Download Current Technologies for Transgenic Poultry

Current Technologies for Transgenic Poultry
James N. Petitte
Departmentof PoultryScience
NorthCarolinaState University,Raleigh, NC, 27603
Introduction:
During the last two decades, significantprogresshas been made in our understanding
of the molecular basis of the genetics of growth and development. This knowledge
coupled with techniques designed to introduce foreign DNA into the genome of poultry
has the potential to enhance current breeding practices for genetic improvement. Gene
transfer into poultry could provide a means of introducing new genetic variation and
changes in genotypes that have increased economic value.
However, before this
becomes a reality the means of producing transgenic poultry must become routine.
Presently, several techniques are currently in use or under development which could
take the production of transgenic poultry beyond the laboratory into the industrial
sphere.
Opportunities for Intervention:
The target for any modification of the avian genome is the germ cell. Thus several
opportunities for genetic intervention include mature oocytes/spermatozoa, the newly
fertilized eggzygote
and early embryo, and primordial germ cells during their
establishment, migration, and colonization of the gonad.
As most people associated with poultry know, fertilization takes place in the
infundibulum of the oviduct.
However, the first cleavage divisions do not appear until
the ovum reaches the shell gland, about 4-5 hours after fertilization. During the 20-odd
hours needed for deposition of the fully calcified shell, the early embryo undergoes
rapid cell division and at the time of oviposition, the blastodisc contains about 40,000
cells.
During this period the embryo acquires its polarity, e.g. anterior/posterior
orientation, yet is visually radially symmetric.
At this time the embryo can be divided
into a peripheral ring of cells attached to the yolk called the area opaca and a central
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more translucent region, the area pellucida, which is suspended above a non-yolky
fluid deposited by the embryo. (Figure 1). The area pellucida can be divided further
divided into the marginal zone at the periphery and the central disk.
At this point the
area opaca will contribute only to extraembryonic structures and the embryo proper will
develop from the area pellucida. Upon incubation, the area pellucida differentiates into
two layers, an upper epiblast and a lower hypoblast. Only the epiblast will give rise to
the embryo proper while the hypoblast contributes to extraembryonic tissues.
This
period of development, i.e. fertilization through hypoblast formation, has been classified
into a series of 14 stages by EyaI-Giladi and Kochav (1976) for the domestic hen and
11 stages by Gupta and Bakst (1993) for the turkey and are indicated using Roman
numerals. Subsequent stages for both species are classified using the staging system
of Hamburger and Hamilton (1951) using Arabic numerals. For the purposes of this
article, all references to stages of development will refer to that for the domestic hen.
The next period of development that is significant for the production of transgenic
poultry is the during the establishment of the germ line. The development of the avian
germ line has been examined for almost a century.
developmental history of avian germ cells.
Figure 2 summarizes the
Swift (1914) was the first to show the
presence of PGCs in an extraembryonic region, referred to as the germinal crescent,
well before the development of the gonad. Swift's observations, which were based on
the morphological
characteristics of PGCs, were later confirmed by several
investigators (Goldsmith, 1928; Willier, 1937; Simon, 1960). The migration of PGCs
from the germinal crescent to the gonadal ridge occurs in two phases. First, PGCs are
carried to the vicinity of the germinal ridge passively through the intra- and
extraembryonic circulation (Swift, 1914; Meyer,1964; Fujimoto et al., 1976ab). Second,
most of the blood-borne PGCs leave the vessels and migrate actively into the germinal
epithelium. In this second and active phase of migration, chemotactic signals released
from the gonad (Dubois and Croisille, 1970; Kuwana et al., 1986), extracellular matrix
components (Urven et al. 1989) and the anatomical arrangement of the vascular
system surrounding the gonadal epithelium (Nakamura et aL, 1988) are thought to be
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important. These various periods of germ cell development represent several windows
of opportunity for direct intervention to produce transgenic poultry.
Current Methodologies:
All methods of producing transgenic poultry rely on techniques designed to stabily
insert novel genetic material into cells that will give rise to germ cells or germ cells per
se. Currently, two methods have been developed that have successfully produced
transgenic poultry:
Retorviral-mediated transfection and DNA microinjection.
Other
methods currently in development use a chimeric intermediate through the transfer of
blastodermal cells or primordial germ cells.
RetroviraI-Mediated Transgenesis:
The use of retroviruses for gene transfer is a common procedure and forms the basis of
gene therapy strategies for humans and transgenesis in laboratory and domestic
animals. For poultry, retroviral gene transfer is the most successful methodology to
date. This is due mainly to the features of the retroviral life cycle (Figure 3).
Retroviruses have an RNA genome encased in a protein core containing integrase,
reverse transcriptase, and protease which is coated by a protein envelope.
For
infection, viral envelope proteins bind to specific proteins on the host cell membrane
and are internalized by receptor mediated endocytosis. The envelope is removed by
cellular enzymes, and viral reverse transcriptase copies the RNA into DNA. The DNA
moves to the nucleus and is integrated into a chromosome of the host cell through the
activity of an integrase on the long terminal repeats (LTRs) at each end.
With
integration, the provirus is replicated with the chromosome and is inherited in a
Mendelian fashion. It is this aspect of the retroviral life cycle that permits successful
transgenesis.
In addition to replication, the proviral DNA can be transcribed into viral RNA for the
synthesis of proteins.
These RNAs encode three classes of proteins, pol for
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polymerases, gag for group-associated antigens, and env for envelope proteins. Once
translated pol and gag proteins associate with the specific packaging sequences on the
RNA and assemble into new viral cores. The env proteins are transported to the host
cell membrane and the viral core buds from the cell through env areas and produces a
new infectious particle.
In the case of replication competent vectors, the viral structural genes and packaging
sequences are intact allowing for the continuous production of infectious particles.
With replication-defective vectors, deletions are made in the structural genes (pol, gag,
env).
This allows the virus to infect a host cell but the provirus will not generate new
infectious virions. To produce infectious particles for transgenesis, helper cell lines
were developed to package the defective-vector. Helper cell lines are generated using
a proviral vector that is missing the encapsidation site but contain the gag, pol, and env
regions of the virus. When this cell line is transfected with the replication-defective
vector containing the exogenous gene of interest, the helper cells can package the
recombinant viral RNA into infectious particles which do not, in turn, produce other
infectious particles.
The most crucial aspect of this process is the development of
helper cells lines that do not produce infectious helper virus.
The need for helper packaging cell lines makes replication-defective retroviral vectors
harder to work with than replication-competent systems. In addition, viral titers are
reduced which often requires considerable concentration to yield sufficient material for
infection.
However, replication-defective vectors allow for larger exogenous genes
(about 10kb vs 2.5 kb) and can encode multiple transgenes.
Despite the limitations of viral vectors,
both replication competent and defective
vectors were the first means available for transgenesis in poultry. Of the various time
periods discussed above for manipulation, viral vectors have been used for infection of
ova Shuman (1984) or PGCs (Simkiss et al., 1990) but are more commonly injected at
the time of oviposition directly into the blastoderm or close to the blastoderm. The first
successful development of transgenic chickens was reported by Salter et al. (1986)
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using reticuloendotheiosis virus (REV) and avian leukosis virus (ALV) replicationcompetent virus injected near the blastoderm, where 25% of viremic males were mosaic
and transmitted the provirus to progeny from 1 to 11% (Salter et al., 1987). Two of the
ALV proviral inserts (alv6 and alv13) were defective in producing virus but expressed
the envelope protein and were resistant to ALV subgroup A infection (Crittenden, et al.,
1989). Chen et al. (1990) subsequently reported that a modified form of the Rous
sarcoma virus (RSV) containing bovine growth hormone could enter the germ line.
Transgenic chickens have been produced using replication-defected REV vectors
(Bosselman et a1.,1989)when injected beneath the blastoderm. About 8% of the male
birds that hatched carried the neomycin resistance gene and transmitted the vector to
progeny at about 2-8%. The same REV vector was used to express chicken growth
hormone constitutively in embryos.
About 50% of the embryos had elevated
concentrations of growth hormone but none hatched. In addition to injection of viral
stocks into the embryo prior to incubation, Vick et al. (1993a) demonstrated that
replication-defective vectors could be used to infect primordial germ cells from the
germinal crescent or blood to produce transgenic chickens.
Despite these successes, there is hesitation to move retroviral-based technology from
the laboratory to the industrial arena for the genetic improvement of commercial stock.
Much of this reluctance is due to fears, real or imagined, associated with using a viral
system. In addition, while retroviral vectors are the most efficient means of producing
transgenic birds, some of the inherent disadvantages such as random-integration,
generation of high titre replication-defective stocks, and packaging size limitations
appear
to preclude
their wide-spread
commercial use.
Overcoming
these
disadvantages is the main reason for the development of nonvirally-based technologies
for gene transfer.
Nevertheless, the use of replication-competent and replication-
defective viral vectors has expanded to the point where they have become common
tools for answering questions associated with molecular aspects of avian biology.
Foremost in this regard is their use in developmental biology where the avian embryo is
a major system for the study of cell lineage analysis, cell migration, and the in vivo
action of expressed proteins.
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DNA Microinjection"
In addition to retroviral vectors, DNA microinjection is the only other means
demonstrated to produce transgenic poultry.
Injection of DNA into the pronucleus of
the newly fertilized egg is a common procedure for the production of transgenic
laboratory animals and mammalian livestock. Unfortunately, these techniques were not
readily adapted to the chicken because of the specific reproductive strategy of birds,
i.e. the large yolky ovum and the production of the amniotic egg.
Before DNA
microinjection could be attempted in birds, a complete ex vivo system from fertilization
to hatch needed to be developed which would yield sufficient numbers of hatchlings to
screen for gene integration. The basic method currently in use is a three stage system
of Perry (1988) using a combination of methods employed by Ono and Wakasugii
(1984), Rowlett and Simkiss (1987) and Rowlett (1991) for post-ovipositional stages of
development.
Newly fertilized eggs surrounded with a capsule of albumen are
removed from the magnum and cultured for about 18-24 hours in synthetic oviductal
fluid without a shell. Stage 2 requires transfer of the egg to an eggshell, completely
sealed with no simulated air space. After 2-4 days, the embryo is transferred to a
larger shell with an upper air space for the remaining period of incubation.
Such
procedures have also been adapted to quail embryos using chicken eggshells (Ono et
al., 1994).
For the production of transgenic poultry, DNA expression vectors are injected into the
cytoplasm of the germinal disk of the ovum upon recovery from the magnum prior to
culture. In most cases, the DNA forms concatemers and remains episomal as seen
after microinjection of mammalian pronuclei
(Sang and Perry, 1989; Naito et al.,
1991b, 1994). However, one mosaic rooster was produced that transmitted a bacterial
beta-gactosidase gene to about 3.4% of its offspring (Love et al., 1994). Transgene
copy number averaged about 6, apparently in a single chromosomal location.
Test
mating of one transgenic rooster showed predictable Mendelian inheritance of the
59
reporter gene. This demonstrated that it is possible to produce transgenic poultry using
DNA injection.
The use of DNA microinjection into newly fertilized ova overcomes some of the
disadvantages of retroviral vectors, namely the production of replication-defective
vectors and the limits on the size of the transgene. Success with the production of
transgenic mice via microinjection of 250kb yeast artificial chromosomes suggests that
a similar-sized construct could be used to produce transgenic birds.
Efficiency of
integration, which is often a limiting variable for the production of transgenic animals
(mammalian or otherwise), appears to be low but is not lower than that for other
agricultural livestock.
Primordial Germ Cells and Blastodermal Cells :
Although retroviral vectors and direct DNA injection can be used to produce transgenic
poultry, neither method can take advantage of current technology using DNA constructs
for "gene targeting", i.e. the ability to make-locus specific modifications to the genome.
For several years, lines of transgenic mice have been produced with relatively precise
changes to particular loci to examine the genetic basis of disease,
as therapeutic
models, and to study various aspects of development. The use of gene targeting in
the production of transgenic animals of commercial importance is particularly attractive
since the chances of obtaining a predictable phenotype appear greater than having to
evaluate several lines of animals produced through random integration of the
transgene.
The basic procedure for the production of any. animal with targeted
modifications to the genome is illustrated in Figure 4.
The first step is to obtain
primordial germ cells or cells that will give rise to the germ line from the embryo and
culture them in conditions that keep the cells in a relatively undifferentiated state.
These cells are transfected with constructs designed to allow for homologous
recombination, and cells in which the correct (and rare) recombination event has
occurred are selected and expanded in vitro.
Finally, the cells are returned to an
embryo where they integrate and become part of the cellular makeup of the recipient.
60
Such individuals, known as chimeras, should have germ cells at sexual maturity that
derive from the transgenic donor cells. When the chimeras are bred, a portion of the
offspring will be transgenic. This technical scheme represents the coordinated effort of
three technologies: 1) the ability to produce germ line chimeras, 2) the development of
suitable DNA constructs for transfection, and 3) the means of culturing germ cells or
cells that will give rise to the germ line. Of the three necessary technologies, only the
latter remains to be established for poultry.
The first germ line chimeric chicken was produced by the transfer of cells from the
unincubated embryo (Petitte et al., 1990). Since that time, the production of germ line
chimeras has become routine using cells from the stage X blastoderm or primordial
germ cells from the germinal crescent or embryonic circulation. (Yasuda et al., 1992;
Vick et al., 1993b;Tajima et al., 1993; Carsience et al., 1993; Thoroval et al., 1994) and
these procedures have been adapted to quail and to the production of interspecific
chimeras (Naito et al., 1991a; Nakamura et al., 1992). In addition, both cell types are
amenable to various transfection methods (Brazolot et al., 1991; Page et al., 1991;
Fraser et al., 1993; Breseler et al., 1994; Allioli et al., 1994; Watanabe et al., 1994; Li
et al., 1995; Rosenblum and Chen, 1995; Ono et al., 1995), and several reports
indicate that homologous recombination is possible in chicken cell lines (Buerstedde
and Takeda, 1991; Li and Dodgson, 1995) or in cells from the stage X blastoderm (Liu,
1995). Hence, the final facet for the use of chimeric intermediates for gene transfer
requires the elucidation of the ideal culture conditions for donor cells.
Culture of Avian Blastodermal Cells:
In the mouse, work on the origin and characteristics of teratocarcinomas led to the
culture of
embryonic stem cells (ESCs) from the inner cell mass out-growths of
blastocysts (see Hooper, 1992).
embryoid bodies,
ESCs are able to differentiate in vitro to form
undergo spontaneous or chemically-induced differentiation, and
when injected into a blastocyst are capable of giving rise to all somatic cell types and in
many cases can give rise to the germ line. In early work, feeder layers of fibroblasts
61
were used to maintain the culture for long periods. One of the first factors shown to
replace the use of a feeder layer was leukemia inhibitory factor (LIF). It is now known
that the effects of LIF and other cytokines such as ciliary neutrophic factor and
oncostatin M, which also can be used to culture ESCs, are mediated through a common
receptor subunit and an intracellular, receptor-associated glycoprotein (gp130) (Yosida
et al, 1994). Given this redundancy, it is not surprising that primary chicken embryonic
fibroblasts or conditioned-media from a chicken liver cell line can also be used to
culture mouse ESCs (Yang and Petitte, 1994). These properties suggested that it
might be possible to culture an avian embryonic stem cell using similar feeder layers or
conditioned-media.
Attempts to culture blastodermal cells of dispersed stage X embryos using chick
embryo fibroblasts, mouse fibroblasts, LMH- or BRL-conditioned media were not
successful.
However, the combination of a mouse fibroblast feeder layer and BRL
conditioned-media sustained the culture of cells from the stage X embryo with a stem
cell-like phenotype (Petitte and Yang, 1993). Such cultures shared several phenotypic
and antigenic characteristics with mouse ESCs (Table 1). In addition, colonies of the
cultured cells expressed the epitopes recognized by the monoclonal antibodies EMA-1
and SSEA-1 which can be used to mark avian PGCs,during development (Urven et al.,
1988; Karagenq et al., 1996).
Although these cells had similar characteristics to ESCs, definitive proof required an
evaluation of pluripotency. This was evaluated in three ways. First, colonies of cells
were grafted onto the chorioallenatoic membrane of 9 day old embryos and incubated
for nine days. Grafts were processed for routine histological examination and were
found to
contain
several
cell
types,
including
smooth muscle,
hematopoieses, epitheilial-lined lumens and karatinization.
regions
of
These results indicated
that the blastodermal cells were at least multipotent when maintained in culture. The
second approach evaluated the ability of the cells to differentiate when transferred to a
stage X embryo. To evaluate this criteria, cultures were initiated with female embryos
62
• sexed using PCR (Petitte and Kegelmeyer, 1995) and injected into stage X recipients.
Phenotypic males were analyzed for the presence of female-specific
(Figure
5).
DNA using PCR
Male embryos were found to contain female DNA in various tissues
indicating that the cultured blastodermal cells were capable of giving rise to several cell
types in ovo.
Although this analysis suggested that the injected cells could give rise
several cell types, it did not provide precise information concerning
the germ cell
lineage. To answer this question, the third approach utilized the fact that chicken germ
cells can be identified within a quail embryo using period acid-Schiff's
colonies of cultured blastodermal
staining.
cells were injected into unincubated
When
quail embryos
and serially sectioned at about 6 days of incubation, it was possible to identify chicken
primordial germ cells in the dorsal mesentery near the region of the germinal ridge
(data not shown).
Therefore,
it would appear that the culture conditions were able to
maintain the pluripotency of the blastodermal cells. In any case, this should provide a
convenient
means of producing
enough transgenic blastodermal
cells to produce a
transgenic bird.
Culture of Primordial
Germ Cells:
In mice, there appears to be a growing connection between the culture of embryonic
stem cells and PGCs.
Recently, it was reported that fibroblast growth factor- 2 (FGF-
2), LIF and stem cell factor (SCF) could be used to culture murine PGCs so that they
took on an embryonic stem cell phenotype (Resnick et al., 1992; Matsui et al., 1992 ).
These cells called embryonic germ cells (EGCs) while not identical in every way to
ESCs are capable of forming somatic and germ line chimeras when injected into a
blastocyst (Labosky et al., 1994).
Reports are now appearing on the culture of avian
PGCs (Allioli et al., 1994; Chang et al., 1995). In some cases, the cultured germ cells
can migrate to the gonadal antage (Chang et al., 1995).
with primordial
However, the main problem
germ cell culture is the occurrence of apoptosis or programmed
death and stimulating
cell division (Dolci et al., 1991, 1993; DeFelici
Pesce et al., 1993; Allioli et al., 1994).
cell
et al., 1992;
The recent cloning and expression of avian
63
stem cell factor should help to define the culture conditions needed for avian PGCs
(Zhou et al., 1993; Petitte and Kulik, 1996).
Summary:
While much work remains to be done before the production of transgenic poultry
becomes more commonplace, significant progress has been made in the last decade.
Reserach applicationsof retroviral vectors for gene transfer in birds are beginning to
emerge in biomedical studies,
techniques for microinjection of DNA have been
demonstrated to be a viable means of producingtransgenic poultry, and the culture of
the avian embryonic stem cells and PGCs should provide the means for gene targeting
in birds. This progress is all the more remarkable since the number of laboratories
world-wide working on avian transgenesis is few compared to those working on
mammalian systems. As various techniques are refined and made more efficient, a
repertoire of methodologies will be available to academic and industrial research
laboratories that wish to answer significant biological questions or improve the genetic
potential of commercial poultry stocks through manipulation of the avian gnome.
Acknowledgments
The research reported here was supported, in part, from USDA-NRI grants #91-372056320 and #94-37205-1031 and NCARS project 01868.
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70
Table 1. Characteristics of Mouse and Avian Embryonic Stem Cells
Mouse
Avian
large nucleus
large nucleus
little cytoplasm
little cytoplasm
prominent nucleolus
single prominent nucleolus
tightly packed colonies
flattened colonies
express specific epitopes
express SSEA-1 and EMA-1
grow almost indefinitely
grow for about 20 passages
undergo spontaneous/induced
differentiation
form somatic and germ line chimeras
spontaneously differentiate in CAM grafts
form somatic and germ line chimeras
71
Stage X Stage XII Stage XIIIStage 2
Stage 4
0--@--@
@@
epiblas't
epibla_t
_ Area opaca
_ Mesoderm
_
Marginal zone
B Primitive streak
_
Hypobiast
primitive
Figure 1. Diagrammatic representation of early avian embryo development from
oviposition to primitive streak development covering about 18 hours of incubation,
selected ventral and cross-sectional views. Hypoblast formation can be detailed from
Stages X-XIII (EyaI-Giladi and Kochav, 1976). Generally, a freshly laid egg contains an
embryo at Stage X characterized by complete formation of the area pellucida
containing clusters of polyingressing cells. Upon incubation, growth of the hypoblast
begins from the posterior marginal zone and by Stage Xll covers about half of the
central disk. At Stage XlII the area pellucida becomes a bi-layered structure with a
distinctive epiblast and hypoblast.
The initial development of the primitive streak
signals the beginning of gastrulation at Stage XIV (E-G &K)/Stage 2 (Hamburger and
Hamilton, 1951). During the formation of the three primary germ layers, the epiblast
cells migrate through the groove of the primitive streak and invade the hypoblast,
displacing it anterio-laterally to form the germinal crescent (Stage 4).
Involution
through the streak leads to the transformation of the epiblast cells to mesoderm and
endoderm.
72
G
St.X
St. XIII
St. 3
St. 6 St. 8
St. 12
H
St. 15
St. 20
Figure 2. A schematic representation of the developmental history of primordial germ
cells (PGCs) in the chick embryo. Committed germ cells have not been identified in the
Stage X (A); however, anti-SSEA-1 marks a population of cells on the hypoblast that
can give rise to germ cells (B) (see also Figure 3). These SSEA-1 positive hypoblast
cells move anteriorly during gastrulation and head fold stages (C-E) to form the
germinal crescent described by Swift (1918). During the formation of blood islands and
the vasculature (F), the germ cells enter the embryonic circulation (G) until they
colonize the gonadal ridge (H). PGCs can be readily identified after gastrulation using
periodic acid-Schiff's staining. The circuitous journey of avian germ cells is thought to
involve periods of passive and active migration guided by morphogenetic movements,
chemotaxis, extracellular matrix components and the vascular configuration (redrawn
after Nieuwkoop and Sutasurya, 1979; with modifications).
73
Retroviral life
cycle:
reverse transcription
integration
mRNAs
structural
provirus
genomic RNA
assembly
infectious virons
Figure 3. Diagram of the life cycle of a retrovirus. Infection of the host cell begins
with the binding of the virus to the cell membrane which is internalized by receptormediated endocytosis. Viral reverse transcriptase copies the RNA into DNA. The
newly generated DNA moves to the nucleus and is integrated into the chromosome of
the host cell. The proviral DNA is then transcribed to generate new genomic RNA and
mRNAs from which viral proteins are translated for viral packaging. New viral cores are
assembled and bud from the host cell to produce new infectious particles. (see text)
74
CultureCellsfromdonorembryo
Transfect
v
_
Blastodermal Cells
or PGCs
Selectionfor
stableintegration
and
homologous
recombination
Injectinto recipientembryo
_,'-_.,_
Screenoffspringfor transgene
Figure 4.
The technical scheme for the production of transgenic poultry using
chimeric intermediates. Blastodermal cells from stage X embryos or PGCs are cultured
using conditions that allow proliferation without differentiation.
Such cultures are
transfected with DNA constructs that can undergo homologous recombination.
Cells
with the correct integration event are expanding using the appropriate selectable
markers and transferred to recipient embryos. The resulting chimeras are bred and the
offspring screened for the presence of the transgene.
75
MALE 1
I
M
wT
MALE 2 FEMALE
II
Brln
II
LvBISkHBrSkHLv
In O_HLvw
I
m f
M
Figure 5. Analysis of chimeric chick embryos after injection of cultured female
blastodermal cells into stage X embryos. Various tissues were harvested from
phenotypic males and assayed for the presence of the the W chromosome Xhol
repetitiveelementsusingPCR (Petitte and Kegelmeyer,1995). Amplificationproducts
were southernblottedonto nylonmembranesand hybridizedwith a probe internalto
the amplificationproduct. W, water blank, T, testis, Br, brain, In, intestine,Lv, liver,BI,
blood, Sk, skin, H, heart, O ovary, m, 5 ng male chicken DNA, f, 5 ng female chicken
DNA.
76
Questions
Question: E. Buss
Has anyone attempted to use a bacterium as a vector?
Answer: J. N. Petitte
No, not that I can recall.
Question: E. Buss
Why did you select the female embryo rather than the male?
Answer: J. N. Pe.titte
We needed a convenient marker for cell lineage analysis of the chimeras made
with cultured blastodermal cells. The repetitive elementsthat make-up the vast
majority of the W chromosome provided a means of determining whether the
donor cells could be incorporated into the recipient embryo. Hence, female
embryos were used to initiate cultures but phenotypic males would provide
information about the extent of chimerism.
Question: A. Emsley
What is the prospect of recovering transgenics from chimeras with low numbers
of donor germ cells?
77
Answer: J. N. Petitte
The efficiency of germ line transmission can be improved dramatically for the
most part by compromising the host embryo (see Carsience et al., 1993; Aige-Gil
and Simkiss, 1991ab). The question remains whether cultured blastodermal
cells or PGCs will retain the same efficiency. If all the cells transferred to the
recipient embryo had the transgene integrated into the genome, then the
prospects of obtaining a transgenic are rather good even if only one germ cell
was transgenic at the time of chimera formation. For example, it is estimated
that 200 PGCs are present in the germinal crescent. These 200 cells will form
the founding population of germ cells for the gonad. If one transgenic germ cell
reaches the germinal ridge, then in a male, the chances of germ line
transmission would appear reasonable.
Question: A. Emsley
Do you expect these functional cells to produce germinal tissue (sperm, egg)
sporadically or continuously during the life of the chimera?
Answer: J. N. Petitte
I would expect normal spermatogenesis and oogenesis. The answer hinges on
whether any given population of sperm is derived from compartmentalization of
spermatogensis in the tesitis or from a continuous production from all tubules at
once. One would expect a more sporadic appearance of transgenic sperm if
spermatogenesis occurs in waves, which is very likely. This would also mean
that when transgenic sperm are present, the proportion would be higher than if
the entire testis were contributing to the ejaculate. This question could easily be
answered by time course analysis of the percentage of offspring derived from
roosters with moderate levels of germ line chimerism.
78