Blackwell Science, LtdOxford, UKASJAnimal Science Journal1344-39412003 Blackwell Publishing Asia Pty LtdJune 2003743157168Review ArticleManipulation of avian embryosM.

NAITO

REVIEW ARTICLE
Development of avian embryo manipulation techniques
and their application to germ cell manipulation
Mitsuru NAITO
National Institute of Agrobiological Sciences, Tsukuba-shi, Japan

ABSTRACT
The development of chicken embryo culture techniques, from single-cell stage to hatching, makes it possible to manipulate
developing embryos at any developmental stage. Production of germline chimeric chickens by the transfer of stage X blas-
todermal cells or primordial germ cells enables the manipulation of germline cells in vitro. Production of transgenic chickens
has been attempted by the retroviral vector method, microinjection of DNA into a fertilized ovum at the single-cell stage,
use of chimeric intermediates produced by the transfer of stage X blastodermal cells or primordial germ cells, manipulation
of spermatozoa, and in vivo manipulation of gonads. So far, the only non-viral method that has successfully produced
transgenic chickens is microinjection of DNA into a fertilized ovum. Manipulation of primordial germ cells could become
an efficient system for producing transgenic chickens by combining it with the highly efficient transfection method or the
in vitro culture system for primordial germ cells. Preservation of avian genetic resources has now become possible by cry-
opreservation of stage X blastodermal cells or primordial germ cells as well as spermatozoa. The development of nuclear
transfer techniques for avian species is necessary.

KEYWORDS: chicken, embryo manipulation, germ cell, germline chimera, transgenesis.

INTRODUCTION applications. Production of pharmaceutical materials

in the eggs of transgenic chickens is the first target
The avian embryo provides an excellent model for application, and success in this production system
studying embryology, especially in the amniotes of could lead to the commencement of a new industry.
higher vertebrates. Manipulation of early avian Manipulation of early chicken embryos has been
embryos has been hampered, however, by their dis- reviewed elsewhere by Naito (1997, 1998, 2003), and
tinctive reproductive characteristics. The chicken manipulation of quail embryos has been reviewed by
ovum is fragile and contains a large amount of yolk. Ono (1997, 2001). In this review, recent studies on
Fertilization takes place within 15 min of ovulation chicken embryo culture and embryo manipulation
and early embryonic development proceeds in the techniques for manipulation of germ cells are
hen’s oviduct during the egg formation process. After described.
oviposition, embryonic development proceeds inside The developmental stages of chicken embryos from
the eggshell. Development of a chicken embryo cul- first cleavage to primitive streak formation (stages I to
ture technique from the single-cell stage to hatching XIV) are designated by roman numerals (Eyal-Giladi &
(Perry 1988) enables the manipulation of early stage Kochav 1976), and from prestreak stage to hatching
embryos which produce viable offspring. Using this
embryo culture technique, various attempts have been
made to produce transgenic chickens as well as germ- Correspondence: Mitsuru Naito, National Institute of Agro-
biological Sciences, Tsukuba-shi, Ibaraki-ken 305–8602,
line chimeric chickens. Japan. (Email: mnaito@affrc.go.jp)
Genetic manipulation of chickens provides great Received 28 October 2002; accepted for publication 16
potential for the basic sciences as well as practical January 2003.

Animal Science Journal (2003) 74, 157–168 157

M. NAITO
(stages 1 to 46) by arabic numerals (Hamburger &
Hamilton 1951).
EMBRYO CULTURE
Chicken embryo development from fertilization to
hatching can be divided into three phases: fertilization
to blastoderm formation (phase I) for one day,
embryogenesis (phase II) for three days, and embry-
onic growth (phase III) for 18 days. During these
periods, the differences in the specific gravity among
the yolk (1.029), thick albumen (1.036) and thin albu-
men (1.040) play a very important role in the normal
development of chicken embryos (Suda et al. 1994).
Perry (1988) devised three discrete culture systems
(systems I, II, and III) to meet the changing demands
at successive stages of development, and obtained a
System I
System II
System III
Fig. 1 In vitro culture system for chicken embryos. A single-cell stage embryo is cultured in vitro using 3-step culture techniques
(systems I, II and III). The embryo develops normally and hatches after 22 days in culture.
Animal Science Journal (2003) 74, 157–168
7% hatching rate by culturing embryos from the
single-cell stage to hatching. This technique can be
applied to culture embryos during the early cleavage
stages (stages I–II), collected from the uterus of the
oviduct, resulting in a 22.5% hatch rate (Naito & Perry
1989). In order to improve the hatch rate of cultured
embryos, Naito et al. (1990) modified Perry’s culture
system, increasing the hatch rate to 34.4% by cultur-
ing embryos from the single-cell stage to hatching. The
modified method of chicken embryo culture from the
single cell stage to hatching is as follows (Fig. 1). A fer-
tilized ovum obtained from the posterior portion of the
magnum of the oviduct at 2.75 h after the preceding
egg is laid is cultured in a glass vessel with a small
amount of thin albumen (system I). At this stage the
yolk is surrounded by a small amount of thick albu-
men. The fertilized ovum is incubated at 41–41.5∞C for
24 h. Then the thick albumen and culture medium are
4-day incubated
embryo
8-day incubated
embryo
22-days at
hatch
158
Manipulation of avian embryos
removed and only the yolk is transferred to the recip-
ient eggshell, filled with thin albumen, covered with
cling film, and secured with plastic rings and elastic
bands (system II). The reconstituted egg is incubated at
38∞C and 50–60% relative humidity for three days
with rocking. Thereafter, the contents of the reconsti-
tuted egg are transferred to a larger recipient eggshell
with air space, and the opening of the eggshell is cov-
ered with cling film (system III). The egg is incubated
at 37.8∞C and 50–60% relative humidity for 14 days
with rocking, and then incubated in stationary pos-
ition at 37.6∞C and 60–70% relative humidity for
another four days until hatching. When embryos are
cultured from the laid egg stage (stage X), embryo cul-
ture systems II and III are used to obtain a 50.0–62.5%
hatch rate (Naito & Perry 1989; Naito et al. 1990).
When three-day incubated embryos (stage 18) are cul-
tured, embryo culture system III is used.
A fertilized ovum with a thin layer of thick albumen
obtained from the anterior portion of the magnum of
the oviduct 60–80 min after the preceding egg is laid
can be cultured in system I, using a piece of gauze
overlaying the whole egg (Naito et al. 1995). The cul-
tured embryo is then incubated using systems II and
III. The hatch rate was reported to be 19.5%. A tech-
nique for in vitro culture of an ovum just after fertili-
zation without thick albumen, or in vitro fertilized ova
has not yet been devised for chickens. However, the
fertilized ovum can be transferred into the oviduct of a
recipient hen (Tanaka et al. 1994). Thick albumen,
eggshell membranes and eggshell are formed around
the ovum and, after laying, the embryo develops nor-
mally inside the eggshell through to hatching.
Development of the chicken embryo culture tech-
nique from the single-cell stage to hatching enables
access to developing embryos at any developmental
stage. Manipulation of chicken embryos from stage X
onward can also be done by opening a small hole in
the eggshell (Etches et al. 1993; Speksnijder & Ivarie
2000). Using the chicken embryo culture technique
increases accessibility of the embryos because the
whole embryo can be exposed.
PRODUCTION OF GERMLINE CHIMERIC
CHICKENS
Development of the chicken germline
From the analysis of germline-specific expression of
the chicken Vasa gene, the chicken germline is thought
to be determined by maternally inherited factors in the
Animal Science Journal (2003) 74, 157–168
germ plasm (Tsunekawa et al. 2000). The Vasa-positive
structure is predominantly localized in the oocytes,
and Vasa protein is restricted to the basal portion of the
cleavage furrow during the early cleavage stages. At
stage X, approximately 30 Vasa-positive cells are scat-
tered in the central disc of the area pellucida. These
Vasa-positive cells, called primordial germ cells (PGC),
are located in the ventral surface of the area pellucida
(Karagenc et al. 1996). Primordial germ cells translo-
cate gradually to the dorsal side of the hypoblast at
stages XI–XIV, and are carried anteriorly to the germi-
nal crescent region. When the blood vascular system
develops, PGC enter the blood vessels and circulate
temporarily throughout the embryo. Finally, PGC
migrate to the germinal ridges, the future gonads
(Nieuwkoop & Sutasurya 1979; Kuwana 1993;
Kuwana & Roglska 1999), where they start to differ-
entiate into male or female gametes. Primordial germ
cells which enter the testes differentiate into sper-
matogonia, primary spermatocytes, secondary sperma-
tocytes, spermatids and spermatozoa. The PGC which
enter the ovary differentiate into oogonia, primary
oocytes, secondary oocytes and ova (Naito 1998).
Blastodermal cell transfer
Blastodermal cells at stage X contain PGC or their pre-
cursors as well as somatic cells with pluripotency.
Stage X blastodermal cells isolated from the yolk of
freshly laid eggs were dissociated and transferred into
the subgerminal cavity of a recipient blastoderm
(Etches et al. 1997a; Naito & Kuwana 2003). The
manipulated embryos were incubated until hatching.
This procedure was used to produce somatic and
germline chimeric chickens (Petitte et al. 1990). The
percentage of donor-derived offspring from chimeric
chickens was, however, very low (0.3%, 2/719)
because recipient embryos possess their own PGC and
the proportion of donor PGC to recipient PGC is low.
In order to increase donor-derived offspring from
germline chimeric chickens, a reduction in the num-
ber of PGC in recipient embryos and an increase in the
number of PGC in donor blastodermal cells is required.
To reduce the number of PGC in stage X recipient
embryos, irradiation by g-ray proved effective in mod-
ifying the recipient embryos (Carsience et al. 1993;
Thoraval et al. 1994). Use of g-ray irradiation delays
development of the treated embryos by approximately
24 h, during which time the injected donor blastoder-
mal cells containing PGC increase in number in the
recipient embryo, and sometimes the number of donor
PGC dominates the recipient PGC.
159
M. NAITO
To increase the number of PGC in donor blastoder-
mal cells, the distribution of PGC in stage X blasto-
derms needs to be analyzed. A stage X blastoderm is
composed of a single-layered area pellucida and a
peripheral area opaca. The area pellucida is subdivided
into a central disc and a peripheral marginal zone.
Ginsburg and Eyal-Giladi (1987, 1989) and Ginsburg
(1994, 1997) analyzed the origin of PGC in stage X
blastoderms. They isolated and cultured various frag-
ments of blastoderms in vitro and then determined the
presence of PGC by periodic acid–Schiff staining. These
studies showed that most PGC originate from the cen-
tral disc. Naito et al. (2001a) also analyzed the distri-
bution of PGC in stage X blastoderms. The dissociated
blastodermal cells derived from the central disc, mar-
ginal zone, and area opaca were transferred into recip-
ient blastoderms and the donor cell contribution to the
recipient germline was assessed. The results showed
that most of the PGC arise from the central disc, but
cells from the marginal zone and area opaca also con-
tain PGC although the number is very small. Accord-
ing to these results, donor blastodermal cells should be
collected from the central disc of stage X blastoderms
for producing germline chimeric chickens.
Removing a cell cluster from the central disc of a
stage X recipient blastoderm and then injecting
donor blastodermal cells, especially blastodermal cells
derived from the central disc, successfully replaced the
recipient PGC with donor PGC (Kagami et al. 1997,
2000). However, Naito et al. (2001a) failed to replace
the recipient PGC with donor PGC efficiently with this
technique in chimeric chickens. Furthermore, twin
embryos were occasionally produced by this method
and the occurrence of twin embryos reduces the num-
ber of embryos available for chimera production as a
result of high mortality and failure to hatch (Naito
et al. 1991a, 1999, 2001a). Although this technique
seems to be effective for producing germline chimeric
chickens, additional experiments are needed to recon-
cile these observations.
Primordial germ cell transfer
Germline chimeric chickens can be produced effi-
ciently by manipulating PGC directly (Naito &
Kuwana 2003). Avian PGC have a unique migratory
pathway and various attempts have been made to pro-
duce germline chimeric chickens by the transfer of
PGC obtained from the germinal crescent region
(Wentworth et al. 1989, 1996; Vick et al. 1993a), from
embryonic blood (Simkiss et al. 1989) or from embry-
onic gonads (Chang et al. 1995, 1997; Tajima et al.
Animal Science Journal (2003) 74, 157–168
1998). The number of PGC in the bloodstream of
developing embryos shows great variation, and
genetic factors may affect this variation (Tajima et al.
1999; Zhao et al. 2003). Primordial germ cells were
successfully transferred into recipient embryos and
differentiated normally into functional gametes and
gave rise to viable offspring. The efficiency of obtain-
ing donor-derived offspring from these chimeric chick-
ens was low, however, because of an insufficient
number of PGC transferred to the recipient embryos.
A method of concentrating PGC from embryonic
blood has been devised (Yasuda et al. 1992) by which
germline chimeric chickens were produced at an
efficiency of donor-derived offspring of up to 12%
(Tajima et al. 1993). To further increase the efficiency
of germline transmission of donor PGC in germline
chimeric chickens, it is necessary to increase the num-
ber of donor PGC and sterilize the recipient embryos.
Naito et al. (1994a) succeeded in producing germline
chimeric chickens with a high transmission rate of
donor-derived gametes by transferring PGC into par-
tially sterilized recipient embryos (Fig. 2). When PGC
were transferred from White Leghorn (WL) embryos
to Barred Plymouth Rock (BPR) embryos, the average
frequency of donor-derived offspring was 81% for
three male chimeric chickens and 96% for one female
chimeric chicken, and approximately 3.5 times that for
transfer from BPR to WL (23% for six male chimeric
chickens). These chimeric chickens continued to
produce donor-derived offspring for a maximum of
146 weeks without any sign of immune rejection
(Naito et al. 1998a).
Several methods have been used to reduce or
eliminate endogenous PGC from recipient embryos,
including ultraviolet irradiation (Reynaud 1976; Aige-
Gil & Simkiss 1991a), laser irradiation (Mims &
McKinnel 1971), application of busulfan (Aige-Gil &
Simkiss 1991b; Hallet & Wentworth 1991; Vick et al.
1993b), application of concanavalin A (Al-Thani &
Simkiss 1991), and excision of the germinal crescent
region (McCarry & Abbott 1982). All these methods
also affect embryonic development and donor PGC.
Naito et al. (1994a) removed embryonic blood from
the recipient embryos prior to donor PGC injection,
which appeared to increase the efficiency of obtaining
donor-derived offspring from the chimeric chickens.
Mixed-sex germline chimeric chickens
In avian species, the male is the homogametic sex
(ZZ), producing spermatozoa with the Z chromo-
some, and the female is the heterogametic sex (ZW),
160
Manipulation of avian embryos
PGCs
Stages 13–15 Stages 14–15
embryo embryos
Fig. 2 Production of germline chimeric chickens by primordial germ cell (PGC) transfer. PGC isolated from embryonic blood are
transferred into recipient embryos and produced germline chimeric chickens. The transferred PGC-derived offspring can be
produced by mating the germline chimeric chickens.
producing ova with either the Z or W chromo-
some. Chicken embryos can be sexed using PCR
(Clinton 1994; Clinton et al. 2001), amplifying the W
chromosome-specific repeating sequences (Mizuno
et al. 1993). Recent development of chicken embryo
manipulation techniques has made it possible to pro-
duce mixed-sex germline chimeric chickens by the
transfer of stage X blastodermal cells or PGC (Etches
et al. 1997a; Naito 1998). The mixed-sex germline chi-
meric chickens provide an excellent system for study-
ing molecular mechanisms of sexual differentiation
and germ cell differentiation (Stevens 1997).
Same-sex and mixed-sex germline chimeric chick-
ens have been produced by the transfer of stage X blas-
todermal cells (Kagami et al. 1995, 1997). In these
experiments, male or female donor blastodermal cells
were transferred into the same or opposite-sex recip-
ient embryos and same-sex or mixed-sex germline
chimeric chickens were produced. Both same-sex
and mixed-sex germline chimeric chickens produced
donor-derived offspring efficiently. The stage X blasto-
dermal cells contain PGC or their precursors, indicat-
ing that PGC at stage X have the ability to differentiate
into both male and female gametes, irrespective of
their genetic sex, giving rise to viable offspring via
germline chimeric chickens. However, Naito et al.
Animal Science Journal (2003) 74, 157–168
Germline chimeric chickens
Transfer
Mating
Donor-derived offspring
(2001a) carried out similar experiments and found
that PGC at stage X differentiated normally in same-
sex germline chimeric chickens, but few donor-
derived offspring were obtained from mixed-sex ger-
mline chimeric chickens. Furthermore, Naito et al.
(1999) transferred PGC obtained from embryonic
blood (2.5-day incubated embryos) into the same and
opposite-sex recipient embryos. When the sex of
donor PGC was the same as the recipient embryos, the
transferred PGC differentiated normally into func-
tional gametes. But when the sex of the donor PGC
was different from the recipient embryos, the differ-
entiation of donor PGC into functional gametes
appeared to be restricted to a greater degree, at some
developmental stages. Thus, the differentiation of PGC
in mixed-sex germline chimeric chickens still remains
unclear, and clarification of this issue is required.
Using PCR, Southern hybridization, and in situ
hybridization, W chromosome-specific repeating
sequences were occasionally seen in DNA extracted
from the semen of male chimeric chickens produced
by transfer of stage X female blastodermal cells, or
PGC obtained from embryonic blood, into male recip-
ient embryos, (Kagami et al. 1995; Simkiss et al. 1996;
Tagami et al. 1997; Naito et al. 1999). The combination
of WL donor and BPR recipient produced W-bearing
161
M. NAITO
spermatozoa more efficiently than the reverse combi-
nation (Kagami et al. 2002). W-bearing spermatozoa
were also identified in sex-reversed chickens (geneti-
cally female and phenotypically male) produced by
injecting aromatase inhibitor into the eggs on day 5 of
incubation (Abinawanto et al. 1998). The fertilizing
ability of these W-bearing spermatozoa is currently
unknown. In vitro fertilization or intracytoplasmic
sperm injection will serve to clarify this issue. If these
W-bearing spermatozoa have the ability to fertilize
ova, the sex ratio of offspring could be altered.
Interspecific avian chimeras
Interspecific avian chimeras are very useful for prolif-
erating endangered avian species and studying
immune rejection of donor cells in the chimeras.
Quail–chicken interspecific chimeras have been pro-
duced by the transfer of stage X blastodermal cells
(Naito et al. 1991b). The injected quail blastodermal
cells differentiate into various tissues and organs
including gonads in the quail–chicken chimeras
(Watanabe et al. 1992), but production of viable off-
spring derived from the donor blastodermal cells has
not been successful. Quail–chicken interspecific chi-
meras have also been produced by the transfer of PGC
isolated from embryonic blood (Yasuda et al. 1992).
The transferred quail PGC settled in the chicken
gonads, and the hatchlings contained two populations
of spermatogonia derived from the transferred quail
PGC and endogenous chicken PGC (Ono et al. 1998a).
Transferred chicken PGC also settled in quail gonads
(Ono et al. 1998b) and chicken-specific DNA was
observed in sperm samples from matured chimeras (Li
et al. 2001). Viable offspring derived from the donor
PGC in interspecific chimeras have yet to be produced
even by the transfer of PGC isolated from embryonic
blood. To produce donor-derived offspring from inter-
specific germline chimeras, factors such as the size of
the eggs, incubation periods, maturation periods and
immunological acceptance need to be considered.
DNA TRANSFER INTO CHICKENS
Various attempts have been made to introduce exog-
enous DNA into chickens, and many transgenic chick-
ens have been produced by retroviral vector methods
(Ronfort et al. 1997; Harvey et al. 2002). For practical
applications, however, non-viral methods for DNA
transfer into chickens are preferable. This section
describes various approaches to DNA transfer into
chickens by non-viral methods (Fig. 3).
Animal Science Journal (2003) 74, 157–168
Single-cell stage
Microinjection of DNA into fertilized ovum
Stage X
Manipulation of blastodermal cells
Stages 14–15
Manipulation of PGCs
Chicks or Adults
Manipulation of gonads in vivo
Manipulation of spermatozoa
Fig. 3 Various approaches for DNA transfer into chickens.
Introduction of exogenous DNA into chickens can be carried
out, such as by microinjection of DNA into fertilized ovum,
manipulation of blastodermal cells or primordial germ cells,
manipulation of gonads in vivo, or manipulation of
spermatozoa.
Microinjection of DNA into fertilized ova
Manipulation of a fertilized ovum at the single-cell
stage has become possible by devising chicken embryo
culture techniques from single-cell stage to hatching
(Perry 1988; Naito et al. 1990). Fertilized ova are
obtained from the magnum of the oviduct 2.75 h after
the preceding egg is laid. At that time, the yolk is cov-
ered by a small capsule of thick albumen. DNA is
injected into the germinal disc of the fertilized ovum
near the female pronucleus, but is not directly injected
into the male or female pronucleus because of the
opaque cytoplasm. The manipulated ova are cultured
until hatching.
The injected DNA, in circular form, is efficiently
expressed in the developing embryos, but it persists
episomally and is gradually lost during embryonic
development. DNA in linear form injected into the
germinal disc is ligated rapidly after injection to form
random concatamers, and the injected DNA is gradu-
ally lost during embryonic development (Sang & Perry
1989; Perry et al. 1991; Sang 1994). In other studies,
injected DNA has been expressed strongly in develop-
ing embryos and has occasionally been detected in
blood cells or spermatozoa of adult chickens (Naito
et al. 1991c, 1994b; Naito 1997; Kino et al. 1998).
162
Manipulation of avian embryos
Integration of the injected DNA into the host chromo-
somes was observed, and the DNA was transmitted to
the offspring (Love et al. 1994). The integration effi-
ciency was enhanced by use of the Drosophila trans-
posable element mariner (Sherman et al. 1998). The
direct microinjection of DNA into the germinal disc is
at present the only non-viral method that has success-
fully produced transgenic chickens.
Manipulation of blastodermal cells
The stage X blastoderm consists of approximately
60 000 cells and contains approximately 30 PGC or
their precursor cells. Their transfer from one embryo
to another makes it possible to produce germline chi-
meric chickens. Freshly collected blastodermal cells
were dissociated and transferred to recipient embryos
of the same developmental stage to produce somatic
and germline chimeric chickens (Etches et al. 1993,
1997b). Stage X blastodermal cells were efficiently
transfected in vitro by lipofection or electroporation
and injected into intact or compromised recipient
embryos (Brazolot et al. 1991; Fraser et al. 1993;
Maruyama et al. 2000). The introduced DNA was
expressed in chimeric embryos cultured for 2–3 days
following injection (Etches et al. 1997b). The intro-
duced DNA was found to be present in an episomal
form and disappeared during the life span of the adult
birds. Transfected blastodermal cells were FACS-
sorted, and transferred to recipient embryos. Offspring
derived from the FACS-sorted blastodermal cells were
efficiently obtained from the chimeric chickens, but
the introduced DNA was not transmitted to the off-
spring (Speksnijder et al. 1999).
To select successfully transfected blastodermal cells,
stage X blastodermal cells were cultured in vitro. Dis-
persed blastodermal cells obtained from the area pel-
lucida can be cultured for at least 48 h and are able to
enter the germline of recipient embryos. The ability of
such cultured cells to contribute to the somatic and
germline lineages has been found to be considerably
reduced compared with freshly collected stage X blas-
todermal cells (Etches et al. 1996). Putative pluripotent
stem cells (ES cells) derived from stage X blastodermal
cells have been identified and maintained in vitro in
long-term culture (Pain et al. 1996, 1999; Acloque
et al. 2001). Although these cells show features similar
to mouse ES cells over a long period, their ability to
contribute to the germline of chimeric chickens was
limited to the first 7 days in culture. They may lose the
ability to differentiate to the germline, or the germline
cells may be lost from the cell population during long-
Animal Science Journal (2003) 74, 157–168
term culture. Gene transfer into chickens using cul-
tured blastodermal cells has not yet succeeded.
Manipulation of primordial germ cells
Manipulation of PGC could ensure the transmission
of genetic modifications to the next generation via
germline chimeric chickens. Primordial germ cells
were transfected in vivo by injecting a liposome-DNA
mixture into the bloodstream of recipient embryos
(Watanabe et al. 1992). The circulating PGC were
transfected and migrated to the germinal ridges. The
introduced DNA was expressed in the PGC that settled
in the gonads, but transfection efficiency was low.
Naito et al. (1998a, 2000a) transfected PGC obtained
from embryonic blood by lipofection and transferred
them to recipient embryos. The introduced DNA was
efficiently expressed in the gonads of the recipient
embryos, but most of the DNA was gradually lost
during embryonic development. The percentage of
embryos with foreign DNA in the gonads was 95.0%
at day 3 of incubation after the injection, but was
reduced to 11.1% at hatching. Although offspring
derived from the transfected PGC were efficiently
obtained from the germline chimeric chickens, the
introduced DNA was not transmitted to the next gen-
eration. Using a gene gun, PGC located in the germinal
crescent region were transfected (Li et al. 1995).
Hatchlings were grown to maturity and exogenous
DNA was shown to be present in their spermatozoa.
Although the DNA was transmitted to the next gen-
eration, the DNA disappeared from the chickens as
they matured, suggesting that the DNA was episomal.
In order to achieve stable incorporation of exoge-
nous DNA in chickens, PGC cultured in vitro have been
used. Primordial germ cells obtained from the gonads
of 5-day incubated embryos were cultured in vitro with
stroma cells and were found to proliferate approxi-
mately 3 times over a few days. These cultured PGC
retained the ability to re-enter the gonads of recipient
embryos and gave rise to viable offspring (Chang et al.
1997; Tajima et al. 1998; Han et al. 2002). The PGC iso-
lated from the gonads of 5-day incubated embryos
were cultured on a feeder layer over 4 months and
established a chicken embryonic germ (EG) cell line
(Park & Han 2000). The chicken EG cell colonies were
uniformly round, multilayered and well delineated.
The chicken EG cells differentiated into various tissues
in chimeric chickens when injected into a stage X blas-
toderm, but germline contribution of these cells has
not been proven.
163
M. NAITO
DNA transfer into chickens via PGC is one of the
most promising methods. Development of high-
efficiency transfection methods for PGC (Hong et al.
1998; Naito et al. 1998b) or in vitro culture of PGC
(Naito et al. 2001b) would make it possible to produce
transgenic chickens routinely in future.
Manipulation of spermatogonia
and spermatozoa
Spermatogonia are stem cells for producing spermato-
zoa. During the prepubertal phase, spermatogonia
increase in number through mitotic division and some
of the spermatogonia are transformed into primary
spermatocytes. If exogenous DNA can be introduced
into the spermatogonia, spermatozoa with exogenous
DNA will be continuously produced. Transfection of
testes in live chickens by lipofection or electroporation
seems to be a promising method for introducing exog-
enous DNA into spermatogonia. However, exogenous
DNA has been transmitted to embryos by transfecting
spermatozoa and artificially inseminating them into
hens (Squires & Drake 1997). The transmitted DNA
was mostly present episomally and disappeared during
embryonic development. Recently, linker based
sperm-mediated DNA transfer has been reported in
pigs and mice and the technique is applicable in chick-
ens (Chang et al. 2002). Sperm microinjection into a
newly ovulated ovum could be applied to introduce
exogenous DNA into chickens.
In vivo electroporation and lipofection
Recent development of lipofection and electroporation
techniques enables us to transfect animal cells effi-
ciently in vitro and in vivo (Muramatsu et al. 1997,
1998; Naito et al. 2000b, 2001c). When transfecting
early chicken embryos, the in vivo electroporation
method is suitable for introducing exogenous DNA
into a limited part of a targeted site in the embryos,
and the lipofection method seems suitable for intro-
ducing exogenous DNA into a relatively broad area of
the embryos (Muramatsu et al. 1997, 1998; Naito et al.
2000b). Transfection of a stage X blastoderm in vivo by
electroporation made it possible to introduce exoge-
nous DNA into early chicken embryos (Naito et al.
2000b; Sano et al. 2003). When electric pulses were
applied horizontally to the blastoderm layer using par-
allel type electrodes, transfection efficiency was varied
and a low expression rate of exogenous DNA in the
embryonic tissues was observed. When electric pulses
were applied vertically to the blastoderm layer using
Animal Science Journal (2003) 74, 157–168
novel electrodes, transfection efficiency was enhanced
and a high rate of expression of exogenous DNA in the
embryonic tissues was observed (Naito et al. 2002). As
PGC are located in the ventral surface of the epiblast of
the stage X blastoderm, efficient transfection of PGC
could be expected by in vivo electroporation applying
electric pulses vertically to the blastoderm layer. The in
vivo electroporation method was applied to the oviduct
of laying hens and resulted in synthesis of human
erythropoietin protein in the oviduct cells (Ochiai et al.
1998). This success may lead to the production of
pharmaceutical proteins in eggs.
PRESERVATION OF AVIAN
GENETIC RESOURCES
Cryopreservation of germ cells from avian species con-
serves genetic material for the improvement of chick-
ens and facilitates the preservation of endangered
species (Naito 2003). In chickens, cryopreservation
of spermatozoa has already been achieved (Lake &
Stewart 1978), but ova or fertilized eggs cannot be pre-
served in the same way because of their large size and
yolk-laden structure. Cryopreservation of PGC pro-
vides an alternative means for conserving both male
and female genetic material, and cryopreserved PGC
can give rise to viable offspring via germline chimeric
chickens. Naito et al. (1994c) first succeeded in pre-
serving PGC isolated from embryonic blood in liquid
nitrogen. The frozen stored PGC were capable of giv-
ing rise to viable offspring via germline chimeric chick-
ens. Primordial germ cells obtained from the gonads of
5-day incubated embryos were also preserved in liquid
nitrogen and were subsequently able to produce viable
offspring via germline chimeric chickens (Tajima et al.
1998). Blastodermal cells at stage X can be frozen in
liquid nitrogen prior to their injection into recipient
embryos to make chimeras (Naito et al. 1992; Petitte
et al. 1993). Viable offspring were successfully pro-
duced from germline chimeric chickens produced by
transfer of frozen-thawed blastodermal cells (Kino
et al. 1997). These techniques for the cryopreservation
of germline cells make it possible to conserve genetic
material in avian species and accelerate the production
of transgenic chickens.
CONCLUSION
The advent of techniques to culture chicken embryos
from the single-cell stage to hatching has accelerated
164
Manipulation of avian embryos
the study of chicken embryo manipulation, and now
germline chimeric chickens are routinely produced by
the transfer of PGC. DNA transfer into chickens will
be acheived by devising highly efficient transfection
methods for PGC. The nuclear transfer technique
enables the use of somatic cell nuclei, and this tech-
nique can be used for many purposes, such as the
proliferation of endangered avian species, and the
production of somatic cell-derived offspring or trans-
genic chickens. The establishment of these techniques
will usher avian developmental biotechnology into a
new era.
ACKNOWLEDGMENTS
I am very grateful to Drs K. Shimada, Y. Maeda and Y.
Tanabe for their encouragement. I also would like to
thank Drs M.M. Perry, T. Kuwana, Y. Yasuda, A.
Tajima, M. Sakurai, E. Sasaki, M. Ohtaki, M. Kinutani,
M. Watanabe, G. Eguchi, K. Agata, K. Otsuka and K.
Kino for their significant advice and collaboration. I
am also thankful to Drs T. Komiyama, T. Ueno, K.
Nirasawa, T. Oishi, H. Hanada, Y. Matsubara, T.
Harumi, T. Tagmi, A. Sano, H. Kagami and K.
Maruyama for helpful advice and collaboration at my
laboratory. This study was performed through Special
Coordination Funds of the Ministry of Education, Cul-
ture, Sports, Science and Technology, and the Ministry
of Agriculture, Forestry and Fisheries of the Japanese
Government.
REFERENCES
Abinawanto, Zhang, C, Saito N, Matsuda Y, Shimada K. 1998.
Identification of sperm-bearing female-specific chromo-
some in the sex-reversed chicken. Journal of Experimental
Zoology 280, 65–72.
Acloque H, Risson V, Birot A-M, Kunita R, Pain B, Samarut
J. 2001. Identification of a new gene family specifically
expressed in chicken embryonic stem cells and early
embryo. Mechanisms of Development 103, 79–91.
Aige-Gil V, Simkiss K. 1991a. Sterilising embryos for trans-
genic chimaeras. British Poultry Science 32, 427–438.
Aige-Gil V, Simkiss K. 1991b. Sterilisation of avian embryos
with busulphan. Research in Veterinary Science 50, 139–144.
Al-Thani R, Simkiss K. 1991. Effects of an acute in vivo appli-
cation of concanavalin A on the migration of avian germ
cells. Protoplasma 161, 52–57.
Brazolot CL, Petitte JN, Etches RJ, Verrinder Gibbins AM.
1991. Efficient transfection of chicken cells by lipofection,
and introduction of transfected blastodermal cells into the
embryo. Molecular Reproduction and Development 30, 304–
312.
Animal Science Journal (2003) 74, 157–168
Carsience RS, Clark ME, Verrinder Gibbins AM, Etches RJ.
1993. Germline chimeric chickens from dispersed donor
blastodermal cells and compromised recipient embryos.
Development 117, 669–675.
Chang IK, Jeong DK, Hong YH, Park TS, Moon YK, Ohno T,
Han JY. 1997. Production of germline chimeric chickens
by transfer of cultured primordial germ cells. Cell Biology
International 21, 495–499.
Chang K, Qian J, Jiang M-S, Liu Y-H, Wu M-C, Chen C-D,
Lai C-K, Lo H-L, Hsiao C-T, Brown L, Bolen J Jr, Huang
H-I, Ho P-Y, Shih P-Y, Yao C-W, Lin W-J, Chen C-H, Wu
F-Y, Lin Y-J, Xu J, Wang K. 2002. Effective generation of
transgenic pigs and mice by linker based sperm-mediated
gene transfer. Biomed Central Biotechnology 2, 5.
Chang IK, Yoshiki A, Kusakabe M, Tajima A, Chikamune T,
Naito M, Ohno T. 1995. Germ line chimera produced by
transfer of cultured chick primordial germ cells. Cell Biol-
ogy International 19, 569–576.
Clinton M. 1994. A rapid protocol for sexing chick embryos
(Gallus g. domesticus). Animal Genetics 25, 361–362.
Clinton M, Haines L, Belloir B, McBride D. 2001. Sexing
chick embryos: a rapid and simple protocol. British Poultry
Science 42, 134–138.
Etches RJ, Carsience RS, Fraser RM, Clark ME, Toner A,
Verrinder Gibbins AM. 1993. Avian chimeras and their
use in manipulation of the avian genome. Poultry Science
72, 882–889.
Etches RJ, Clark ME, Toner A, Liu G, Verrinder Gibbins AM.
1996. Contributions to somatic and germline lineages of
chicken blastodermal cells maintained in culture. Molecu-
lar Reproduction and Development 45, 291–298.
Etches RJ, Clark ME, Verrinder Gibbins AM, Cochran MB.
1997a. Production of chimeric chickens as intermediates
for gene transfer. In: Houdebine LM (ed.), Transgenic Ani-
mals: Generation and Use, pp. 75–82. Harwood Academic
Publishers, The Netherlands.
Etches RJ, Clark ME, Zajchowski L, Speksnijder G, Verrinder
Gibbins AM, Kino K, Pain B, Samarut J. 1997b. Manip-
ulation of blastodermal cells. Poultry Science 76, 1075–
1083.
Eyal-Giladi H, Kochav S. 1976. From cleavage to primitive
streak formation: a complementary normal table and a
new look at the first stages of the development of the
chick I. General morphology. Developmental Biology 49,
321–337.
Fraser RA, Carsience RS, Clark ME, Etches RJ, Verrinder
Gibbins AM. 1993. Efficient incorporation of trans-
fected blastodermal cells into chimeric chicken embryos.
International Journal of Developmental Biology 37, 381–
385.
Ginsburg M. 1994. Primordial germ cell formation in birds.
In: Marsh J, Goode J (eds), Ciba Foundation Symposium
182, Germline Development, pp. 52–67. John Wiley and
Sons, Chichester, UK.
Ginsburg M. 1997. Primordial germ cell development in avi-
ans. Poultry Science 76, 91–95.
Ginsburg M, Eyal-Giladi H. 1987. Primordial germ cells of
young chick blastoderm originate from the central zone
of the area pellucida irrespective of the embryo-forming
process. Development 101, 209–219.
Ginsburg M, Eyal-Giladi H. 1989. Primordial germ cell devel-
165
M. NAITO
opment in cultures of dispersed central discs of stage X
chick blastoderms. Gamete Research 23, 421–428.
Hallett JS, Wentworth BC. 1991. The effects of busulfan on
gonadal differentiation and development in Japanese
quail (Coturnix coturnic japonica). Poultry Science 70, 1619–
1623.
Hamburger V, Hamilton HL. 1951. A series of normal stages
in the development of the chick embryo. Journal of Mor-
phology 8, 49–92.
Han JY, Park TS, Hong YH, Jeong DK, Kim JN, Kim KD, Lim
JM. 2002. Production of germline chimeras by transfer of
chicken gonadal primordial germ cells maintained in vitro
for an extended period. Theriogenology 58, 1531–1539.
Harvey AJ, Speksnijder G, Baugh LR, Morris JA, Ivarie R.
2002. Expression of exogenous protein in the egg white
of transgenic chickens. Nature Biotechnology 19, 396–399.
Hong YH, Moon YK, Jeong DK, Han JY. 1998. Improved
transfection efficiency of chicken gonadal primordial
germ cells for the production of transgenic poultry. Trans-
genic Research 7, 247–252.
Kagami H, Clark ME, Verrinder Gibbins AM, Etches RJ.
1995. Sexual differentiation of chimeric chickens con-
taining ZZ and ZW cells in the germ-line. Molecular Repro-
duction and Development 42, 379–387.
Kagami H, Iwata J, Nakata A, Tagami T, Matsubara Y,
Harumi T, Tachi C, Okabayashi H, Kashiwazaki N, Shino
M, Naito M. 2000. Substantial evidence to localize the
developmental origin of primordial germ cells in the
chicken. Animal Science Journal 71, 38–41.
Kagami H, Iwata J, Yasuda J, Ono T. 2002. Strain preference
in donor and recipient for production of W-bearing sperm
in mixed-sex germline chimeric chickens. Comparative
Biochemistry and Physiology. Part A, Molecular & Integrative
Physiology 131, 287–292.
Kagami H, Tagami T, Matsubara Y, Harumi T, Hanada H,
Maruyama K, Sakurai M, Kuwana T, Naito M. 1997. The
developmental origin of primordial germ cells and the
transmission of the donor-derived gametes in mixed-sex
germline chimeras to the offspring in the chicken. Molec-
ular Reproduction and Development 48, 501–510.
Karagenc L, Cinnamon Y, Ginsburg M, Petitte JN. 1996. Ori-
gin of primordial germ cells in the prestreak chick
embryo. Developmental Genetics 19, 290–301.
Kino K, Noda K, Miyakawa H, Ohtsuka K, Ono T, Mochii M,
Eguchi G, Agata K. 1998. A low frequency of chromo-
somal integration of cloned DNA after microinjection into
the cytoplasm of fertilized eggs of chickens. Japanese Poul-
try Science 35, 356–366.
Kino K, Pain B, Leibo SP, Cochran M, Clark ME, Etches RJ.
1997. Production of chicken chimeras from injection of
frozen-thawed blastodermal cells. Poultry Science 76, 753–
760.
Kuwana T. 1993. Migration of avian primordial germ cells
toward the gonadal anlage. Development Growth and Differ-
entiation 35, 237–243.
Kuwana T, Roglska T. 1999. Migratory mechanisms of chick
primordial germ cells toward gonadal anlage. Cellular and
Molecular Biology 45, 725–736.
Lake PE, Stewart JM. 1978. Preservation of fowl semen in
liquid nitrogen – an improved method. British Poultry Sci-
ence 19, 187–194.
Animal Science Journal (2003) 74, 157–168
Li HC, Kagami H, Matsui K, Ono T. 2001. Restriction of pro-
liferation of primordial germ cells by the irradiation of
Japanese quail embryos with soft X-rays. Comparative Bio-
chemistry and Physiology Part A, Molecular & integrative phys-
iology 130, 133–140.
Li Y, Behnam J, Simkiss K. 1995. Ballistic transfection of
avian primordial germ cells in ovo. Transgenic Research 4,
26–29.
Love J, Gribbin C, Mather C, Sang H. 1994. Transgenic birds
by DNA microinjection. Biotechnology 12, 60–63.
Maruyama K, Kagami H, Matsubara Y, Harumi T, Tagami T,
Naito M. 2000. Transfection of blastodermal cells with
reporter genes and expression in early chick embryos.
Japanese Poultry Science 37, 142–153.
McCarry JR, Abbott UK. 1982. Functional differentiation of
chick gonads following depletion of primordial germ cells.
Journal of Embryology and Experimental Morphology 68, 161–
174.
Mims MF, McKinnel RG. 1971. Laser irradiation of the chick
embryo germinal crescent. Journal of Embryology and
Experimental Morphology 26, 31–36.
Mizuno S, Saitoh Y, Nomura O, Kunita R, Ohtomo K, Nish-
imori K, Ono H, Saitoh H. 1993. Sex-specific DNA
sequence in Galliformes and their application to the study
of sex differentiation. In: Etches RJ, Verrinder Gibbins
AM (eds), Manipulation of the Avian Genome, pp. 257–274.
CRC Press, Boca Raton.
Muramatsu T, Mizutani Y, Ohmori Y, Okumura J. 1997.
Comparison of three nonviral transfection methods for
foreign gene expression in early chicken embryos in ovo.
Biochemical and Biophysical Communications 230, 376–380.
Muramatsu T, Nakamura A, Park H-M. 1998. In vivo elec-
troporation: a powerful and convenient means of nonvi-
ral gene transfer to tissues of living animals (Review).
International Journal of Molecular Medicine 1, 55–62.
Naito M. 1997. The microinjection of DNA into early chicken
embryo. In: Houdebine LM (ed.), Transgenic Animals: Gen-
eration and Use, pp. 69–73. Harwood Academic Publishers,
The Netherlands.
Naito M. 1998. Manipulation of primordial germ cells for
avian transgenesis. Agbiotech News and Information 10,
397–404.
Naito M. 2003. Cryopreservation of avian germline cells and
subsequent production of viable offspring. Journal of Poul-
try Science 40, 1–12.
Naito M, Agata K, Otsuka K, Kino K, Ohta M, Hirose K, Perry
MM, Eguchi G. 1991c. Embryonic expression of b-actin-
lacZ hybrid gene injected into the fertilized ovum of the
domestic fowl. International Journal of Developmental Biol-
ogy 35, 69–75.
Naito M, Kuwana T. 2003. Production of chimeric chickens.
In: Schatten H (ed.), Germ Cells, Methods in Molecular Med-
icine. [In press]. Humana Press, Totowa.
Naito M, Matsubara Y, Harumi T, Tagami T, Kagami H,
Sakurai M, Kuwana T. 1999. Differentiation of donor
primordial germ cells into functional gametes in the
gonads of mixed-sex germline chimaeric chickens pro-
duced by transfer of primordial germ cells isolated from
embryonic blood. Journal of Reproduction and Fertility 117,
291–298.
Naito M, Matsubara Y, Harumi T, Tagami T, Sakurai M,
166
Manipulation of avian embryos
Kuwana T. 2000a. Foreign gene expression in the gonads
of chimaeric chicken embryos by transfer of primordial
germ cells transfected in vitro by lipofection for 24 hours.
Animal Science Journal 71, 308–311.
Naito M, Nirasawa K, Oishi T. 1990. Development in culture
of the chick embryo from fertilized ovum to hatching.
Journal of Experimental Zoology 254, 322–326.
Naito M, Nirasawa K, Oishi T. 1992. Preservation of quail
blastoderm cells in liquid nitrogen. British Poultry Science
33, 449–453.
Naito M, Nirasawa K, Oishi T. 1995. An in vitro culture
method for chick embryos obtained from the anterior
portion of the magnum of oviduct. British Poultry Science
36, 161–164.
Naito M, Perry MM. 1989. Development in culture of the
chick embryo from cleavage to hatch. British Poultry Sci-
ence 30, 251–256.
Naito M, Sakurai M, Kuwana T. 1998b. Expression of exog-
enous DNA in the gonads of chimaeric chicken embryos
produced by transfer of primordial germ cells transfected
in vitro and subsequent fate of the introduced DNA. Jour-
nal of Reproduction and Fertility 113, 137–143.
Naito M, Sano A, Matsubara Y, Harumi T, Tagami T, Sakurai
M, Kuwana T. 2001a. Localization of primordial germ
cells or their precursors in stage X blastoderm of chickens
and their ability to differentiate into functional gametes
in opposite-sex recipient gonads. Reproduction 121, 547–
552.
Naito M, Sano A, Matsubara Y, Harumi T, Tagami T, Sakurai
M, Kuwana T. 2001c. Fate of donor blastodermal cells
derived from the central disc, marginal zone and area
opaca transferred into the recipient embryos. Journal of
Poultry Science 38, 58–61.
Naito M, Sano A, Tagami T, Harumi T, Matsubara Y. 2000b.
Efficient transfection of chicken blastoderms in vivo by
lipofection and electroporation using green fluorescent
protein gene as a marker. Animal Science Journal 71, 377–
385.
Naito M, Sano A, Tagami T, Harumi T, Matsubara Y, Kuwana
T. 2001b. Effect of mytomycin C treatment on feeder cells
and the proliferation of cultured primordial germ cells
isolated from embryonic blood. Journal of Poultry Science
38, 343–347.
Naito M, Sano A, Tagami T, Harumi T, Matsubara Y, Kuwana
T. 2002. Efficient gene transfer into early chicken
embryos by electroporation of stage X blastoderms in vivo,
applying electric pulses vertically to the blastoderm layer.
Journal of Poultry Science 39, 292–301.
Naito M, Sasaki E, Ohtaki M, Sakurai M. 1994b. Introduction
of exogenous DNA into somatic and germ cells of chick-
ens by microinjection into the germinal disc of fertilized
ova. Molecular Reproduction and Development 37, 167–171.
Naito M, Tajima A, Tagami T, Yasuda Y, Kuwana T. 1994c.
Preservation of chick primordial germ cells in liquid nitro-
gen and subsequent production of viable offspring. Jour-
nal of Reproduction and Fertility 102, 321–325.
Naito M, Tajima A, Yasuda Y, Kuwana T. 1994a. Production
of germline chimeric chickens, with high transmission
rate of donor-derived gametes, produced by transfer of
primordial germ cells. Molecular Reproduction and Develop-
ment 39, 153–161.
Animal Science Journal (2003) 74, 157–168
Naito M, Tajima A, Yasuda Y, Kuwana T. 1998a. Donor pri-
mordial germ cell-derived offspring from recipient germ-
line chimaeric chickens: absence of long term immune
rejection and effects on sex ratios. British Poultry Science
39, 20–23.
Naito M, Watanabe M, Kinutani M, Nirasawa K, Oishi T.
1991b. Production of quail-chick chimaeras by blasto-
derm cell transfer. British Poultry Science 32, 79–86.
Naito M, Watanabe M, Nirasawa K, Oishi T. 1991a. Devel-
opmental ability of twin embryos produced by microin-
jection treatment into chick blastoderm. Poultry Science 70,
1261–1264.
Nieuwkoop PD, Sutasurya LA. 1979. Primordial Germ Cells in
the Chordates. Cambridge University Press, Cambridge.
Ochiai H, Park H-M, Nakamura A, Sasaki R, Okumura J-I,
Muramatsu T. 1998. Synthesis of human erythropoietin
in vivo in the oviduct of laying hens by localized in vivo
gene transfer using electroporation. Poultry Science 77,
299–302.
Ono T. 1997. The complete in vitro development of quail
embryo. In: Houdebine LM (ed.), Transgenic Animals: Gen-
eration and Use, pp. 61–67. Harwood Academic Publishers,
The Netherlands.
Ono T. 2001. Ex ovo culture of quail embryos and its appli-
cation for embryo manipulation. Animal Science Journal
72, 361–371.
Ono T, Yokoi R, Maeda S, Nishida T, Aoyama H. 1998a. Set-
tlement of quail primordial germ cells in chicken gonads.
Animal Science and Technology (Jpn.) 69, 546–555.
Ono T, Yokoi R, Maeda S, Nishida T, Aoyama H. 1998b.
Transfusion of chick primordial germ cells into quail
embryos and their settlement in gonads. Animal Science
and Technology (Jpn.) 69, 911–915.
Pain B, Chenevier P, Samarut J. 1999. Chicken embryonic
stem cells and transgenic strategies. Cells Tissues and Organs
165, 212–219.
Pain B, Clark ME, Shen M, Nakazawa H, Sakurai M, Samarut
J, Etches RJ. 1996. Long-term in vitro culture and char-
acterisation of avian embryonic stem cells with multiple
morphogenetic potentialities. Development 122, 2339–
2348.
Park TS, Han JY. 2000. Derivation and characterization of
pluripotent embryonic germ cells in chicken. Molecular
Reproduction and Development 56, 475–482.
Perry MM. 1988. A complete culture system for the chick
embryo. Nature 331, 70–72.
Perry M, Morrice D, Hettle S, Sang H. 1991. Expression of
exogenous DNA during the early development of the
chick embryo. Roux’s Archives of Developmental Biology 200,
312–319.
Petitte JN, Brazolot CL, Clark ME, Liu G, Verrinder Gibbins
AM, Etches RJ. 1993. Accessing the genome of the
chicken using germ-line chimeras. In: Etches RJ, Ver-
rinder Gibbins AM (eds), Manipulation of the Avian Genome,
pp. 81–101. CRC Press, Boca Raton.
Petitte JN, Clark ME, Liu G, Verrinder Gibbins AM, Etches
RJ. 1990. Production of somatic and germline chimeras in
the chicken by transfer of early blastodermal cells. Devel-
opment 108, 185–189.
Reynaud G. 1976. Reproductive capacity and offspring of
chickens submitted to a transfer of primordial germ cells
167
M. NAITO
during embryonic life. Roux’s Archives of Developmental
Biology 179, 85–110.
Ronfort C, Legras C, Verdier G. 1997. The use of retroviral
vectors for gene transfer into bird embryo. In: Houdebine
LM (ed.), Transgenic Animals: Generation and Use, pp. 83–
94. Harwood Academic Publishers, The Netherlands.
Sang H. 1994. Transgenic chickens – methods and potential
applications. Trends in Biotechnology 12, 415–420.
Sang H, Perry MM. 1989. Episomal replication of cloned
DNA injected into the fertilized ovum of the hen, Gallus
domesticus. Molecular Reproduction and Development 1, 98–
106.
Sano A, Tagami T, Harumi T, Matsubara Y, Naito M. 2003.
Introduction of exogenous DNA into gonads of chicken
embryos by lipofection and electroporation of stage X
blastoderms in vivo. British Poultry Science 44, 36–39.
Sherman A, Dawson A, Mather C, Gilhooley H, Li Y, Mitchell
R, Finnegan D, Sang H. 1998. Transposition of the Droso-
phila element mariner into the chicken germ line. Nature
Biotechnology 16, 1050–1053.
Simkiss K, Luke G, Behnam J. 1996. Female chromosomes
in cockerel ejaculates. Proceedings of the Royal Society of Lon-
don B 263, 1245–1249.
Simkiss K, Rowlett K, Bumstead N, Freeman BM. 1989.
Transfer of primordial germ cell DNA between embryos.
Protoplasma 151, 164–166.
Speksnijder GJ, Etches RJ, Verrinder Gibbins AM. 1999.
Germline chimeric chickens from FACS-sorted donor
cells. Molecular Reproduction and Development 52, 33–42.
Speksnijder G, Ivarie R. 2000. A modified method of shell
windowing for producing somatic or germline chimeras
in fertilized chicken eggs. Poultry Science 79, 1430–
1433.
Squires EJ, Drake D. 1997. Transgenic chickens by liposome-
sperm-mediated gene transfer. In: Houdebine LM (ed.),
Transgenic Animals: Generation and Use, pp. 95–99. Har-
wood Academic Publishers, The Netherlands.
Stevens L. 1997. Sex chromosomes and sex determining
mechanisms in birds. Science Progress 80, 197–216.
Suda T, Abe E, Shinki T, Katagiri T, Yamaguchi A, Yokose S,
Yoshiki S, Horikawa H, Cohen GW, Yasugi S, Naito M.
1994. The role of gravity in chick embryogenesis. FEBS
Letters 340, 34–38.
Tagami T, Matsubara Y, Hanada H, Naito M. 1997. Differen-
tiation of female chicken primordial germ cells into
spermatozoa in male gonads. Development Growth and Dif-
ferentiation 39, 267–271.
Tajima A, Hayashi H, Kamizumi A, Ogura J, Kuwana T,
Chikamune T. 1999. Study on the concentration of
Animal Science Journal (2003) 74, 157–168
circulating primordial germ cells (cPGC) in early chick
embryos. Journal of Experimental Zoology 284, 759–764.
Tajima A, Naito M, Yasuda Y, Kuwana T. 1993. Production of
germ line chimera by transfer of primordial germ cells in
domestic chicken (Gallus domesticus). Theriogenology 40,
509–519.
Tajima A, Naito M, Yasuda Y, Kuwana T. 1998. Production of
germ-line chimeras by transfer of cryopreserved gonadal
primordial germ cells (gPGC) in chicken. Journal of Exper-
imental Zoology 280, 265–267.
Tanaka K, Wada T, Koga O, Nishio Y, Hertelendy F. 1994.
Chick production by in vitro fertilization of the fowl
ovum. Journal of Reproduction and Fertility 100, 447–449.
Thoraval P, Lasserre F, Coudert F, Dambrine G. 1994.
Somatic and germline chicken chimeras obtained from
Brown and White Leghorns by transfer of early blasto-
dermal cells. Poultry Science 73, 1897–1905.
Tsunekawa N, Naito M, Sakai Y, Nishida T, Noce T. 2000.
Isolation of chicken vasa homolog gene and tracing the
origin of primordial germ cells. Development 127, 2741–
2750.
Vick L, Li Y, Simkiss K. 1993a. Transgenic birds from trans-
formed primordial germ cells. Proceedings of the Royal Soci-
ety of London B 251, 179–182.
Vick L, Luck G, Simkiss K. 1993b. Germ-line chimaeras can
produce both strains of fowls with high efficiency after
partial sterilization. Journal of Reproduction and Fertility 98,
637–641.
Watanabe M, Kinutani M, Naito M, Ochi O, Takashima Y.
1992. Distribution analysis of transferred donor cells in
avian blastodermal chimeras. Development 114, 331–338.
Wentworth BC, Tsai H, Hallet JH, Gonzales DS, Rajcic-
Spasojevic G. 1989. Manipulation of avian primordial
germ cells and gonadal differentiation. Poultry Science 68,
999–1010.
Wentworth BC, Tsai H, Wentworth A, Wong E, Proudman J,
El Halawani M. 1996. Primordial germ cells for genetic
modification of poultry. In: Miller RH, Pursel VG, Nor-
man HD (eds), Biotechnology’s Role in the Genetic Improve-
ment of Farm Animals, pp. 202–227. American Society of
Animal Science, Savoy.
Yasuda Y, Tajima A, Fujimoto T, Kuwana T. 1992. A method
to obtain avian germ-line chimaeras using isolated pri-
mordial germ cells. Journal of Reproduction and Fertility 96,
521–528.
Zhao D-F, Yamashita H, Matsuzaki M, Takano T, Abe S-I,
Naito M, Kuwana T. 2003. Genetic factors affect the num-
ber of circulating primordial germ cells in early chick
embryos. Journal of Poultry Science 40, 101–113.
168