A genetically modified animal with the heterologous gene of interest being inserted for the purpose of biopharming or make a diseased model to study the consequences of disease and its probable therapy

1. TRANSGENIC ANIMALS
[Production & Application]
K.K.Gupta

2. Back ground
• Improving livestock animals was an old age
practice for given genetical traits like milk
production, Egg laying efficiency , wool
characteristics through mating and selection.
(selective breeding methods
• But this traditional method was replaced by
nuclear transfer technique and nuclear cloning
techniques by which a single functional gene or
clusters are transferred into chromosomal DNA
of higher organism. .

3. INTRODUCTION
• Nowadays, breakthroughs in molecular biology are happening
at an unprecedented rate.
• One of them is the ability to engineer animals. Transgenic
animals, i.e., engineered to carry genes from other species,
have the potential to improve human welfare .
• The first genetically modified organism was a bacteria created
in 1973 by Stanley N. Cohen and Herbert Boyer.
• These bacteria contained genetic information from a variety
of different species.
• The technology has now produced transgenic animals such as
mice, rats, rabbits, pigs, sheep, and cows.
• Although there are many ethical issues surrounding
transgenesis,
• It has applications in agriculture, medicine, and industry.

4. 1980s –beginning era
• The idea of introducing gene in to egg came in
to reality.
• Thus animal whose genetic composition was
altered by addition of exogenous DNA of
interest is called transgenic and overall
process is called transgeneisis and transfer of
gene ix s called transgene

5. • Transgenic animals are those animals that
have a foreign gene deliberately inserted
or deleted from their genome.
The first transgenic animals
were mice created in 1974 by
Rudolf Jaenisch, a professor of
biology at Massachusetts
Institute of Technology.

6. Transgenic animals
Evi pig
capability of digesting
plant phosphorus by phytase
enzymes
Herman bull
for lactoferrin

7. WHY --------?
• To enhance growth of an animal like fish
which require less food and fast growth so as
to lower the cost of pork or beef or broiler
• Increasing the quality of milk
• Generating fluorescence ornamental fish
• Bio-farming for desired protein production of
medical importance .
• Generation of animal model for research and
study of human disease.

8. Transgenic animal
Transgene Promoter Transgenic
species
Purpose
Longer acting
tissues
plasminogen
Whey acidic
protein
Goat Biopharming of Tissue plasminogen activator is a
protein involved in the breakdown of blood clots
α1 Antitrypsin β Lactoglobulin Sheep Alpha-1 antitrypsin deficiency (AAT deficiency) is
an inherited condition that raises risk for lung
and liver disease. Alpha-1 antitrypsin (AAT) is
a protein that protects the lungs.(Biopharming)
Clotting Factor
IX
β Lactoglobulin sheep Heamophilia treament
Soluble CD4
protein
Whey acidic
protein
Mouse CD4 is a co-receptor of the T cell receptor (TCR)
and assists the latter in communicating with
antigen-presenting cells
Lactoferrin α Casein Cattle, for treating stomach and intestinal ulcers,
diarrhea, and hepatitis C
Urokinase αCasein Mouse a thrombolytic (THROM-bo-LIT-ik) drug,
sometimes called a "clot-busting" drug.
CFTR β casein mouse Treatement of CF
Interleukin 2 β casein Rabbit increase production of several different

9. Strategy
• Insertion of cloned gene into nucleus of
fertilized egg
• Implantation of such fertilized egg into
receptive female
• Some of offspring cells may may have this
newly introduced gene
• Animal with this gene in the germs cells are
allowed to breed for the establishments of
new genetic lines .

10. Methods of Production
TRANSGENIC
METHODS
Retroviral
method
Cloning by
nuclear
transfer
Engineered
embryonic
stem cell
method
Yeast artificial
chromosome
transgensis
DNA
microinject
ion method

11. Method at glance
Retroviral vector
with transgene
Method III
Method III

12. Retroviral
methods
• Gene are introduced by
replicative defective retrovirus
vector
Effective means of integrating small
piece of transgene of about 8 Kb
Drawbacks as some times entire genome
of the vector gets integrated in the same
nucleus as transgene so transgenic
animal/plant to be used as food is not
recommended

13. Retrovirus vector
• . To introduced gene throgh retrovirus vector
(Ex.Maloney Murine Leukemia Virus –MMLV) is made
replication defective .
• The retrovirus is designed as follows by deleting the
gag ,Pol & env gene and is replaced by therapeutic
gene.
This virus can infect both mouse and human rapidly dividing cells. However a
packaging cell is generated in order to introduced therapeutic gene.
A packaging cell line is developed as follows
5’LTR contains transcriptional
signal for all gene
Gag-capsid
pol- reverst transcriptase and
integrase
Env-envelop protein

14. Construction
• Retrovral DNA is cloned
• By restriction endoclease and limited
exonuclease 3’end of gag and all pol and env
is dleted while 5’end of gag and 5 & 3 ; LTR
were retained
• Transgene xpression directed bt propmoter
within

15. In vito
packaging
of
replicative
defective
retroviral
vector
Production of
packaged
retrovirus

16. DNA micro-injection methods
• Retroviral vector mediated delivery has has limitation in size as well
as immunogenic . So this option.
• By injecting pregnant mares serum for super ovulation followed by
HCG .
• Produced 35 eggs instead of normal 5-10 eggs
• Super ovulated females are mated and then sacrificed fertilized
eggs are flushed

17. procedure
Fertilized eggs are obtained by super-
ovulation by giving pregnant mare’s
serum and HCG after 48 hrs.
Mating with male
Female sacrificed for egg
Micro injection of transgene in to large
male pro nucleus .
I Implantation of eggs in foster mother and
made pseudo pregnant by mating with
castrated male .
A tail piece was tested for transgene by
southern blotting or PCR
Such mice mated with normal mice to
check germ line transgene presence

18. Overall efficacy of microinjection
Draw backs : Transgenesis only 5% , random site integration. Multiple copies
of trangene injection leads to over expression and disturbing physiology

19. Transgenesis by
engineered embryonic
stem cell
ESC can differentiate in to
any type of cell including
germ line cells , so as
balstocyst . Functional
Transgene is introduced at
specific sites by
transfection in blasotcyst
embryo during culture.
Selected blastocyst is
implanted for the
production. Retro viral
vector method and
microinjection are avoided

20. Transfection vector
• Allow specific sites integration at sites not coding for essential product otherwise
integration may hamper its normal physiology .Also trascription of transgene is
not prevented .Ignore spurious integration
• To enrich , a positive negative selection of blastocyst is performed
Vector- consists HB1 & HB2- homologous sequence for target site
Transgene for desired function
G-418 ( neor)
HSV tk1 &HSV tk2 outside each homologous seqence block

21. Positive and negative selection
Rely on arrangement/order of the genes in the vector
If homologous recombination involves homologous sequens then proper
integration (positive )
If outside the homologous sequence , then no n specific integration as shown
When gown in Neor , cells with no tk1 & tk2 gene will survive and when present
I is is killed intgration will be killed and those with integrated will not be killed
If grown in ganciclovir . Only those lacking ( Tk1 gene convert ganciclovir into
toxic substances
Killed in ganciclovir but
not in Neor
Survive in ganciclovir
and also in Neor

22. PCR -Another way to detect blastocyst
with specific site integration
• Such vector has two blocks of homologous sequence
to target sites consisting transgene and cloned
bacterial or synthetic unique DNA sequence.
PCR can not generate bands due to no hybridization of primer as it is made
complementary to sequence adjacent to homologous site (HB1 & HB2 are
Complementary o CS 1 & CS2 sites

23. Knock out gene
• Transgenesis is not only for gain of
gene but also disruption (knock) of
gene function. This is done to know
the developmental and
physiological consequences of
knock out gene For example
rhodopsin gen knocking cause no
rod cells like retinitis pigmentosom
in human
A nude mouse is a laboratory mouse from a strain with a
genetic mutation that causes a deteriorated or absent
thymus, resulting in an inhibited immune system due to a
greatly reduced number of T cells. The phenotype (main
outward appearance) of the mouse is a lack of body hair,
which gives it the "nude" nickname.

24. Disruption of gene

25. Cloning by
nuclear transfer
Wilmut et al. 1997
• Nucleus of Ovum is removed
and Go nucleus of mammary
epithelial cells are introduced
in the enucleated ovum by
cell fusion methods
• Implantation into foster
mother . 1 out of 29
transferred at early stage
embryo produced a live
lamb Scottish black face – Cytoplasmic
Donor
Fin Dorset – Nucleus Donor
Ewe -Surrogate mother

26. YEAST ARTIFICIAL TRANSGENESIS
• Transgenes are generally cDNA </= to 20kb of part of genes .
Typicall cDNA poorly expressed or genomic DNA fragments
lack upstream sequence , so not exppressed.
• Complete gene more than 100 kb is not suitable for
conventional vector so YAC can 100- >1000kb. It is micro
injected or transfected with yac that carry multi gen or large
gen. Eg human beta globin gen cluster of 250 kb bearing five
cluster gen. Als has promoter and regularory sequence.
• Unfortunetly Mab produced in mice is immunogenic to
humans

27. Ig gene
• H chain consist -95 VH,30DH.6JH and 5 main
constant for heavy chain alpha, beta , gamma,
epsilon and mu
• K gene has 76 Vk , 5Jk, and 1 constant (Ck)
• To creaste a transgenic mice for the
production of this antibody , the endogenous
such region is inactivated and yAC of 1000 kb
bearing 66 VH , 30 DH 6JH and mu, delta and
gama gene is introduced .

28. Application
• Transgenic mice – made as model to study disease like Alzisheimer,
muscular dystrophy, tumourogenesis, hypertension
neurodenerative disorders endocrinological disorders, coronary
diseas etc. in order to study its etiology and for production of
therapeutic agent
• APP gene ( β amyloid precursor protein )with platelet derived
growth factor promotor , complete construct -PDAPP minigene is
responsible for plaques formation, , neuronal cell death and
memory loss due to production of Aβ42 which lyse the Asn and
leucine instead of lysine & methionine at APP67/671 position
• Similarly CFTR transgene has been introduced for its secretion in the
milk to treat cystic fibrosis patient