This presentation deals with the scope and technique of avian genome manipulation by using avian primordial germ cells to obtain the pharmaceuticals using chicken egg as a bioreactor system and also to enhance the overall poultry production, and disease resistance, etc.

1. Major credit seminar
Primordial Germ Cells:
A Tool for Avian Genome Manipulation
Dr. Vispute Mayur M.
PhD Scholar
Roll No- P-2065

2. Introduction: Avian PGCs
 Primordial Germ Cells :
 Are pleuripotent embryonic stem cells
 Originated from epiblast region of the embryo
 Have an ability to differentiate into male and female gametes in adult life
 Localized in:
 The central disc of area pellucida of
stage- X embryos (30 PGCs approx)
 Unique migration pattern :
 Permits collection from donor and transfer to recipient at various stages of
embryonic development
PGCs in
Area pellucida
(Chojnaka-Puchata et al., 2012)

3. (Bellairs et al.,1978 )
Avian embryo: Morphological difference
Discoidal meroblastic cleavage with a
large amount of yolk and small germinal disc
Manipulation is difficult than
mammalian embryos

4. Stages of avian embryo and their nomenclature
 From 1st cleavage to primitive streak formation:
stage I to XVI - Designated by Roman numerals
e.g. EGK-stage X embryo - At the time of oviposition-
Contains around 60000 cells
(Eyalgiladi and Kochav, 1976)
 From pre-streak to hatching:
Stage 1to 46- Designated by Arabic numerals
(Hamburger and Hamilton, 1951)

5. Migration pattern of PGCs
EGK-
(Stage-X)
No. of PGCS:
 Stage- X : 30
 Stage- 4 : 250
 Stage- 31 : 1000
Left gonad may have more PGCs than right one
(Chang Guobin et al. 2010)
Entry into blood vessels
(Stage 10-12)
Dorsal hypoblast
(Stage- XI- XIV)
Entry into gonadal
primordium
(Stage-20-24)
Germinal
crescent
EGK-
(Stage-X)

6. Genome manipulation
 Genome editing:
Manipulation (insertion, deletion, modification and replacement)
of genomic DNA of living organism to produce transgenic/chimeric
organism for several applications like -
 To improve productivity of birds
 Producing pharmaceutical and therapeutic proteins
 Studies of disease resistance and immunity
 Cancer studies in human etc.
 Conservation of endangered species
(Johnson and Giles,2013; Anderson and Georges, 2004)

7. History of avian transgenesis
Naked DNA in
early embryo
Replication defective
viral vector
Blastodermal cells
PGCs
1987
1990
1993
1994
(Salter et al., 1987; Petitte et al., 1009; Vick et al., 1993 and Love et al., 1994)

8. Methods of avian genome manipulation
Sr. Method References
Earlier methods
1
Replication defective viral vectors
eg. Retrovirus, Lentivirus, Equine
infectious anemia virus (EIAV)
Eyalgiladi and Kochav, 1976
2 Direct naked DNA/ gene injection Love et al., 1994
Alternative recent methods
1 Blastodermal cell transfer Wang et al., 2006
2 Primordial germ cell transfer Naito and Kuwana, 2003
3 Spermatogoinal manipulation Wu et al., 2006 and Jung et al., 2007
4 In-vitro-electroporation and lipofection
Muramatsu et al., 1997 & 1998 and Naito
et al., 2000b & 2001c

9. Sr. Method Remarks
1 Replication defective viral vectors Difficulty in identification of pronuclei, and
culturing of treated embryo till hatching
2 Direct naked DNA/ gene injection  Mostly episomal transfer
Less success rate
3 Blastodermal cell transfer May lose ability to differentiate
Low success rate
2 Primordial germ cell transfer One of the most promising method
Successfully produced germline chimeras
4 Spermatogoinal manipulation Mostly episomal DNA and disappearsduring
embryonic development
5 Electroporation, In-vitro transfection in limited area of target site
of embryo
6 Lipofection In-vitro transfection In broader area of target
site of embryo

10. PGC mediated Avian genome
manipulation

11. Journey so far….

12. continued….

13.  Collection of PGCs from donor embryo
 Isolation and Purification of collected PGCs
 Transfection of PGCs
 Culturing of transfected PGCs
 Depletion of endogenous PGCs (24 hrs before injection)
 Injection of PGCs in recipient embryo
 Incubation of embryo s and obtaining germline chimeras
 Transgene expression constructs: For assessment of expression of
transgenes
The Technique

14. Collection of PGCs
Blood of 2.5- 3 day old embryo
13-16 H H Stage
Gonads of 5.5-6 days old embryo
Unincubated blastoderm
EGK Stage- X embryo
27-29 H H Stage

15. Isolation and purification of PGCsa
Sr. Method Principle
1 Morphology based
 Glycogen and yolk rich cytoplasm- PAS
staining
2
Density gradient dependent
centrifuge
 Low yield, less purity and viability
3 Surface Markers
A) Stage specific Embryonic
Antigen (SSEA-1)
 Surface glycoprotein, expressed only in PGCs
 Identified by anti-SSEA-1 antibody
B) Chicken vasa homologue  Presesnt at ovipositional stage
 Selective germline expression
C) Quail Germ Cell-Specific
markers (QCR-1)
 Specific surface antigen for quail
4
Magnetic Activated Cell sorting
(MACS)  Specific antibody based-
 Highly purified PGCs can be obtained
 High germline transmission rate5
Fluoroscence Activated Cell
Sorting (FACS)
6
Transwell mediated size dependent
isolation
Size based isolation of PGCs of various avian
species at stage- 14-16 of embryonic blood

16. PGCs under bright field microscopy
(MacDonald et al., 2010)
Doublets: Dividing PGCs

17. SSEA-1 Mito PAS
1.CVH - Chicken vasa homologue
2.CDH - Chicken dead end homologue
3.SSEA-1 – Surface specific embryonic antigen
Identification of PGCs
by staining
characteristics
(MacDonald et al., 2010)

18. Transfection
of PGCs
Electoporation
Lipofection
TALEN mediated
knockout
CRISPR/Cas9
PiggyBac
transposons
In-vivo
Nucleofection
Direct in-vivo
transfection
Viral vectors
Transfection: A deliberate alteration in DNA

19. Methods of Transfection of PGCs
Sr. Method Germline transmission Reference
1 Viral vectors-
Lentivirus, HIV-virus
3.6-6% Shin et al., (2008)
Montono et al., (2009)
2 Electroporation <1-86%. Microinjection of DNA
constructs
Van de Lavoir et al.,
(2004)
3 Lipofection 17% .For linear plasmid DNA Naito et al., (2007)
4 TALEN mediated gene
knockout
> 33% Parek et al., (2014)
5 CPISPR/Cas9 mediated
transfer
> 53%. Highly efficient Oshi et al., (2016)
6 PiggyBac transposons
system
Site specifc cut-paste meHigh
germiline transmission
Lee et al., (2016)
7 In-vivo Nucleofection Circular or linear DNA plasmids Naito et al., (2007)
8 Direct in-vivo transfection For the species of which PGCs can not
be manipulated in-vitro
Tyack et al., (2013)

20. Culturing of PGCs
Culturing of PGCs
On Feeder Cells
Chicken embryonic
fibroblasts
Gonadal chicken feeder
cells
Buffalo-Rat Liver (BRL)
feeder cells
Knockout Dulbeco’s
modified Eagles medium
Growth media with
LIF, bFGF, SCF
Inhibits differentiation &
enhances proliferation
(Park and Han, 2000; Van de Lavoir et al., 2004; Tang et al., 2007 and Naito et al. ,2010)
To achieve stable incorporation of exogenous DNA and in-vitro
proliferation

21. Depletion of endogenous PGCs
 Low germline transfer rate due to :
 Presence of recipient’s own PGCs
 Low Donor: Recipient PGC ratio (Naito. et al., 2007)
 Depletion of endogenous PGCs is must to improve germline transfer
rate (Carsience, 1993 and Thorawal, 1994)
Stage-X recipient
embryo
γ-irradiation
Delayed development of
treated embryos by 24 hrs
approx.
Increased donor PGC (may
exceed than recipient PGCs
Improved germline
transmission

22. Other methods to deplete the endogenous PGCs
Sr. Method Reference
1 UV irradiation (Reynaud, 1976)
2 Laser irradiation (Mims & McKinnel, 1971)
3
Application of busulfan
(Efficiency: 99% vs. 6% in treated and untreated embryos)
(Aige-Gill & Simkiss,
1991)
4 Excision of germinal crescent region (McCarry & Abott, 1982)
5
Removal of embryonic blood prior to injection of
donor PGCs (Stage 14-15)
(Naito et al., 1994a)

23. Embryo transfer of transfected PGCs
 By microinjection in the embryonic blood
 Shall be done in the recipient embryos at the same developmental stage at which
the PGCs were collected from donor embryos
 Genetic sex of donor and recipient:
Similar :
 Significantly improved survival and reproducing ability
Different (Heterosexual) :
Gonad development- with cells of opposite sex to that of the resident
cells and causes gonadal abnormalities

24. (Chojnaka-Puchata et al., 2012)
Sexing of embryos
By PCR
with primers targeting W-chromosome
specific XhoI repetitive sequences
Absence of W-specific marker
ZZ- genotype
(Male)

25. In-vitro culturing of avian embryo
(System I)
(41-41.5°C-24 hrs )
(System II)
(38°C & RH- 50-60%- 3 days)
(System III)
1) 37.8°C & RH- 50-60%- 14 days
2) 37.6°C & RH- 60-70%- 4 days
(Perry and Naito, 1989, Naito, 1990)

26. Transfect with the gene
of interest
Viral vectors
TALEN
CRISPR/Cas9

27.  Enhanced Green
fluorescent Protein :
 A jelly fish derived protein
with luminescence upon
exposure to UV spectrum
 Used as a reporter of
expression in transgenic
organisms.
(Montono et al., 2009)
EGFP: A Transgene expression construct
Extensive colonization of a Day 2 testis by
female G23 GFP positive cells
(Song et al., 2014)

28. Expression of EGFP
A & A’: Validation of PGC donor-derived chicken by monitoring the EGFP
expression using fluorescence microscopy
TG : Transgenic type
WT : Wild type
(American Society for Experimental Biology)

29. Other expression constructs
Sr. Principle Construct Type of expression References
1
Steroid responsive
promoters
Ov 100 and
Ov 900
Cell specific expression
Park and
Muramatsu
(1999)
2
Estrogen Response
Element (ERE)
regulatory sequence
ERE
Tissue specific response
of heterologus proteins in
oviduct
Lilico et al.,
(2010)
3
Chimeric ScFv-Fc
miniantibody
miR24
Synthesis of recombinant
components in egg white`
Lilico et al.,
(2010)
4 Human interferon β1 hIFNβ1a --- Do----
Lilico et al.,
(2010)
5
Oviduct specific
regulatory elements
Chicken oviduct
specific and
enhancer like region
(COSE)
Tissue specific ovalbuin
coding sequence
Park et al.,
(2006)

30. Mixed sex germline chimeras
 Produced by transfer of donor PGCs to the recipient of opposite sex-
 Excellent system for studying molecular mechanism of sexual
differentiation
 In opposite sex PGC transfer :
1) Kagami et al. (1997) :
– Stage-X PGCs have ability to differentiate into both gametes
irrespective of their genetic sex
2) Naito et al. (2001a) :
– Great amount of restriction of differentiation into functional
gamete

31. W-bearing spermatozoa
 Produced by transfer of PGCs from female donor to the male recipient
embryos.
 Also found in sex reversed chickens (Genetic female and phenotypic
male), produced by aromatase inhibitor at 5th day of incubation
(Abhinawanto et al., 1998)
 W-chromosome identified by PCR (W-specific sequences), in situ-
hybridization and Southern hybridization etc.
 Fertilizing ability is unknown
 If fertile : The sex ratio of offspring could be altered

32. Interspecific chimeras
 Produced by interspecies PGCs transfer
 Useful for proliferating endangered avian species and studying
immune rejection of donor cells into chimeras
 Quail-Chicken interspecies chimera
(Naito et al., 1991b)
 Successful differentiation of injected blastodermal cells in various
tissues
 Populations of both chicken and quail spermatozoa in gonads
(Ono et al., 1988)
 But, no viable offspring produced yet
(Watanabe et al., 1992)

33. Scope and applications of PGC
mediated genome editing

34.  Chicken and turkey genome sequencing project- completed- 2004
(Dalloul et al., 2010)
 Earth genome sequencing project- Launched to sequence DNA of all life of
earth- 1.5 million species
(Zhang et al., 2015)
Will reveal genomic information of avian species
Infinite possibilities to use this information in
developing genome-edited birds
Scope

35. 1. As a bioreactor system
 Hens- Produce valuable egg white
proteins in large quantity
 Production of biopharmaceuticals and
therapeutic proteins
Integration of target protein
sequence into ovalbumin locus
Via Homology Direct Repair
(HDR) mediated gene editing
Can produce > 1 gm target
protein/egg in low cost

36. 2. Removal or enhancement of specific nutrient
• By knocking out the allergen-
related genes like ovomucoid and
ovalbumin
Allergen free meat
and eggs
• By editing muscle-related genes
like myostatin
Muscle hypertrophy-
chickens
(Park et al., 2014 ;Crispo et al., 2015 and Oshi et al., 2016)

37. 3. Avian disease-resistant model
 Development of Avian disease-resistant birds
for high risk infectious poultry diseases
e.g. MD, RD and Avian influenza
By elimination of host factors or
receptors for avian viruses
(Biggs and Nair, 2012; Longs et al., 2016)

38. 4. Oncological studies
 Birds- Lay large no. of eggs in relatively short ovulation cycle
Thus, more susceptible to develop ovarian cancer
One of the best animal models to
study human ovarian cancer pathogenesis
 Creation of avian models like human ovarian cancer
 By precise gene editing in ovarian cancer related genes
(Johnson and Giles, 2013)

39. 5. Other avian models
Zebra finches
 Investigating biological basis of speech learning
 Neurobehavioral research
 Huttington’s disease model
Exclusive non-human model for many diseases
that can not be studied in other animal models
(Spierings and Ten Cate, 2016)

40. 6. Restoration of endangered avian species
 PGCs can be cryo-preserved in LN2 for longer duration like
other genetic resources (but chicken ova are difficult to preserve)
(Naito et al., 1994c)
Will help to restore and proliferate
the endangered species of birds

41. Restoration of Heath Hen/ Greater Prairie chicken
(Tympanuchus cupido)-
A Project by ‘Revive and Restore’: A California based NGO
World’s first genetically
engineered Greater Prairie
Chicken germ cells

42. Challenges and concern
 Efficient germline- competent cell culture system is yet to be
successful for many avian species
 Public concern for safety of GMO-derived food
Conventional GMOs
Has foreign genes or uncontrolled
random mutations
May have unknown allergens and
cause antibiotic resistance
PGC mediated Genome-edited
chickens
By precise genome editing
technology- Like natural mutations
SAFE than conventional GMOs
(Tizard et al., 2016)

43. Conclusion
 Currently PGC mediated genome manipulation is most promising way than
conventional methods
 It can be applied in creation of many avian models like egg-based bioreactor,
avian and human disease resistance, and allergen free poultry model, etc.
 There are some challenges for practical application, which can be overcome
by comprehensive research and providing technological inputs
 Thus, the PGC mediated avian transgenesis is expected to create more
possibilities for the future research and ultimately for the betterment of
mankind

44. Future perspectives
 Shall be directed to explore full potential of transgenic technology
 To develop highly efficient transfection methods and embryo culturing
system to improve germline transfer efficiency
 Introduction of in-vitro models based on chicken PGCs
 Exploration and utilization of genetic potential of diversified as well as
wild avian species

46.  Chojnacka-Puchta, L, K. Kasperczyk, K., Płucienniczak, G., Sawicka, D., Bednarczyk.
M. (2012). Primordial germ cells (PGCs) as a tool for creating transgenic chickens. Polish
Journal of Veterinary Sciences Vol. 15, No. 1 181-188
 Han J. Y. and Park, H. Y. (2018). Primordial germ cell-mediated transgenesis and genome
editing in birds. Journal of Animal Science and Biotechnology 9:19 DOI 10.1186/s40104-018-
0234-4
 Joni Macdonald, James D. Glover, Lorna Taylor, Helen M. Sang, Michael J. McGrew*
(2010). Characterization and Germline Transmission of Cultured Avian Primordial Germ
Cells. www.plosone.org.. Volume 5: (11). E15518
 M. Farzaneh, F. Attari, P. E. Mozdziak & S. E. Khoshnam (2017) Theevolution of chicken
stem cell culture methods, British Poultry Science, 58:6, 681-686,
DOI:10.1080/00071668.2017.
 Naito, M. (2003). Development of avian embryo manipulation techniques and their
application to germ cell manipulation. Animal Science Journal 74, 157–168
References