Different transgenesis method and devolopment in livestock sector.

2. Introduction
Brief history
Conventional methods
Recent techniques
Comparison techniques
Developed models in livestock
Pros & Cons
Ethical issues
Conclusion
Overview

3. What is a Transgenesis????
•Transgenesis is the process of introducing an exogenous gene into a living
organism
• which exhibit a new property
•transmit that property to its offspring
tomato
Growth hormone gene
Genetically modified tomato

4. 1985
1986
2000
First transgenic sheep and pigs
Hammer et al.
Embryonic cloning of
sheep Willadsen et al.
Somatic cloning of sheep
Wilmut et al.
Transgenic cattle Cibelli et al.
MMLV transgenic cattle Chan et
al. 1998
Gene targeting in Sheep
McCreath et al.
1997

5. 2006
2010
2007
2002Trans-chromosomal cattle
Kuroiwa et al.
Heterozygous knock-out in pigs
Dai et al., Lai et al.
Transposon transgenesis in
pigs
Jakobsen et al.
Gene knock-out in
cattle Richt et al.
Conditional
transgenesis in
pigs
Kues et al.
Homozygous gene
knockout in pigs
Phelps et al.
Lentiviral transgenesis in
pigs
Hofmann et al.
2003

6. • Classic method of gene transfer in farm animals
(Hammer et al., 1985)
Pronuclear DNA microinjection
Doyle, A., McGarry, M.P., Lee, N.A. and Lee, J.J., 2012. The construction of transgenic and gene
knockout/knockin mouse models of human disease. Transgenic research, 21(2), pp.327-349.

7. Species specific modifications are necessary
• Rabbits, pigs, sheep and goats- Embryo transfer (Brem, 1993)
• Cattle – IVM & IVF (Krimpenfort et al. 1991)
For visualisation of pronuclie
1. Cattle & pig: centrifugation prior to injection
2. Sheep : Nomarski phase contrast optics
Problem: discriminating between integrated and non integrated sequences,
(Seo et al. 1997)
• Neomycin phospho transferase (neo) (Bondioli & Wall, 1996)
• or green fluorescent protein (GFP) expression as selectable markers
Takada et al. 1997)

8. Pronuclear DNA microinjection in farm animals.
A, microinjection into a porcine zygote
B: efficiency of the DNA microinjection technique in various species

9. • Retroviral infection was the first method used to produce transgenic mice
(Jaenisch, 1976)
Viral Vectors
RNA
DNA
Integration in host genome
• Retroviruses can only integrate in dividing cells
Reverse transcriptase
Retro viral integrase

10. Chan
et.al.,
1998
• Moloney murine lukaemia virus with pseudo capsid glycoprotien
of stomatitis virus
Jaenisch,
1976
• Retroviral infection to produce transgenic mice .
Tsukui et
al., 1996
• Replication defective Adeno virus for transgenic mice
Whitelaw
et al.,
2004
• Lentiviruses for production of porcine zygotes
Ritchie et
al., 2009
• Lentivirus-mediated gene transfer in livestock

11. • mosaic foetuses were obtained (Haskell, 1995)
• limited size(< 10 kb) insertion (Brem, 1993)
• (LTRs) flanking interfere with mammalian promoters (Wells et al., 1999)
Other problems:
• gene silencing by DNA methylation due to the presence of viral sequences
• oncogene activation or insertional mutagenesis
(Hofmann et al., 2006)
Contd..
Disadvantages

12. • Sperm cells are incubated with DNA used for AI
• Plasmid DNA internalised in spermatozoa with the nuclear scaffold
recombination with genomic DNA (Spadafora, 1997)
• Generation of human decay accelerating factor (hDAF)transgenic pigs by
sperm mediated gene transfer (Lavitrano et al., 1999)
Sperm mediated gene transfer and
Intra-cytoplasmic sperm injection
Miao, X., 2013.
Recent advances in
the development of
new transgenic
animal
technology. Cellular
and Molecular Life
Sciences, 70(5),
pp.815-828.

13. • Intracytoplasmic sperm injection (ICSI)
• sperm cell membranes are damaged
• then immobile spermatozoa are used for ICSI
• Intratesticular transfection of germ cells (Kim et al. 1997)
• Male germ cells treated with busulfan
• LacZ gene introduced into each seminiferous tubule
• Transmission of the donor haplotype to the next generation after germ-cell
transplantation has been achieved in goats
(Honaramooz et al. 2003)
Contd..

14. • In mammals, the results of sperm mediated gene transfer has
 low efficiency
 repeatability (Gandolfi, 2000)
• Major obstacles of this strategy are the
 lack of efficient in vitro culture methods for prospermatogonial cells
 the lack of efficient gene transfer techniques into these cells.
(Keskintepe et al. 1997)
Disadvantages

15. Pluripotent Cells

16. • Pluripotent embryonic stem (ES) cells have the ability to participate in
organ and even germ cell development following injection into blastocysts
(Rossant, 2001)
• Targeted alterations of the genome can be induced in cultured cells by
homologous recombination
(Ledermann, 2000)
• General or tissue specific inducible gene knockouts site directed insertions
into defined loci
(Stacey et al., 1994)
• Possibility of engineering large chromosomal rearrangements
(Zheng et al., 1999)
Embryonic stem (ES) cells

17. Doyle, A., McGarry, M.P., Lee, N.A. and Lee, J.J., 2012. The construction of
transgenic and gene knock-out/knock-in mouse models of human
disease. Transgenic research, 21(2), pp.327-349.

18. • Nuclear transfer technology involves the transfer of donor nucleus into the
cytoplasm of a zygote or a metaphase II enucleated oocyte.
• Done by electrofusion of karyoplast and cytoplast
• The proof of this strategy was the generation of human factor IX transgenic
sheep by using nuclear transfer
Nuclear transfer technology
(Wilmut et al., 1997)

19. Nuclear transfer in cattle.
a—c: enucleation of a recipient oocyte to produce a cytoplast
d—f: transfer of a nuclear donor cell (karyoplast) under the zona
pellucida of the cytoplast
g: clone of four Simmental calves produced following electrofusion of
karyoplast—cytoplast complexes

20. Wilmut et al., 1997
Kato et al., 1998; Wells et al.,1999;
Zakhartchenko et al., 1999
Baguisi et al., 1999
Betthauser et al. 2000; Onishi et al.
2000; Polejaeva et al., 2000

21. Genetic modification of farm animals by DNA microinjection vs. nuclear
transfer using transfected donor cells

22. • Homologous recombination (HR) in the donor cells
(Kues, W.A. and Niemann, H. 2011)
• Gene knockout is feasible in large mammals. (Richt, J.A. et al. 2007)
Problem
• Uncontrolled, ‘passive’ nature of genomic insertion.
• Short life span of transgenic animal
Advantage and Disadvantage

23. Precision in Expression

24. • Small interfering RNA (siRNA) molecules 19–27 base pairs in length
• Transient gene knockdown, synthetic siRNAs can be transfected into cells
or early embryos
(Clark and Whitelaw, 2003; Iqbal et al., 2007)
• RNAi knockdown of porcine endogenous retrovirus (PERV) has been
demonstrated in porcine primary cells (Dieckhoff et al., 2007)
and in cloned piglets (Dieckhoff et al., 2008)
• siRNA mediated knockdown of the prion protein (PRNP) gene has been
accomplished in bovine embryos
(Golding et al., 2006).
RNA interference mediated gene
knockdown

26. • Carry very large pieces of DNA that are maintained as episomal entities
(Niemann and Kues 2003)
• Expression of human immunoglobulin in bovine
Human Ig
heavy and
light chain
Bovine
fibroblast
Trans-
chromosomal
bovine
offspring
Artificial chromosomes as transgene
vectors

27. • “Jumping genes” – 1st discovered in maize
• Active transposons : Piggy Bac,Tol2 (Clark et al., 2007)
• Transposons reactivated from defective element :
Sleeping beauty (Ivics et al., 1997)
Frog prince, Hsmar1. (Clark et al., 2007)
• Use cut-and-paste mechanism
• first transposon transgenic pigs (Kues et al.,2010; Garrels et al., 2010)
Contains a transgene
flanked by inverted
terminal repeats
Transposons

28. Co-injected
transposon and
helper plasmid
Chromosome
Expressed
SB100Xtransposase
will
catalyze the
transposition of
Venus-transposon
Stably integrated
transposon, backbone
and transposase
plasmid will become
degraded/diluted
pT2/Venus
RMCE
pCMV-SB100X
SB100X-catalyzed
transposition
CMV-SB100X
CAGGS Venus
SB100X SB100X
SB100XSB100X
CAGGS Venus
ITRITR
ITR ITR

29. • All animals are phenotypic positive
• No evidence of gene silencing
• No variegated transgenic expression
• F1 & F2 offspring shows no change in transposon mediated expression
Advantages
Contd..

30. Gene knock out mediated by
designer nuclease
• Target modification by homologus recombination : SCNT
• For non homologus recombination
Designer
nuclease
ZFN Meganuclease TALEN

31. Recognise
predetermined
genome
2 subunits of
recombinant
nuclease bind to
target locus
Each subunit nicks
one complementary
strand
Introduce a DSB
DSB repair by non-
homologus end
joining

32. +
Chemical or physical mutagen
UV-light, free radicals, irradiation, etc
3′-OH
5′-P OH-3′
P-5′
Transposase, viral integrase or ZFN
Depending on sequence specificity
one or more binding sites may exist
Ku70, Ku80 end binding factors,
DNA dependent protein
kinase,
XRCC4/DNA ligase IV
ZFNs create a DSB, in
combination with HR this may
result in a targeted integration
Passive DNA-Integration Active DNA-Integration
Exogenous protein
Double strand break (DSB)
Foreign DNA Cellular repair and
illegitimate recombination
Ectopic enzyme-catalyzed
Foreign DNA integration
Concatemeric arrays, Monomeric copy of transgene
antibiotic marker,
plasmid backbone

33. Comparison techniques
PNI SCNT ICSI Artificial
chromosom
e
Transposo
n systems
Retro- and
lentiviral
infection
ZNF
Integration Passive Passive Passiv
e
No
integration
the vector
is episomal
Active Active Active
Mechanism DSB
repair
DSB
repair
/HR
DSB
repair
Episomal
persistence
Transpo
sase
catalyse
d
Viral
integrase
catalysed
Binding
and
cleavage
of target
DNA + HR

34. PNI SCNT ICSI
mediat
ed
transg
enesis
Artificial
chromosom
e
Transposo
n systems
Retro- and
lentiviral
infection
ZNF
Transgenic
status
Stable,
often
concatem
eres
Stable,
often
concate
meres
Stable Stable in
founder
Stable,
majority
monom
eric
Stable,
monomeri
c
Stable
knock-in
Max.
construct
size (kb)
20–(500) 20–
(250)
20–
(250)
500 8–12
(60)
6–7 n.a.
Ratio of
transgenic
offspring
per born
offspring
3–10% 70–
100%
6% n.a 40–60% 50–90% n.a.
Contd..

35. PNI SCNT ICSI
mediat
ed
transg
enesis
Artificial
chromosom
e
Transposo
n systems
Retro- and
lentiviral
infection
ZNF
Preference
of
integration
sites
Random Random
or
targeted
for HR
Rando
m
n.a. Transpo
sasedep
endent,
random
or
semi-
random
Semi
random
preference
s
for
transcribed
genes, risk
of
insertional
mutagenes
is
Target
sequence/
off-target
effects
possible
Transgene
silencing
or
variegated
expression
Frequent Frequen
t
Frequ
ent
n.a. Rare Frequent Rare

36. Pigs for
xenograft
Bovine
lactalbumin in
sow milk
Enviro pig
Pigs given
spinach gene
Transgenic pig

37. Flatulant cow Herman the bull
Mastitis resistant
cow
Cattle over
expressing milk
protien
BSE resistant cow

38. Production
of anti
thrombin 3
Spider silk
Good
quality
wool
production
Tracy(α1
antitrypsin)
Transgenic sheepTransgenic goat

39. • Research human disease
• Produce consumer products
• Human therapeutic use
• Enhance production
• Improve animal health
• Creation of neo organs
• Unpredictable outcome
1. Mutagenesis
2. Functional disorder
• Destruct natural breeding
• Disruption of natural genetic
information
• Low survival rate
CONSPROS

40. • Consequences of genetic modification
• Environment safety issue
• Crossing species boundries
• Xenotransplantation issue
Ethical issues

41.  Since discovery of 1st transgenic mice all conventional techniques were
playing role.
 But for precise integration new techniques in this field evolved.
 Now transgenesis presenting difficult challenges for 21st century scientist
and ethicists
 Two major consideration
 How much a transgenic animal benefits human???
 How much pain does it cause to the animal???
Summary

42. Reference
1. Garrels, W., Ivics, Z. and Kues, W.A., 2012. Precision genetic engineering in large
mammals. Trends in biotechnology, 30(7), pp.386-393.
2. Niemann, H. and Kues, W.A., 2007. Transgenic farm animals: an
update. Reproduction, Fertility and development, 19(6), pp.762-770.
3. Wolf, E., Schernthaner, W., Zakhartchenko, V., Prelle, K., Stojkovic, M. and Brem,
G., 2000. Transgenic technology in farm animals–progress and
perspectives. Experimental Physiology, 85(6), pp.615-625.
4. Kues, W.A. and Niemann, H., 2011. Advances in farm animal
transgenesis. Preventive veterinary medicine, 102(2), pp.146-156.
5. Miao,X.,2013. Recent advances in the development of new transgenic animal
technology. Cellular and Molecular Life Sciences,70(5), pp.815-828
6. Forsberg, C.W., Meidinger, R.G., Liu, M., Cottrill, M., Golovan, S. and Phillips, J.P.,
2013. Integration, stability and expression of the E. coli phytase transgene in the
Cassie line of Yorkshire Enviropig™. Transgenic research, 22(2), pp.379-389.