from primordial germ cells of foetal gonads); the
pluripotent cells containing the foreign gene are injected into an
early embryo to generate chimeric animals harbouring the
foreign gene DNA; (6) 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. Methods 1, 2, 3 and 4 allow random
gene addition whereas methods 5 and 6 allow random gene
addition and targeted gene integration via homologous
recombination for gene addition or gene replacement including
gene knockout and knocking.

19. DNA Microinjection
• Gordon and Ruddle, 1981
• The young virgin mice (4-5weeks age) are
subjected to ovulation
• Administration of FSH (pregnant mare’s serum)
• After 2 days admistration of HCG
• Super ovulated mice produce 30-35 eggs
• Above female mice mated with males
• Fertilized eggs are removed from the fallopian
tubes
• By micromanipulation using microinjection needle
&holding pipette the DNA is injected into the
male pronuclear of the fertilized egg
• Trans genes are kept overnight in an incubator
• The foster mother delivers pups after 3 weeks of
implantation

20. Retro Virus-
Mediated Gene Transfer
• Jaenisch, 1976
• The transfer of small pieces (8kb) of DNA can
be effectively carried out by retroviruses
• This method however is unsuitable for
transfer of larger genes
• Further even of small genes there is a risk:
losing some regulatory sequences .
• Drawback:-Risk of retroviral contamination in
the products(in foods for human
consumption).
• Not regularly use for transgenesis

21. Embryonic Stem Cell-
Mediated Gene Transfer
• Gossler et al., 1986
• This method involves prior insertion of the desired DNA
sequence by homologous recombination into an in vitro
culture of embryonic stem (ES) cells.
• These cells are then incorporated into an embryo at the
blastocyst stage of development.
• ES cell-mediated gene transfer is the method of choice
for gene inactivation method (knock-out method).
• This technique is of particular importance for the study
of the genetic control of developmental processes.
• This technique works particularly well in mice.
• It has the advantage of allowing precise targeting of
defined mutations in the gene via homologous
combination

22. 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.

23. • Panel A, depicts the oocyte ready
for enucleation.
• Panel B, depicts the nuclear
material being removed from the
cytoplast. The bottom portion of
the figure shows the karyoplast
transfer into the enucleated
cytoplast.
• Panel C, depicts the insertion of
the karyoplast transfer needle into
the zona pellucida of the
enucleated oocyte.
• Panel D, depicts the karyoplast
after insertion into the zona prior
to cell fusion into the cytoplast
(Photographs courtesy of Jane H.
Pryor and Charles R. Long, Texas
A&M University).

24. “Dolly” 1996, first living offspring derived from a differentiated cell.
(Ian Wilmut and Keith Campbell,1996)

25. Use of Transposons
• Transposons are short genomic DNA regions which are replicated and
randomly integrated into the same genome.
• The number of a given transposon is thus increasing until the cell
blocks this phenomenon to protect itself from a degradation of its
genes.
• Foreign genes can be introduced into transposons in vitro. The
recombinant transposons may then be microinjected into one day old
embryos.

26. • The foreign gene becomes integrated into the embryos with a yield of
about 1%.
• All the transgenic insects are being generated by using transposons as
vectors. Transposons also proved to be efficient to generate
transgenic fish, chicken and mammals (Ding et al. 2005).
• Transposons are efficient tools but they can harbour no more than 2–
3 kb of foreign DNA.

27. Efficient Generation of Transgenic Cattle Using
the DNA Transposon
• Here, efficiently generated transgenic
cattle using two transposon systems
(Sleeping Beauty and Piggybac).
• Sleeping Beauty is preferred for insertion
into “TA” sites in the host genome, while
Piggybac is preferred for insertions into
“TTAA” sites
• Blastocysts derived from microinjection
of DNA transposons were selected and
transferred into recipient cows.
• Nine transgenic cattle have been
generated and grown-up to date without
any health issues except two.
• Some of them expressed strong
fluorescence and the transgene in the
oocytes from a superovulating one were
detected by PCR and sequencing.
(Yum et. al., 2017)

28. Birth of a transgenic (tg) cow with the YFP gene
via Sleeping Beauty (SB) and its analysis.
(a) After 60 days of embryo transfer, pregnancy
was confirmed by ultrasonography. The calf was
delivered without assistant (b) and grew to 5-
months (c) and 16 months (d) old without any
health issue. (e) When ultraviolet light was
exposed to nose of tg cattle, YFP expression was
found (arrow). To determine YFP expression in
primary skin and endometrial cells, the cells were
cultured and captured by confocal image
equipment ((f-1) skin cells from a wild type, (f-2)
skin cells from a tg cattle, (f-3) endometrial cells
from a tg cattle, upper: brightness, lower:
fluorescence). The primary skin cells from tg or
non-tg were reprogrammed and developed into
blastocysts (f-4) blastocysts from skin cells of
non-tg cattle, (f-5) blastocysts from skin cells of
the tg cattle; upper: brightness, lower:
fluorescence). The tg integration was confirm by
PCR (g) and sequencing (h).
(Yum et. al., 2017)

29. Birth of a transgenic (tg) cattle with the rox-GFP-
rox-RFP gene via Piggybac (PB) and its analysis
(a) After 45 days of embryo transfer, pregnancy was
confirmed by ultrasonography. (b) The calf was
delivered without assistant. (c) When ultraviolet
light was exposed to nose of tg cattle, GFP
expression was strongly observed. And the tg cattle
grew up to 12 months old without any healthy issue
(d). To determine GFP or RFP expression in a piece
of tissue or primary skin cells via recombination, the
tissue and cells were cultured and transfected with
Dre recombinase mRNA by nucleofection ((e) a
piece of tissue from tg cattlebrightness, (e`) before
Dre recombinase transfection (GFP), (e``) after Dre
recombinase transfection (RFP)). The primary skin
cells from the tg cattle were isolated, cultured and
transfected with Dre recombinase mRNA. Before
transfection, only GFP expression was observed, RFP
expression were observed via GFP gene excision by
recombination ((f–f``) before transfection
brightness, fluorescence, and merged, respectively;
(g–g``) after transfection brightness, fluorescence,
and merged, respectively). The transgene
integration and recombination were confirmed by
genomic DNA PCR ((h)
(Yum et. al., 2017)

30. 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.

31. 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.

32. 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.

33. 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.

34. Some Examples
of
Transgenic Animals

35. Transgenic Fish
Super fish
• Growth hormone gene inserted into fertilized egg.
• Increased growth and size.
• Transgenic fish grows about 10-11 time faster than normal fish.
Glo fish
• Genetically modified zebra fish.
• Produced by integrating a fluorescent protein gene from jelly fish into
embryo of fish.

36. 27
Super fish
Glo fish

37. Transgenic Mouse
Onco mouse
• Mouse model to study cancer.
• Made by inserting activated oncogenes.
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.

39. Transgenic Pig
Enviro pig
• Pigs have trouble fully digesting a compound known as phytate in many
cereal grains used to feed them.
• Transgenic pigs are created by introducing phytase gene of Ecoli.
• Phytase enzyme is thus produced in the salivary gland of pig.
• It degrades indigestible phytase 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.

41. Transgenic Sheep
• For good quality wool.

42. Transgenic Monkey
• “ANDI” was the first transgenic monkey, born in 2000.
• ANDI proves that transgenic primates can be created and can express
a foreign gene delivered into their genome.

43. Transgenic Cow
• “ROSIE” was the first transgenic cow , born in 1997.
• Produced human protein enriched milk at 2.4g/lt contains human
gene Alpha lactalbumin.

44. Importance of Transgenic Animals
• Industrial importance –
Toxicity sensitive transgenic animals to test chemicals.
Spider silk in milk of goat.
• Medical importance –
Disease model.
Xenotransplantation.
• Agriculture importance –
Disease resistant animals.
For improving quality & quantity of milk, meat, eggs & wool production.

45. Applications
• In medical research, transgenic animals are used to identify the
functions of specific factors in complex homeostatic systems.
• In toxicology: as responsive test animals (detection of toxicants);
• In mammalian developmental genetics;
• In molecular biology:
the analysis of the regulation of gene expression makes use of the
evaluation of a specific genetic change at the level of the whole animal.

46. • In the pharmaceutical industry, targeted production of
pharmaceutical proteins, drug production and product efficacy
testing;
• In biotechnology: as producers of specific proteins;
• Genetically engineered hormones to increase milk yield, meat
production.
• Developing animals specially created for use in Xenografting

47. 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.

48. Issues related to Transgenic Technology
• 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.
• Abnormalities suffered are more.
• Reduced fertility.
• Respiratory and circulatory problems.
• Weak immune system.

49. Ethical Concerns pertaining to Biotechnology
• Introduction of a transgene from one species into another species violates
the “integrity of species”.
• Biotechnology may pose unforeseen risks to the environment, including
risk to biodiversity.
• Transfer of human genes into animals ( and vice – versa ) dilutes the
concept of “humanness”.
• When animals are used for the production of pharmaceutical proteins,
they are virtually reduced to the status of a “factory”.
• Use of animals in biotechnology causes great suffering to them.
• Biotechnology is disrespectful to living beings , and only exploits them for
the benefit of human beings.

50. How the Ethical Issues can be Overcome:
• PATENTS…..!
• Avoid unnecessery repetition of experiments.
• Use immunochemical system to replace bioassays for detecting
bacterial toxins.
• The scientists should not view animals as mere machines.
• India enacted an animal law as early as 1960's called the `Prevention
of Cruelty to Animals Act' amended in 1982 which provided for the
prevention of cruelty to animals in general.
• It also provides that the Animal Welfare Board constitute a
Committee for the Control and Supervision of Experiments on
Animals.

51. THANK YOU