Download Chimeras and Transgenics: From Greek Mythology

Chimeras and Transgenics: From Greek Mythology to Poultry Breeding
Robert J. Etches
Department of Animal & Poultry Science
University of Guelph
Guelph, Ontario, Canada N 1G 2W 1
Introduction
In Greek mythology, a chimera is an imaginary fire-breathing animal with the head of
a lion, the body of a goat and the tail of a serpent. While the fanciful creation that was killed
by the hero Bellepheron as he rode his winged horse Pegasus is not generally contemplated as
a research objective by modem biologists, the idea of an animal with parts derived from
different sources has broad appeal. In the case of poultry breeding, the appeal of chimeric
technology lies in the possibility of combining cells of differing genotypes to obtain chickens
that can transmit unique genetic characteristics to their offspring. The unique characteristics
may be from cells that have been genetically modified in specific ways using transgenic
technology, or from cells that have been cryopreserved for many years.
Somatic and germline chimerism
Most of the cells in an adult organism are in somatic tissues such as muscle, liver,
spleen, pancreas, brain, etc. A much smaller population of cells belongs to the germline,
which includes spermatogonia, cells in all stages of spermatogenesis, functional sperm,
oocytes and ova. From the point of view of poultry breeding, germline chimerism is much
more important that somatic chimerism because only traits that are incorporated into sperm
and ova can be transmitted to the next generation.
Germline-competent
cells
The fate of cells during embryonic development can be viewed as an irreversible cascade
that begins with a newly fertilized egg. At the one-celled zygote stage, the embryo is
described as pluripotential because the lineage of every cell in an adult organism can be traced
back to the zygote. As development proceeds, however, cells become committed to
specialized functions, such as muscle, liver, spleen, pancreas, brain, etc., and the acquisition
of commitment to a specific developmental fate precludes contribution of that cell to alternate
developmental fates. Commitment is a gradual process that occurs during the first day of
embryonic development in birds.
The first day of embryonic development occurs as the egg descends through the
reproductive tract and receives its investments of egg white, shell membranes, and shell.
During this interval, the embryo divides from a single cell into an embryo containing
approximately 50,000 - 60,000 cells in a newly-laid, unincubated egg. At this stage of
development, which has been arbitrarily designated as stage X (Eyal-Giladi and Kochav,
1976; Eyal Giladi et al., 1980), the embryo is a radial dome of cells overlying a subgerminal
cavity which separates the embryo from the yolk (Fig. 1). The central disk of the embryo is
the area pellucida, which is surrounded by the area opaca. Transfer of cells from the area
pellucida of stage X embryos to the subgerminal cavity of recipient embryos at the same stage
of development produces chimeras (Fig. 2) that are comprised of both donor and recipient
cells in somatic tissues and thegermline (Petitte et al., 1990; 1993; Carsience et al, 1993;
Thoraval., 1994; Kagami et al., 1995; Kino et al., 1997). The technical details of making
chimeras are illustrated in Fig. 3.
Mixed-sex chimeras
The genome of male chickens includes two copies of the Z chromosome whereas the
genome of the female contains one copy of the Z and a W chromosome. There have been
many attempts to convert female chickens into males and vice versa to meet the aspirations of
the broiler and egg industries, respectively (Etches and Kagami, 1997). All of these attempts
have focused on differentiation of the gonad, which begins at about the fifth day of
incubation. An alternative strategy to achieve sex reversal is to alter sex determination,
which occurs when the egg and sperm unite to form a zygote. This situation can be achieved
by injecting male cells into female recipients and vice versa (Fig. 4).
When male donor cells are injected into female recipients approximately one half of the
chimeras are male and one half are female (Kagami et al., 1995). When female donor cells
are injected into male chimeras, however, all of the chimeras are male (Fig. 4). Regardless of
the sex of the donor and the recipient, male chimeras derived from male and female
blastodermal cells contain genetically female cells in a male environment. This combination
provides a powerful experimental model to study sex determination in birds that can be used
to determine (1) if genetically female cells can function as spermatogonia in a testis and (2) if
genetically male cells can function as ova in females. In brief, when male blastodermal cells
are injected into female recipients and a female chimera is produced, the male blastodermal
cells are processed normally as ova. When the same combination yields a male chimera,
however, genetically female (ZW) spermatogonia can only produce Z-bearing spermatozoa.
The W-beating spermatids fail to develop into functional sperm, indicating that there are
genes on the Z chromosome that are required for the completion of spermiogenesis. This
series of genes may be analogous to those situated on the long arm of the Y chromosome that
are essential for complete spermatogenesis in mammals. From the perspective of poultry
breeding, the observation that ZZ male cells can become functional female cells indicates that
sex reversal from males to females should be possible. The converse may be much more
difficult because it is clearly more complicated to produce functional W-bearing sperm. In the
future, transgenic technology might be employed to bring together the appropriate genes into
parental lines that would yield only male or only female offspring. While this strategy has
been applied in plant breeding, its execution on poultry breeding will require more
information about sex determination and differentiation in birds.
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i
i
Creating inbred lines of chickens
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While highly inbred lines of chickens have not been used in commercial poultry breeding
programs, they have found widespread application in studies of the genetic control of the
immune system of chickens (Lamont and Dietert, 1990). In contrast, plant breeding strategies
have relied heavily on the creation and hybridization of inbred lines. The difficulty of
obtaining inbred lines of chickens has been an impediment to their use in poultry breeding
programs, but their development could be greatly facilitated using chimeric technology.
Conventional breeding strategies for producing highly inbred lines use 6 to 10 generations
of full-sib matings to yield animals that are at least 90% homozygous. However, it is possible
to achieve the same level of inbreeding in 3 generations from inter se matings of male and
female germline chimeras derived from a single donor embryo (Fig.5). By colonizing the
germline of male and female birds with genetically identical cells, it is possible to achieve
self-fertilization (Fig.5A). The genome of offspring that result from the first generation of
this scheme contains DNA that is derived only from the donor embryo and the only
rearrangement of the genome is introduced by segregation. To produce highly inbred
populations of chickens, eggs from the offspring of chimeras produced from the same donor
embryo would be used as donors for a second cycle of self fertilization. Reiteration of this
cycle for 3 generations could provide large populations of highly inbred birds (Fig 5B).
Cryopreservation
of genetic resources
Hundreds of defined breeds, strains and lines of chickens have been developed by research
institutes, univerisities, private breeding companies and poultry enthusiasts. Unfortunately,
financial constraints prevent the retention of all of these genetic resources as live populations
and alternate methods for storing potentially valuable genotypes is required. Semen can be
frozen and stored at -196C, and reasonable levels of fertility can be obtained from sperm after
thawing and insemination. However, the precise combination of genes cannot be recovered
from frozen semen because the line must be outcrossed while inseminating hens with
cryopreserved semen. The entire genome can be cryopreserved, however, by freezing
blastodermal cells (Fig. 6)i When the line is required in the future, the frozen cells can be
thawed and injected into recipient embryos (Fig. 6). Inter se matings of the male and female
germline chimeras that are produced yield offspring that are an exact reconstitution of the
stock from which blastodermal cells were frozen (Kino et al, 1997). A gene bank has been
established at the Univeristy of Guelph that currently holds a number of the lines that were
produced by Agriculture and Agri-Food Canada in Ottawa. Expansion of this concept could
provide a vast genetic resource from which specific genes or genotypes could be recovered in
the future.
Creating transgenic chickens via chimeric intermediates
The production of transgenic chickens requires modification of the genome of cells that
can enter the germline and transmit the genetic modification to the next generation. Since
blastodermal cells contribute to the germline, they are excellent candidates for genetic
modification. The strategy that is currently being developed at the University of Guelph for
the production of transgenic chickens via chimeric intermediates is illustrated in Fig. 7.
Briefly, blastodermal cells from donor embryos are dispersed into a single cell suspension and
transfected with the gene of interest and a gene conferring antibiotic resistance. Since
transfection is usually an inefficient process, cells that have incorporated the genetic
modification must be separated from those that remain unaltered. Selection is accomplished
by culturing the cells in the presence of the appropriate antibiotic for several days. The
selected cells are then transferred to an irradiated recipient embryo to form a transgenic
chimera from which founder offspring carrying the transgene are derived. The utility of the
transgene would then be evaluated in subsequent breeding programs.
Genes that would be inserted into the germline via chimeric intermediates fall into three
categories according to their intended use. Genes that might be considered for the poultry
breeding industry include those encoding disease resistance and those that might alter the
nutritional capabilities of the birds. For example, alleles at the major histocompatability
complex might confer partial resistance to Marek's Disease (Bacon and Witter, 1993, 1994a,
1994b) and, therefore, are potentially useful in commercial breeding programs. The
incorporation of genes encoding phytase, which would yield chickens that could utilize plant
phosphorus more efficiently, would reduce the level of phosphorus in diets and therefore,
reduce level of phosphorus in poultry waste. While it may be possible to increase levels of
production per se using transgenic technology, it is unlikely that execution Of this strategy
will find public acceptance. On the other hand, changes that confer a welfare benefit to the
bird, or are perceived as improvements to food safety and quality, are likely to be widely
accepted by consumers.
The chicken is a widely used experimental model in developmental biology, but its
usefulness is currently limited by the lack of ability to create transgenic animals. However,
there are many examples of genes that are of interest to developmental biologists who require
the ability to make transgenic chickens to execute experimental paradigms that reveal
information about gene products and their function. While this use oftransgenic technology
lies outside the commercial poultry industry, unexpected benefits to the industry may arise
from the rapid increase in knowledge that will be provided by the biomedical research
community.
A new industry that uses the egg as a repository for proteins encoded by transgenes may
become popular if transgenic chickens can be produced routinely. For example, if genes
encoding human immunoglobulins (hlg) can be introduced into chickens and if the hlg is
deposited in egg yolk, it should be possible to harvest these pharmaceutically important
proteins from eggs. The application of transgenic technology to poultry, therefore, has the
potential to provide a new set of tools for industrial poultry breeding industry and to spawn
new opportunities for poultry breeders that lie outside the traditional domain.
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References
.-
Bacon, L.D., Witter (1993). Influence of B-haplotype on the relative efficacy of Marek's disease
vaccines of different serotypes. Avian Diseases 37, 53-59
Bacon, L.D., Witter (1994a). B haplotype influence on the relative efficacy of Marek's disease
vaccines in commercial chickens. Poultry Science 73, 481-487.
Bacon, L.D., Witter (1994b). Serotype specificity of B-haplotype influence on the relative
efficacy of Marek's disease vaccines. Avian Diseases 38, 65-71.
Carsience, R.S., Clark, M.E., Verrinder Gibbins A.M. and Etches, R.J. (1993). Germline
chimeric chickens from dispersed donor blastodermal cells and compromised recipient
embryos. Development 117, 669-675
Etches, R.J. (1995). Reproduction in Poultry. CAB International, Wallingford, England.
Etches, R.J., Clark, M.E., Toner, A., Liu, G. and Verrinder Gibbins, A.M. (1996). Contributions
to somatic and germline lineages of chicken blastodermal cells maintained in culture.
Molecular Reproduction and Development 45, 291-298
Etches, R.J. and Kagami, H. (1997). Genotypic and phenotypic sex reversal. In: Perspectives in
Avian Endocrinology. Edited by S. Harvey and R.J. Etches, Journal of Endocrinology
Ltd., Bristol.
Eyal-Giladi, H., and S. Kochav, 1976. From cleavage to primitive streak formation: a
complementary normal table and a new look at the first stage of the development of the
chick. I. General morphology. Dev. Biol. 49: 321-337.
Kagami, H, M. E. Clark, A. M. Verrinder Gibbins, and R. J. Etches, 1995. Sexual
differentiation of chimeric chickens containing ZZ and ZW cells in the germline. Mol.
Reprod. Dev. 42:379-388.
Kino, K., B. Pain, S. P. Liebo, M. Cochran, M. E. Clark and R. J. Etches, 1997. Production of
chicken chimeras from injection of frozen-thawed blastodermal cells. Poultry Sci. 76:
in press.
Kochav, S., M. Ginsburg and H. Eyal-Giladi, 1980. From cleavage to primitive streak
formation: a complementary normal table and a new look at the first stage of the
development of the chick I. General morphology. Dev. Biol. 79:296-307
Lamont S.J. and Dietert, R.R. (1990). Immunogenetics. In Poultry Breeding and Genetics,
edited by R.D. Crawford Elsevier, Amsterdam.
Pain, B., Clark, M.E., Nakazawa, H., Sakurai, M. Samarut, J. and Etches, R.J. (1996). Long
term culture and characterization of avian embryonic stem cells with multiple
morphogenetic capabilities. Development 122, 2339-2348
Petitte, J.N., M. E. Clark, G. Liu, A. M. Verrinder Gibbins and R. J. Etches, 1990. Production
of somatic and germline chimeras in the chicken by transfer of early blastodermal cells.
Development 108:185-189
Petitte, J.N., Brazolot, C.L., Clark, M.E., Liu, G., Verrinder Gibbins, A.M. and Etches, R.J.
(1993). Accessing the chicken genome using germline chimeras. In Manipulation of
the Avian Genome. Edited by R.J. Etches and A.M. Gibbins. CRC Press, Boca Raton.
Thoraval, P., F. Lasserre, F. Coudert and G. Dambrine, 1994. Production of germline chimeras
obtained from Brown and White Leghorns by transfer of early blastodermal cells.
Poultry Sci. 73:1897-1905.
J.,
4.4 mat
Area opaca i--Area
pcHucida_
--I
Area#,_
sphcr_
Subgcrminal fluid
Blastodermal cells
Fig. 1. The structure of an embryo at the time of laying illustrated as a cross section through
the embryo perpendicular to the surface of the yolk (from Etches, 1995).
Fig. 2. Three chimeras produced by injecting approximately 500 cells from a stage X Barred
Plymouth Rock donor embryo into a White Leghorn recipient embryo at the same stage of
development.
Somatic chimerism is evident from the patches of donor-derived and recipientderived feather pigmentation.
Germline chimerism is assessed by mating chimeras to Barred
Rocks and examining the down color of the offspring. Black chicks are derived from donor
cells that contributed to the germline whereas yellow chicks are derived from recipient
contributions to the germline.
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_ '_'_
....................
•_
Recipient
(II)
White Leghorn
Plymouth
Donor
Barred (ii) Roc
eggs irradiated
chimeric
chick
,
/
and drilled
dispersed
'_
rejected
into
stage X cells
recipient
chimeric
rooster
Barred
Rock hen
Fig. 3. A schematic representation of the procedure for making somatic and germline
chimeras. Donor cells are harvested by enzymatic digestion from Barred Plymouth Rock
embryos that arehomozygous
for the recessive allele at the I locus. Recipient embryos are
obtained from White Leghorn eggs that are homozygous for the dominant allele at the I locus
and. Within two hourprior to injection of donor cells, development of recipient embryos is
arrested for about 24 h by exposing the eggs to approximately 500 rads of), irradiation.
Donor cells divide rapidly after injection into the quiescent recipient to yield a chimera that
contains substantial contributions from the donor cells. Chimerism in somatic tissues can be
assessed from donor-derived down color. Germline chimerism is determined by mating
chimeras to Barred Rocks. The proportion of black chicks from this mating is proportional to
the donor contribution to the gerrnline.
Fig. 4. Schematic diagram for making mixed-sex chimeras. Blastodermal cells from a donor
embryo are dispersed and injected into a series of recipient embryos. The sex of the donor
embryo is determined by PCR or in situ hybridization of DNA in a sample of donor cells.
The sex of the chimera is determined by PCR or in situ hybridization of erythrocytes.
Comparison of the sex of the donor embryo and the chimera reveals the sex of the recipient.
Injection of male (ZZ) cells into a female (ZW) recipient produces approximately equal
numbers of male and female chimeras whereas injection of female cells into male recipients
always yields males. ZZ cells in a female chimera are processed in the germline as female
cells. ZW cells in a male chimera form spermatogonia and spermatids, but spermiogenesis of
W-bearing spermatozoa is blocked. Z-bearing spermatozoa, however, are processed normally
into functional spermatozoa. See Kagami et al. (1995) for additional details.
(A)
X
#2
CHIMERIC#3
-'_
CHIMERIC
#4
(B)
1
0.9
0.8
g 0.7
_
,Q_
(t)
0.6
0.5
0.4
_Selfing
I
_
FullSibs
!--&--- Half Sibs r
I
0.3
¢= 0.2
==
0.1
o
0
1
2
3
4
5
Generation
Fig. 5. (A) Highly inbred lines of chickens can be produced by dispersing blastodermal cells
from a single embryo into a series of recipient embryos. Some of the resulting chimeras will
be male and others will be female. Inter se matings between these birds is the equivalent to
selfing in plants and produces offspring in which 50% of the genes are homozygous(B).
Reiteration of this cycle 4 times produces birds that are 94% homozygous. In comparison to
matings between full sibs or half sibs, the level of homozygosity increases much more rapidly
when chimeras derived from the same embryo are mated.
10
o
Recipient (II)
White Leghorn
Donor (ii)
Barred
Plymouth Rock
eggs irradiated
and drilled
"_
dispersed
stage X cells
thawed cells
__'"
chimeric
chicks
injected into _
recipient eggs
chimeric
rooster
cells frozen
in liquid
_,iiiiii,iiiiiii_ii.m]m!ii
i
_i-Jiiiiiiiiiiiiii_
_
nitrogen
chimeric
hen
Barred Rock
chick
Fig. 6. Populations of chickens can be maintained in a cryopreserved state for an indefinite
period by freezing blastodermal cells. When the population is required, blastodermal cells are
thawed and injected into recipient embryos. To demonstrate this principle, cells from Barred
Plymouth Rocks were frozen, stored, thawed and injected into White Leghorn recipients to
form chimeras. Inter se matings between the chimeras yielded offspring that were faithful
representatives of the Barred Rock population that provided the blastodermal cells for
freezing. See Kino et al. (1997) for additional details.
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Hiiiiiiiii _iiiiiiiiiH
!iiiiii!!!_ni.iiiiii_iiii
ii!!!_!_iiiiE_
!ii!iiii!i!
Gene of interest
injected into
recipient
egg
.
stage X donor
_
modified cells
Genetically
selected
in culture
_'"
= development of subline homozygous
for the genetic modification
Fig. 7. A schematic representation of the strategy for producing transgenic chickenS at the
University of Guelph. The gene of interest and a gene conferring antibiotic resistance are
transfected into dispersed blastodermal cells. The genetically modified cells are then selected
by culturing the cells in the presence of the antibiotic. After selection in culture, genetically
modified cells are transferred into recipient embryos to form germline chimeras. Donorderived offspring from the chimeras are screened for the presence of the gene of interest.
Birds carrying the gene of interest become the founders for a line of chickens that express the
desirable trait.
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•
Questions and answers:
Dr. A. Tajima
Q. Is there any possibility of improving the incorporation rate of donor blastodermal cells
into the germ line?
A. It may be possible to identify cells within the stage X blastoderm that have the ability to
enter the germline. If populations of donor cells that were comprised exclusively
germline competent cells was obtained, a higher rate of germline transmission would be
anticipated.
Dr. M. Delany
Q. What is the level of cell death and embryonic death in irradiated receipients?
A. We have not estimated cell death. Embryonic mortality after exposure to approximately
500 rads is about 20% (see Carsience et al., 1993).
Q. What is the mutation rate in recipient embryos and is that important for developoment of
the chimeras and their offspring?
A. We have reared irradiated recipients and examined their offspring for evidence of
mutation. Fertility and hatchability of irradiated recipients is normal and no morphological
abnormalities were observed among the offspring. From these data we have concluded
that there are no major effects of the low dose of irradiation. However, the recipient is
only a vehicle for transferring cells that have been genetically modified from a culture
vessel into the germline of a reproductively active bird that can produce transgenic
offspring. Recipient embryos do not contribute to the transgenic gene pool and, therefore,
any potentially detrimental effects of irradiation would be excluded from the subsequent
development of a line of birds expressing the transgene.
Q. Have you tried to make chimeras with turkey embryos?
A. No, we have not.
Dr. Steve Stice
Q. For how long can you maintain blastodermal cells in culture?
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A. We have been able to make somatic and germline chimeras from cells that have been in
culture for up to 7 days, but have kept the cells in vitro for much longer periods. See
Etches et al. (1996) and Pain et al. (1996) for additional details.
Q. Is this long enough to get selection of the gene for making transgenic animals and possibly
gene knockouts?
A. Seven days provides sufficient time to obtain significant enrichment of the population of
transfected cells and may provide sufficient time to make a knockout. This is an area that
we are actively pursuing at the moment.
Dr. Douglas Rhoads
Q. Have you tried sex reversal of the chimera based on recipient genotype?
A. All combinations of male and females as donor and recipients have been investigated but
the combinations have been identified retrospectively. See Kagami et al. (1995) for
additional details.
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