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RECENT TRENDS IN

POULTRY
PRODUCTION
S, P.TIWARI O, P.DINANI
Chapter 21

Advances in Avian Transgenesis

Dr. R. Richard Churchil, Ph.D
Professor, Department of Poultry Science, Madras Veterinary College,
Tamil Nadu Veterinary and Animal Sciences University, Chennai-Z
E-mail : drchurchil@)zahoo. com

lntroduction
Transgenic chickens hold promise in many commercial and
industrial applications beyond the intrinsic value as model systems in
biological research. Chickens deposit large amounts of proteins into
their eggs. A transgenic chicken could conceivably function as a bio-
reactor for the production of commercially or pharmaceuticaliy
important proteins, which are difficult or impossible to produce
economically in prokaryotic systems. Gene transfer technology may
also prove to be a useful complement to traditional poultry breeding
methods in the improvement of commercially important production
traits, such as growth and disease resistance. Both intra and inter-
species transfer could be used in poultry to introduce genes controlling
desirable physiological functions unavailable in the existing pool of
genetic variation. The quality of the product can be modified by
biotechnolory; a transgenic layer chicken can produce more eggs with
less cholesterol or a genetically engineered broiler chicken can produce
lean meat or with a favourable lipid profile to reduce health risk to the
consumer.

What is transgenic chicken?

A transgenic chicken is carrying recombinant DNA molecules that
were intentionally introduced by human intervention in contrast to
spontaneous mutation. The introduced DNA may or may not derive
from the same species as the host genome; and it may or may not be
targeted to an intended site of incorporation in the genome. Although
the term'transgenic animal' fully represents all such cases, more specific
terms, such as 'knockout animals' or 'mutant animals' are used to
274 Recent Trends in Poultry Production

notify the targeted disruption or mutagenesis of selected endogenous

gene sequences in the gerilr line animals. Classically, transgenesis
entailed the stable integration of the transgene within the host animal's
genome and its transmission to progeny through normal breeding
Programmes.

H istory of Avian Transgenesis

Gordon and Ruddle (1981) first demonstrated that the genetic
material can be successfully transferred into the genomes of newborn
mice by injection of that foreign material into pronuclei of fertilized
eggs. The first report in chicken was described after eight years, in
which the transgenic chicken resistant to pathogenic Avian Leucosis
Virus sub-group A infection was generated using an avian retrovirus
proviral insert (Salter and Crittenden, 1989). Lentiviral vectors were
proved to produce chimeric chickens and quail from eggs, but
integration is random and leaves a viral DNA footprint in the chicken
genome (McGrew et a1.,2004). Later, in2006, the ability of Primordial
Germ Ce1l (PGC) to grow and to be maintained in culture, then injected
PGCs into embryos can migrate to the gonad and can form functional
gametes (van de Lavoir et a1.,2006) was studied. In the yeat 2012,
Macdonald et nl. (2012) and Park and IHan (2012) demonstrated the
integration of a transgene using vectors with transposable elements
and a transposase enzyme. Immediateiy after that, Schusset et al. (2013)
described gene targeting by homologous recombination in cultured
PGCs to generate a specific gene knockout bird. Later, precise
alterations to the genome including gene knockout and gene edits in
PGCs mediated by TALENs have been demonstrated by Park et al.
(2014). The development of avian influenza resistant chicken (Lyall
et nl., 2011) and biopharming (Herron et al., 2018) are the latest
developments in avian transgenesis.

Advantages of avian species as a transgenic bio-reactor

The advantages of using chicken oviduct as a bio-reactor for the
production of therapeutic bio-molecules have been described by Ivarie
(2003). The mammary gland as bio-reactor has several disadvantages
like long generation interval and difficulties of protein purification
from a large liquid volume containing many milk proteins and fat.
Contamination with potential human pathogens in raw milk is also a
concern. The use of the hen can circumvent many of these problems
and has additional advantages. More than half of the egg white protein
Advances in Avian Transgenesis 275

content derives from the ovalbumin gene with four other proteins
(lysozyme, ovomucoid, ovomucin and conalbumin) present at levels
of 50 milligrams or greater (Gilbert, 1984). Modern layers produce 330
eggs per year so three or four hens producing one gram per egg would
yield a kilogram of raw product annually. The naturally sterile egg
also contains egg white protein at high concentration allowing for a
long shelf-life of recombinant protein without loss in activity (Harvey
et al., 2002).

Difficu lties i n avian transgenesis

Single celled ouum/ zygote is not nccessible: A laying hen ovulates
once a day and the oocyte (the yolk) is immediately taken up by the
oviduct and fertilised within approximately 15 min of ovulation. The
embryo develops rapidty inside the oviduct, reaching the blastodermal
stage of development by the time the egg is laid. DNA microinjection
is performed in one celled ovum; therefore, not possible ln chicken.
However, the newly fertilized zygotemay be recovered from the oviduct
by sacrificing a laying hen within two to three hours of the previous
oviposition (Sang, 1994). But this is a complicated procedure'
Structure of oaum hinders manipulation: DNA microinjection into
the pronucleus of ovum is the most efficient method of transgenesis
(Brinster et a1.,1981). However, the production of transgenic birds by
this method has been hampered by the yolk-laden structure of the
ovum and their unique reproductive system. Also, identification of
the male pronucleus among the supernumerary spermatozoa, as well
as the return of the ovum to the oviduct of a fistulated hen are very
difficult (Pancer et aI.,1989). Visualisation of the pronuclei is not readily
possible in live material, as they are located approximately 50mm below
the surface of the yolk and the cytoplasm is opaque with yolk particies.
Further, the embryo in chicken is also surrounded by severai layers of
albumin, sheli membrane and shell, which limits easy access to embryo.
Nuclear transfer is not possible in aoian species: Nuclear transfer is
the other available gene transfer strategy for generating transgenic
livestock in which the nucleus is transferred into the enucleated
ovum or into the enucleated one-cel1ed embryo. This is the technique
by which the clones (like dolly) are produced. Nuclear transfer involves
the manipulation of single-celled zygote. Nuclear transfer is a
difficult procedure in chicken, since the presence of yolk material,
albumin, shell membrane and shell makes the single celled embryo
inaccessible.
276 Recent Trends in Poultnz Production

Methods of transgenesis
A number of strategies designed to allow the manipulation of the
avian genome have been investigated with varying degrees of success.
They are:

1. Viral mediated gene transfer

This is the nature's natural gene delivery system. Viral vectors
have a RNA genome, which is transcribed through the virus's own
reverse transcriptase into DNA in infected cells and subsequently
integrated into the genome of the cell. Retroviral vectors can either be
replication-competent or replication-defective. Replication-defective
vectors are the most common choice in studies because the viruses
have had the coding regions for the genes necessary for additional
rounds of virion replication and packaging replaced with other genes,
or deleted. These virus are capable of infecting their target cells and
delivering their viral payload, but then fail to continue the typical lytic
pathway that leads to cell lysis and death. Conversely, replication-
competent viral vectors contain all necessary genes for virion synthesis,
arLd continue to propagate themselves, once infection occurs. Because
the viral genome for these vectors is much lengthier, the length of the
actual inserted gene of interest is limited compared to the possible
length of the insert for replication-defective vectors. Depending on
the viral vector, the typical maximum length of an allowable DNA
insert in a replication-defective viral vector is usually about 8-10 kB
These retroviruses were replication-competent vectors derivedfrom
avian leucosis virus (ALV) (Salter and Crittenden, 1989), replication-
defective vectors derived from reticuloendotheliosis virus (Bosselman
et a1.,1989), an ALV replication-defective vector (Harvey and Ivarie,
2003) and an avian spleen necrosis virus vector (Mozdziak et a1.,2003)
are used in production of transgenic birds. The efficiency of production
of transgenic chickens using these vectors was very low (- 0.5 % of G\
progeny); however, they were capable of generating germline modified
birds including transgenic hens that express low levels of a therapeutic
protein in egg white (Harvey and Ivarie, 2003). Germ line transgenic
quails were produced using a VSV-pseudotyped retroviral vector based
on Moloney murine leukemia virus pseudotyped vesicular stomatits
virus G protein (Mizuarai et al., 2001). The production of transgenic
chickens carrying theTacZ marker gene using the avian spleen necrosis
virus (SNV) based retroviral vector has also been reported
(Borwornpinyo et al., 2001).
Advances in Avian Transgenesis 277

There are some important limitations to this method. The lentiviral

vectors have three main limitations (reviewed by Bednarczyk,2016):
(i) restriction of the size of the vector genome to less than 8 to 10 kb
(Wu ef al., 201,0); however, the promoters necessary to drive specific
expression in some cells exceed this size; (ii) vector insertion can cause
disruption of endogenous genes by insertional mutagenesis or the
transactivation of neighbouring endogenous genes (Li and Lu,20L0);
and (iii) integrated lentiviral vectors are subject to positional effects
(Yi et nl., 201-1). Moreover, the viral transduction technique is
characterized by high embryonic lethality rates, and relatively low
and unpredictable rates of germline transmission and production of
transgenic chickens (McGrew et a1.,2004). Further, technical and safety
considerations limit the usefulness of retrovirus mediated gene transfer,
particularly in agricultural applications (Salter et nl., 1986; Harvey
et a1.,2002).
2. lVicroinjection
DNA microinjection is very expensive and laborious to obtain
zygote by superovulation and surgical collection. However, this method
requires special equipments and specific ski1l and is not useful for some
species specifically the avian, the fertilized one-cell embryo of which
is not accessible. To circumvent these problems, Love et al. (1994) used
the eggs removed from the oviduct of laying hens approximately 2.5
hours after the time of lay of the preceding egg. The eggs were already
fertilized but the pronuclei which were located in the germinal disc
have not yet fused. The egg is she1l-less at this stage but the zygote can
be cultured ex aiao in a host shell for the entire embryonic period
through to hatch as per the procedure described by Perry (1988).
In chicken, microinjection is successfully combined with stem cell
or viral mediated mediated gene transfer. In this strategy,
microinjection of viral particies is done beneath the blastoderm into
the sub-germinal cavity (Salter et a1.,1986). The egg after microinjection
is then sealed and allowed to hatch. Virus infected the blastodermal
cells and ALV proviruses integrated at random into the genomes of
some germline progenitor cells. This led to G0 mosaic founders from
which G1 hemizygotes containing the viral sequences in all cells were
bred.
3. Embryonic stemi primordialcell-mediated gene transfer
Embryonic Stem (ES) cells are undifferentiated cells that have the
potential to differentiate into any type of cell (somatic and germ cells)
278 Recent Trends in Poulhy Production

and therefore, to give rise to a complete organism. The PGC are the
precursors of the cells, which compose the germline. They are excellent
candidates for genetic modification. This cell mediated method involves
prior insertion of the desired DNA sequence by homologou.s
iecombination into an in aitro co-culture of ES or PG cell along with
the vector having a desirable gene sequence. Genetically, modified ES
and PG cells can also be produced by infection with replication-
defective retroviral vectors, by lipofection and by electroporation. The
transfected cells can be separated by markers and then incorporated
into a recipient embryo at the blastocyst stage of development. The
result is a chimeric chicken in the founder generation. If some of the
transfected embryonic stem cells also contribute to the germline of the
chimera, it can serve as the founder of a transgenic line. A chimeric
intermediate produces both transgenic and normal birds in the first
generation. The transgenic offspring breed true from the second
generation onwards.
Origin of Primordial Germ Cells: ln chicken embryos, PCC arise in
the epiblast at the centre of the Stage X blastoderm and migrate to the
hypoblast at Stage 12. During gastrulation, they continue to migrate
anteriorly and laterally until they arrive at the germinal crescent after
18 h of incubation. With the establishment of the embryonic vascular
system, they migrate into the circulating blood and reach the peak
number of PGC at Stage 13 to 14 (about 50 h of incubation). Then,
PGC migrate through the blood vessels (Ginsburg and Eyal-Giladi,
1986) and finally arrive at the germinal ridge and begin to proliferate.
In adults, PGC differentiate into oogonia or spermatogonia (Fujimoto
et nl., 1976).
Isolntion and culture: The PGCs can be isolated from different
embryonic tissues, such as the germinal crescent at HH stage 4,
extraembryonic blood vessels at embryonic day 2.2 and embryonic
gonads at embryonic days 5 to 6. Because of their extra-embryonic
origin and specific migration routes, PGC could be collected and
transferred at four regions: the centre of blastoderm at Stage X, the
germinal crescent during gastrulation, the early vascular system at
Stage 13 to L4, and the germinai ridge after 72 h of incubation.
Applicntion of genome-editing in aaian trnnsgenesis: The genome
editing tools and their applications in avian transgenesis is reviewed
by Hwang and Han (2018). The total germ-line transrnission efficiency
from targeted PGCs is approximately 0.1% due to natural homologous
recombination; the efficiency can be improved to 8% by using TALEN
Advances in Avian Transgenesis 279

technology (Park et a\.,2014). This case is the first reported

programmed DNA nuclease-mediated knockout chicken. Later, the
CRISPR/ Cas9 system was used to efficiently generate ovomucoid
gene-targeted chickens by transferring transiently drug-selected PGCs
into recipient embryos using gamma-ray irradiation to deplete
endogenous PGCs (Oishi et nl., 2016). More recently, CVH gene-
targeted chickens via the TAlEN-mediated HDR system were
produced using 2-week-recovered PGCs with GFP transgene knockin
at the CVH locus with 8.1% efficiency (Taylor et a1.,2017). A recent
method, called sperm transfection-assisted gene-editing based on direct
delivery of the CRISPR/ Cas9 complex, is a potential alternative for
avian transgenesis and genome editing without culturing PGCs, despite
the low efficiency of genome editing and germ-line transmission
(Cooper et al., 2017).

Production of germline chimera and transgenic birds

a) Embryonic Stem cell manipulntion and transfection: When chicken
ESCs were transplanted after in aitro transfection, transgene-
expressing chimeric chickens were produced (Zhu et al.,
2005). Despite several efforts witl-tin aitro culttre of chicken
ESCs, germline transmission capacity was extremely low and
has not been repeated experimentally so far (Lavial and Pain,
2010).
b) Virus mediated Germline chimera prodtLction: Another method of
chimera production is by infecting the blastodermal cells of stage
X embryos with retroviral or lentiviral vectors. Transgene
expression was observed in various tissues (including germ cells)
after further development, indicative of the contribution of
blastodermal cells to somatic and germ cells (Bosselman et al,
1989; McGrew et a1.,2004). However, compared with the somatic
chimerism rate, germline transmission efficiency of blastodermal
cells was too low.
c) lntra-species chimerns: Petitte et al. (1990) produced chimeras by
injecting dispersed blastodermal cells of Barred Plymouth Rock
chicken embryos into White Leghorn chickens at the same stage
of development as the recipients. By transferring circulating
PGC (cPGC) Tajima et al. (1993), and Han et al. (1997) produced
germ-line chimeras between White Leghorn and Rhode Island
Red chickens and Silky fowl and White Leghorn chickens,
respectively.
280 Recent Trends in Poultry Production

d) Interspecies germline chimera for restoration of endangered species:

PGC isolation have been attempted not only in chicken, but also
in other avian species, such as pheasant, quail, turkey, duck and
Guinea fowl, and have been produced using PGCs for the
purpose of restoring endangered birds. Many studies have been
conducted on the production of interspecies germline chimeras
by transplantation of PGCs, blastodermal cells and SSCs,
including quail-to-chicken (Watanabe et al., 1992), turkey-to-
chicken (Reynaud, 1,969), duck-to-chicken (Li et a|.,2002)
chicken-to-duck (Liu et a\.,201.2) and chicken-to-Guinea fowl
(van de Lavoir et a1.,2012). Recently, donor-derived progeny of
wild birds, including pheasant (Kang et a1.,2008) and Houbara
bustard (Wernery et nI.,2010) using PGCs and gonadal cells,
respectivellu w€re produced successfully using the interspecies
germline chimera system. Endangered species, which increase
every year, ate an important and global problem that can be
addressed.

4. Testis mediated gene transfer

Testis Mediated Gene Transfer (TMGT), involves the in

oiao transler of genes into the testes in order to deliver DNA into eggs
via the spermatozoa as an alternative method of transgenic animal
production (Sato ef al., 2002).In this method, exogenous genes are
introduced into spermatozoa and spermatogonia in the testis by using
lipofection reagents and spermatozoa ejaculated from the testis are
used for artificial insemination. Although the expression of their
transgene was detectable in morula-stage embryos, the ratio of
offspring carrying the transgene was very low.

5. Sperm mediated gene transfer (SIVGT)

Spermatozoa could interact with exogenous DNA and could
convey such DNA into the egg via fertilization is the basic principle in
this method. The precise mechanisms mediating integration of a
transgene into the sperm genome remain largely unknown. Some
groups have suggested that an endogenous enzymatic system, which
might be activated for DNA repair, could be involved in the integration
of foreign DNA (Spadofora, 1998). SMGT, when applied to chicken,
has additional advantages: low cost and ease of use. In this method,
the sperm cells are made transgenic first using various methods, like
transfection with nude DNA, electroporation, restriction enzyme-
Advances in Avian Transgenesis 281

mediated inte gration (REMD (Churchil, e t al., 2011), receptor-mediated

gene transfer, intracytoplasmic sperm injection (ICSI) and lipofection
(Churchil, et al., 2011). Though there has been few works on the
production of other species of transgenic animals, virtually no report
is available for chicken. One such work was conducted at Central
Avian Research Institute, lzatnagar by the author (Churchil and
Sharma, 2013). Transgenic sperms were efficiently produced and
internalization to the genomic DNA was tested. For this transgenic
study, pVIVO2-GFP/LacZ vector was used. Three type of inserts i.e.
5317 bp fragment containing LacZ gene,2152bp fragment containing
GFP gene and 9620 bp linearized plasmid were used as exogenous
DNA. In the second phase, transfection experiments were conducted
using purified LacZ/ GFP / whole vector gene constructs as exogenous
DNA by nude transfection, lipofection and Restriction Enzyme
Mediated Integration (REMI) methods (Churchil and Sharma,2013).
The integration of foreign DNA was confirmed by amplification of
specific reporter gene (s) by PCR and by hybridizalion of dot blots
using biotinylated DNA probes. Lipofection and REMI were found to
increase the DNA uptake by the spermatozoa compared to nude DNA
transfection (ChurchTl, et al., 2011).
The efficiency of SMGT is low. This is mainly due to the poor
uptake of exogenous DNA by the spermatozoa, thereby reducing the
number of fertilized oocytes with transfected spermalozoa (Anzar and
Buhr, 2006).In addition, interspecies and intraspecies success
variability remains a problem (Garcia Yazqtez et a1.,2009). The poor
reproducibility of SMGT was suggested to be due to the activation of
defense mechanisms in the spermatozoa and seminal plasma, resulting
in degradation of the exogenous DNA (Sato et a1.,2003; Kang et al.,
2008). Celebi et al. (2002) suggested that the transferred plasmid was
lost during the numerous germ cell divisions required to make
functional sperm. The other factors include the obstacle created by
the plasma membrane of the spermatozoon itself and the potential
damage to the sperm genome caused by membrane permeabilization.
Transgenesis for Developing Disease Resistant Poultry
The first transgenic chicken resistant to pathogenic Avian Leucosis
Virus sub-group A infection was generated using an avian retrovirus
(Salter and Crittenden, 1989). Rapid advances in genomics and gene
editing suggest that disease resistant, genetically modified chickens
could represent a viable solution to the problem of diseases, particularly
avian influenza in the future. Lyall et al. (2011) generated transgenic
282 Recent Trends in Poultry Production

chickens expressing a short-hairpin RNA designed to function as a

decoy that inhibits and blocks influenza virus polymerase and hence,
interferes with virus propagation.

Transgenesis for prod uctivity im provement

Transgenesis can improve genetic features of domesticated
Animals: Improved food quality-manipulation of biosynthetic
pathways. Transgenesis can bring in reduced or improved fat/
composition, overall body composition (lean/fat ratio) in poultry.
Improved food quantity-feed efficiency, rate of gain, overall body
composition and size in poultry can be obtained.

Biopharming
Transgenic poultry has the potential to be used for the production
of pharmaceutical and industrial proteins in eggs (Ivarie, 2003) and
several successfully generated transgenic chickens as a bioreactor have
been reported (Zhu et al., 2005; Lillico et al., 2007). Recently, genome-
editing tools, such as transcription activator-like effector nuclease
(TALEN) and clustered regularly interspaced short palindromic repeat
(CRISPR)/ CRlSPR-associated protein (CRISPR/ Cas) have been
adapted as promising methods for developing avian model (Park et
nl., 2014; Oishi et al., 201,6; Taylor et nl., 2017). Chicken bioreactors
provided, among others, human erythropoietin (Koo et a1.,2010),
interferon alpha- 2b (Rapp et al., 2003), interferon beta-1a (Lillico ef
al., 2007), monoclonal antibodies (Kamihira et al., 2005) and
granulocyte colony stimulating factor (G-CSF) (Kwon et a1.,2008).
Since 2011, the U.S. Food and Drug Administration Centre for
Drug Evaluation and Review (CDER) and the Centre for Bioiogics
Evaluation and Review (CBER) combined have approved 62
recombinant therapeutic proteins (Lagass6 et al., 20L7). Many
biopharmaceuticals are produced using bacterial, yeast or mammalian
cell culture expression systems. Current mammalian cell-culture based
systems are increasingly optimised and industrialized (Shukla et al.,
2017). The first drug produced in a transgenic animal, antithrombin
III in goat milk, was approved for use by the European Medicines
Agency in 2006 and the FDA in 2009 (Kues and Niemann, 2011).
Almost seven years later, the FDA has approved a second
biopharmaceutical product made using a transgenic chicken. The
product is Alexion Pharmaceuticals' 'Kanuma' (sebelipase alfa) for
the treatment of patients with the rare disease lysosomal acid lipase
Advances in Avian Transgenesis 283

(LAL) deficiency. Kanuma is made using GE chickens containing an

rDNA construct responsible for producing rhLAL - a protein that
functions in place of the missing or inactive LAL protein in the patient
- in their egg whites. The whites are then refined and the rhLAL protein
extracted and purified. As a result of these successes, an established
regulatory framework to approve therapeutic protein produced using
transgenic approaches is now in place, in both the EU and USA.
Researchers have for a number of years being perfecting an approach
to produce transgenic hens that can lay eggs that are rich in a
specifically targeted therapeutic protein. More recently, transgenic
chicken expressing IFNalpha2a (human cytokine interferon 6.2a), a
powerful antiviral and anti-cancer molecule and macrophage-CSF, a
therapy that stimulates damaged tissues to repair themselves (Herron
et a1.,2018) are developed.

Conclusions
The benefits of biotechnology are plenty. Animal transgenesis raises
some ethical problems as embryo manipulation and transgenes
themselves may reduce animal welfare. Thoughtful ethical decision-
making is needed in the biotechnology industry, scientists, policy-
makers, and the public. The safety of the product should be ensured
which should be approved through a science-based regulatory process.
Another important question is whether human welfare the only
consideration? What about the welfare of other life forms? Rather,
should scientists focus oninaitro (cultured in a lab) transgenic methods
to limit the use of using live animals/ birds to maximum extent possible
to alleviate animal suffering? Efficient methods for producing
transgenic animals expressing recombinant proteins have been
developed over the past 20 years. On positive side, transgenic animals
offer opportunities to significantly reduce costs in producing mAb and
human recombinant proteins with post-translational modifications that
closely match those of human proteins. Recent technical advances,
namely gene editing has opened new avenues. We can anticipate many
developments on efficiency of techniques in avian transgenesis that
will provide a critical tool to increase productivity in the poultry and
biopharmaceutical industries in the future.

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