Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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Title: Methods for Selecting Germ-line Competent Cells in Chicken Embryos, and
the Use of the Cells in the Production of Chimeras
This application claims benefit from United States provisional application
serial no. 60/039,488 filed on February 28, 1997.
FIELD OF TH . INVENTIO~1V
The invention relates to methods for selecting germ-line competent cells, and
methods for producing germ-Line chimeras in avian species.
BACKGROUND OF THE INVENTION
The poultry industry has traditionally relied on crossbreeding of pure lines
to
provide chickens, turkeys and other poultry with desirable characteristics.
The genetic
rearrangement that occurs at each generation usually results in offspring that
bear only a
small proportion of the attributes of superior individuals in the parental
population.
Ideally, breeders would like more control over the manipulation of the genome.
There have been many studies to try to manipulate the genome during the
embryonic stage of development to introduce favourable characteristics in
poultry. Methods
have been used to introduce DNA into the pronuclei or newly fertilized ovum
but these
methods suffer from many disadvantages. The methods are expensive since the
avian
species must be killed to obtain each ovum or zygote; it is difficult to
identify the male and
female pronuclei among the supernumary spermatozoa that enter at
fertilization; exogenous
DNA does not integrate into the genome at a high rate when injected into newly
fertilized
zygotes; and it is technically difficult to return the manipulated ovum to the
oviduct of a
fistulated hen, or to maintain it in a surrogate egg. Therefore, more current
methods
involve the manipulation of blastodermal cells contained in the newly laid
egg.
Fertilization of chicken embryos occurs within 15 minutes following ovulation
in the infundibulum of the reproduction tract. The embryo, which is situated
on the surface
of the yolk, develops during the next 18-23 hours as egg formation is
completed by the
secretion of albumen, membranes and shell around the yolk. The first cell
division occurs
approximately 5 hours after fertilization as the egg enters the shell gland.
During the next
13-18 hours, embryonic divisions continue rapidly to yield an embryo
containing 40,000 -
60,000 cells (Eyal-Giladi & Kochav, 1976; Kochav et al., 1980; Watt et al.,
1993). When
egg formation is completed, the egg is expelled from the shell gland at
oviposition. At
oviposition, the embryonic structure which contains 40,000-60,000 cells is
designated as a
stage X (E-G & K) embryo.
When a sample of up to 1000 cells from a population of cells harvested from
stage X (E-G & K) embryos is transferred to recipient embryos at the same
stage of
development, the donor cells contribute to both somatic tissues and the germ-
line of the
resulting chimera (Petitte et al., 1990; Carsience et al., 1993, Fraser et
al., 1993; Thoraval et
al., 1994; Kagami et al., 1995; Etches et al., 1996a; Kino et al., 1997). The
identification of
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cells that contribute to the germ-line and somatic tissues, however, has not
been made. The
production of somatic and germ-line chimeras after the injection of cells from
stage X (E-G &
K) embryos may indicate that each cell in a stage X (E-G & K) embryo can
contribute to
ectoderm, mesoderm, endoderm and the germ-line and, therefore, each cell from
a stage X
(E-G & K) embryo is pluripotent. However, some indirect evidence suggests that
the
population of cells in stage X (E-G & K) embryos contains cells with different
functional
properties. For example, the EMA-1 epitope, which is believed to be expressed
uniquely on
primordial germ cells which are committed to the germ-line, is expressed on
some cells in
stage XI (E-G & K) embryo (Urven et al., 1988; Karagenc et al., 1995). The
epitope SSEA-1,
which is expressed by mouse embryonic stem cells, is expressed by some cells
in stage XIII (E-
G&K) embryos (Petitte and Karagenc, 1996). Evidence supporting the presence of
morphologically unrecognizable primordial germ cells in stage X (E-G&K)
chicken embryos
may be inferred from the presence of committed primordial germ cells in
cultures derived
from the central disc, but not the area opaca, of stage X blastoderms
(Ginsburg and Eyal-
Giladi, 1987). The presence of cells that are destined for the germ-line can
also be inferred
from the observation that somatic and germ-line chimeras are produced more
frequently
from cells taken from the central disc rather than the area opaca of stage X
(E-G&K)
embryos {Petitte et al., 1993). In the quail, the QH-1 epitope, which is
believed to be
expressed by cells committed to the germ-line in quail, is expressed in
embryonic cells at the
time of oviposition (Pardanaud et al., 1987). Although these data provide
indirect
evidence indicating that the precursors of primordial germ cells are present
in stage X (E-
G&K) embryos, it is not yet clear if (1) these cells are committed to the germ-
line, (2) if
they retain the ability to enter both the somatic and germ-line lineages arid
(3) by what
means they can be identified.
The production of chimeric avian species such as chickens would be greatly
enhanced if the location of germ-line competent cells within stage X (E-G&K)
embryos were
identified, and if populations of germ-line committed cells could be isolated
from the entire
population of cells that comprise a stage X (E-G&K) embryo. For example, it
would be
possible to extirpate the endogenous germ-line competent cells within a
recipient embryo to
eliminate any contribution to the germ-line except that of the donor embryo.
Using current
technology, the endogenous contribution is reduced, but not eliminated, by
irradiating the
recipient embryo (Carsience et al., 1993). Irradiation impedes growth of the
recipient
embryo for approximately 24 hours while the donor-derived contributions to the
chimera
proliferate (Carsience et al., 1993). While this approach has proved to be
useful, the
extent of the donor-derived contribution to the germ-line is neither
predictable nor
consistent. The lack of a predictable and consistent contribution to the germ-
line is
particularly important when the donor-cell population contains a small number
of
genetically unique and rare donor cells. Examples of rare cells might include
genetically
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modified cells (see Brazolot et al., 1991; Fraser et al., 1993) or cells
derived from
cryopreserved stocks that are extinct (see Kino et al., 1997).
The production of chimeric chickens would also be enhanced if cell surface
epitopes expressed by germ-line competent cells within stage X (E-G&K) embryos
were
identified. The identification of the epitopes would facilitate development of
methods to
identify and isolate germ-line competent cells.
SUMMARY OF THE INVFNTrnrr
The present inventor has identified cell surface epitopes expressed by germ
line competent cells within stage X(E-G&K) embryos of chickens. In particular,
the present
inventor has identified the EMA-1 and SSEA-1 epitopes in stage X (E-G & K)
chicken
embryos and in cells derived from them in culture. The identification of the
epitopes
facilitated the identification and isolation of cells within stage X (E-G&K)
chicken
embryos that are capable of replication without differentiation in vitro and
which retain
the ability to enter the germ-line following injection into a recipient
embryo. These cells
are referred to herein as "germ-line competent cells". Isolated germ-line
competent cells
from donor embryos were introduced into recipient embryos to produce chimeric
embryos.
The extent of chimerism was increased when donor cells were injected into
recipients from
which cells had been extirpated from the central region of the recipient
embryo and when
recipient embryos are irradiated prior to the injection of the donor cells.
Broadly stated the present invention relates to a method for selecting germ-
line competent cells in stage X (E-G&K) chicken embryos comprising separating
from a stage
X (E-G&K) chicken embryo cells that have an epitope expressed by germ-line
competent
cells associated with their cell surface. In particular, germ-line competent
cells may be
isolated by reacting cells obtained from a stage X (E-G&K) chicken embryo with
a substance
which binds directly or indirectly to an epitope expressed by germ-line
competent cells. In a
preferred embodiment, the epitope expressed by germ-line competent cells is an
EMA-1 or an
SSEA-1 epitope.
The invention also relates to a method for producing a cell preparation
enriched for germ-line competent cells of chickens comprising (a) providing
cells from a
stage X (E-G&K) chicken embryo; and (b) selecting cells that have an epitope
expressed by
germ-line competent cells associated with their cell surface.
Germ-line competent cells identified and isolated using the methods of the
invention may be genetically modified by introducing a recombinant expression
vector into
the cells. Therefore, the methods of the invention may additionally comprise
transfecting
the germ-line competent cells with a recombinant expression vector containing
an exogenous
gene and the necessary elements for the transcription and translation of the
gene.
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The invention additionally relates to a cell culture comprising germ-line
competent cells or genetically modified germ-line cells obtained using the
methods of the
invention.
The invention still further relates to a method for identifying a region in an
embryo which contains germ-line competent cells comprising
(a) treating the embryo with a substance which directly or indirectly binds to
an epitope expressed by germ-line competent cells wherein the substance is
labelled with a
detectable marker; and
(b) detecting the detectable change produced by the detectable marker to
identify a region in the embryo which contains germ-line competent cells.
In a preferred embodiment, the epitope expressed by germ-line competent
cells is an EMA-1 or an SSEA-1 epitope and the substance that binds to the
epitope is an
antibody.
The germ-line cells identified in accordance with the methods of the
invention may be used to produce chimeric chicken embryos. Therefore, the
present
invention provides a method for producing a chimeric chicken embryo comprising
(a)
isolating from a donor stage X (E-G&K) chicken embryo cells that have an
epitope
expressed by germ-line competent cells associated with their cell surface; (b)
optionally
transfecting the cells with a recombinant expression vector containing an
exogenous gene and
the necessary elements for the transcription and translation of the gene; (c)
introducing the
cells into a recipient stage X (E-G&K) chicken embryo from which a portion of
the central
disk of the embryo has been removed; and (d) incubating the recipient embryo
to produce a
chimeric embryo. The chimeric embryo may be grown to term to produce chimeric
chickens.
The invention further provides chimeric embryos and chimeric chickens
produced by the methods of the invention.
Still further the present invention relates to kits for performing the methods
of the invention.
Other objects, features and advantages of the present invention will become
apparent from the following detailed description. It should be understood,
however, that
the detailed description and the specific examples while indicating preferred
embodiments
of the invention are given by way of illustration only, since various changes
and
modifications within the spirit and scope of the invention will become
apparent to those
skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described in relation to the drawings in which:
Figure 1 shows the structure of a stage X embryo illustrated as a cross-
section
through the embryo perpendicular to the surface of the yollc;
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Figure 2 shows a phase contrast illumination micrograph illustrating the
expression of EMA-1 epitopes on stage X blastodermal cells.
Figure 3 shows a fluorescence micrograph of Figure 2;
Figure 4 shows a phase contrast illumination micrograph illustrating the
expression of SSEA-1 epitopes on stage X blastodermal cells;
Figure 5 shows a fluorescence micrograph of Figure 4;
Figure 6 shows a phase contrast illumination micrograph illustrating the
SSEA-1 expression on stage X whole mount;
Figure 7 shows a fluorescence micrograph of Figure 6;
Figure 8 shows a phase contrast illumination micrograph illustrating HNK-
1 /NC-1 expression on stage X blastodermal cells; and
Figure 9 shows a fluorescent micrograph of Figure 8.
DETAILED DES RIPT10N OF THE INVENTIOr;f
I. Selection of Germ-line Competent Cells
As hereinbefore mentioned, the present invention relates to a method for
selecting germ-line competent cells in stage X (E-G&K) chicken embryos
comprising
separating from a stage X (E-G&K) chicken embryo, cells which have an epitope
expressed
by germ-line competent cells associated with their cell surface. The invention
also relates
to a method for producing a cell preparation enriched for germ-line competent
cells of
chickens comprising (a) providing cells from a stage X {E-G&K) chicken embryo;
and (b)
selecting cells that have an epitope expressed by germ-line competent cells
associated with
their cell surface. In a preferred embodiment, the epitope expressed by germ-
line competent
cells is an EMA-1 or an SSEA-1 epitope.
A stage X (E-G&K) chicken embryo is an embryo which is expelled from the
shell gland at oviposition and it is characterized by containing between
40,000 - 60,000 cells
(Eyal-Giladi & Kochav, 1976; Kochav et al., 1980; Watt et al., 1993). The
architecture of a
stage X (E-G & K) embryo is illustrated in Figure 1. Morphologically, none of
the cells in a
stage X embryo demonstrate the characteristics of differentiated cells. The
central disc,
which includes the area pellucida and the marginal zone, is surrounded by the
area opaca.
The area pellucida is situated above a layer of subgerminal fluid that
separates the
embryo from the underlying yolk. The area opaca is directly in contact with
the
surrounding yolk.
Stage X (E-G&K) embryos may be isolated from eggs using conventional
methods, For example, embryos may be isolated from freshly laid unincubated
eggs by
separating albumen from the yolk as described by Carsience et al (1993)
Prior to selection of the germ-line competent cells, cells from a stage X (E-
G&K) chicken embryo are preferably dissociated from the embryo using
conventional
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techniques For example, blastoderm cells may be dispersed from an embryo by
treating
with a substance which digests the extracellular matrix such as trypsin.
Cells that have epitopes expressed by germ-line competent cells associated
with their surface may be selected using substances which directly or
indirectly bind to the
epitopes. In an embodiment of the invention, the epitope is EMA-1 or SSEA-1
and the
substance is an antibody specific for a EMA-1 or SSEA-1 epitope. Therefore,
the invention
provides a method for selecting germ-line competent cells in stage X chicken
embryos
comprising reacting cells obtained from a stage X (E-G &K) chicken embryo with
one or more
antibodies to an epitope expressed by germ-line competent cells; forming
conjugates between
the antibodies and the germ-line competent cells having the epitope associated
with their
cell surface; and isolating the conjugates to obtain a cell preparation
containing germ-line
competent cells. The method may employ more than one antibody, for example one
antibody to EMA-1 and one antibody to SSEA-1.
The term "antibody" includes polyclonal antisera or monoclonal antibodies.
Conventional methods can be used to prepare the antibodies. For example, by
using a EMA
1 and SSEA-1 epitope, polyclonal antisera or monoclonal antibodies can be made
using
standard methods. A mammal, (e.g., a mouse, hamster, or rabbit) can be
immunized with an
immunogenic form of a EMA-1 or SSEA-1 epitope which elicits an antibody
response in the
mammal. Techniques for conferring immunogenicity on an epitope include
conjugation to
carriers or other techniques well known in the art. The progress of
immunization can be
monitored by detection of antibody titers in plasma or serum. Standard ELISA
or other
immunoassay procedures can be used with the immunogen as antigen to assess the
levels of
antibodies. Following immunization, antisera can be obtained and, if desired,
polyclonal
antibodies isolated from the sera.
To produce monoclonal antibodies, antibody producing cells (lymphocytes) can
be harvested from an immunized animal and fused with myeloma cells by standard
somatic
cell fusion procedures thus immortalizing these cells and yielding hybridoma
cells. Such
techniques are well known in the art, [e.g., the hybridoma technique
originally developed
by Kohler and Milstein (Nature 256, 495-497 (1975)]. Other techniques such as
screening of
combinatorial antibody libraries can be employed (Huse et al., Science 246,
1275 (1989)].
Hybridoma cells can be screened immunochemically for production of antibodies
specifically reactive with the epitopes and the monoclonal antibodies can be
isolated.
The term "antibody" also includes antibody fragments which also
specifically react with an epitope expressed by germ-line competent cells.
Antibodies can
be fragmented using conventional techniques and the fragments screened for
utility as
described above. For example, F(ab')2 fragments can be generated by treating
antibody with
pepsin. The resulting F(ab') 2 fragment may be treated to reduce disulfide
bridges to produce
Fab' fragments.
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Chimeric antibody derivatives, i.e., antibody molecules that combine a non-
avian variable region and an avian constant region are also within the scope
of the
invention. Chimeric antibody molecules include, for example, the antigen
binding domain
from an antibody of a mouse, rat, or other species, with avian constant
regions. Standard
methods may be used to make chimeric antibodies containing the immunoglobulin
variable
region which recognizes an epitope expressed by germ-line competent cells
(See, for
example, Morrison et al., Proc. Natl Acad. Sci. U.S.A. 81,6851 (1985); Takeda
et al.,
Nature 314, 452 (1985), Cabilly et al., U.S. Patent No. 4,816,567; Boss et
al., U.S. Patent
No. 4,816,397; Tanaguchi et al., European Patent Publication EP171496;
European Patent
Publication 0173494, United Kingdom patent GB 2177096B).
Similarly, binding partners may be constructed utilizing recombinant DNA
techniques to incorporate the variable regions of a gene which encodes a
specifically
binding antibody. Within one embodiment, the genes which encode the variable
region
from a hybridoma producing a monoclonal antibody of interest are amplified
using
nucleotide primers for the variable region. These primers may be synthesized
by one of
ordinary skill in the art, or may be purchased from commercially available
sources. The
primers may be utilized to amplify heavy or light chain variable regions,
which may then
be inserted into vectors such as ImmunoZAPT" H or ImmunoZAPT"' L (Stratacyte),
respectively. These vectors may then be introduced into ~. ~oli for
expression. Utilizing
these techniques, large amounts of a single-chain protein containing a fusion
of the VH and
VL domains may be produced (See Bird et al., Science 242:423-426, 1988).
Antibodies against an epitope may also be obtained from commercial sources.
For example, monoclonal antibodies to SSEA-1 and EMA-1 may be obtained from
Immunotech, Westbrook, ME, USA.
The antibodies may be labelled with a detectable marker including various
enzymes, fluorescent materials, luminescent materials and radioactive
materials.
Examples of suitable enzymes include horseradish peroxidase, biotin, alkaline
phosphatase, Q-galactosidase, or acetylcholinesterase; examples of suitable
fluorescent
materials include umbeiliferone, fluorescein, fluorescein isothiocyanate,
rhodamine,
dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an
example of a
luminescent material includes luminol; and examples of suitable radioactive
material
include S-35, Cu-64, Ga-67, Zr-89, Ru-97, Tc-99m, Rh-105, Pd-109, In-111, I-
123, I-125, I131,
Re-186, Au-198, Au-199, Pb-203, At-211, Pb-212 and Bi-212. The antibodies may
also be
labelled or conjugated to one partner of a ligand binding pair. Representative
examples
include avidin-biotin and riboflavin-riboflavin binding protein. Methods for
conjugating or
labelling the antibodies with the representative labels set forth above may be
readily
accomplished using conventional techniques.
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The antibodies may be labelled with a detectable marker as described herein
or indirect methods may be employed. In an indirect method, the primary
antigen-antibody
reaction may be amplified by the introduction of a second antibody, having
specificity for
the antibody reactive against an epitope. By way of example, if the antibody
having
specificity against an EMA-1 or SSEA-1 epitope is a mouse IgG antibody, the
second
antibody may be rat or rabbit anti-mouse gamma-globulin labelled with a
detectable
marker as described herein.
The antibodies may be bound to a carrier such as agarose, cellulose, dextran,
Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-
exchange
resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-
malefic acid
copolymer, amino acid copolymer, ethylene-malefic acid copolymer, nylon, silk,
etc. The
carrier may be in the shape of, for example, a tube, test plate, beads, disc,
sphere etc.
The antibodies may be conjugated to a matrix. Examples of matrices are
magnetic beads, which allow for direct magnetic separation (Kernshead 1992),
panning
surfaces e.g. plates, (Lebkowski, J.S, et al., (1994), J. of Cellular
Biochemistry supple.
18b:58), dense particles for density centrifugation (Van Vlasselaer, P.,
Density Adjusted
Cell Sorting (DACS), A Novel Method to Remove Tumor Cells From Peripheral
Blood and
Bone Marrow StemCell Transplants. (1995) 3rd International Symposium on Recent
Advances in Hematopoietic Stem Cell Transplantation-Clinical Progress, New
Technologies and Gene Therapy, San Diego, CA), adsorption columns (Berenson et
al. 1986,
Journal of Immunological Methods 91:11-19.), and adsorption membranes (Norton
et al.
1994). The antibodies may also be joined to a cytotoxic agent such as
complement or a
cytotoxin, to lyse or kill the targeted germ-line competent cells.
The antibodies may be directly coupled to a matrix. For example, the
antibodies may be chemically bound to the surface of magnetic particles for
example, using
cyanogen bromide. When the magnetic particles are reacted with a sample
containing
germ-line competent cells having an epitope expressed by germ-line competent
cells on the
their cell surfaces, conjugates will form between the magnetic particles with
bound
antibodies and the germ-line competent cells.
Alternatively, the antibodies may be indirectly conjugated to a matrix using
antibodies. For example, a matrix may be coated with a second antibody having
specificity
for the antibodies to a SSEA-1 or an EMA-1 epitope. By way of example, if the
antibodies
to the SSEA-i or EMA-1 epitope are mouse IgG antibodies, the second antibody
may be
rabbit anti-mouse IgG. The antibodies may also be incorporated in antibody
reagents which
indirectly conjugate to a matrix. Examples of antibody reagents are bispecific
antibodies,
tetrameric antibody complexes, and biotinylated antibodies.
Bispecific antibodies contain a variable region of an antibody specific for an
epitope expressed by germ-line competent cells, and a variable region specific
for at Least
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one antigen on the surface of a matrix. The bispecific antibodies may be
prepared by
forming hybrid hybridomas. The hybrid hybridomas may be prepared using the
procedures
known in the art such as those disclosed in Staerz & Bevan, (1986, PNAS (USA)
83: 1453)
and Staerz & Bevan, (1986, Immunology Today, 7:241). Bispecific antibodies may
also be
constructed by chemical means using procedures such as those described by
Staerz et al.,
(1985, Nature, 314:628) and Perez et al., (1985 Nature 316:354), or by
expression of
recombinant immunoglobulin gene constructs.
A tetrameric immunological complex may be prepared by mixing a first
monoclonal antibody which is capable of binding to at least one antigen on the
surface of a
matrix, and a second monoclonal antibody specific for an epitope expressed by
germ-line
competent cells. The first and second monoclonal antibody are from a first
animal species.
The first and second antibody are reacted with an about equimolar amount of
monoclonal
antibodies of a second animal species which are directed against the Fc-
fragments of the
antibodies of the first animal species. The first and second antibody may also
be reacted
with an about equimolar amount of the F(ab')2 fragments of monoclonal
antibodies of a
second animal species which are directed against the Fc-fragments of the
antibodies of the
first animal species. (See U.S. Patent No. 4,868,109 to Lansdorp, which is
incorporated
herein by reference for a description of tetrameric antibody complexes and
methods for
preparing same).
The antibodies may be biotinylated and indirectly conjugated to a matrix
which is labelled with (strept) avidin. For example, biotinylated antibodies
may be used
in combination with magnetic iron-dextran particles that are covalently
labelled with
(strept) avidin (Miltenyi, S. et al., Cytometry 11:231, 1990). Many
alternative indirect
ways to specifically cross-link the antibodies and matrices would also be
apparent to those
skilled in the art.
In a preferred embodiment of the invention, the cell-antibody conjugates are
removed by magnetic separation using magnetic particles. Suitable magnetic
particles
include particles in ferrofluids and other colloidal magnetic solutions.
Examples of
ferrofluids and methods for preparing them are described by Kemshead J.T.
(1992} in J.
Hematotherapy, 1:35-44, at pages 36 to 39, and Ziolo et al. Science (1994)
257:219 which are
incorporated herein by reference. Colloidal particles of dextran-iron complex
may be used
in the process of the invention. (See Molday, R.S. and McKenzie, L.L. FEBS
Lett. 170:232,
1984; Miltenyi et al., Cytometry 11:231, 1990; and Molday, R.S. and MacKenzie,
D.,
J.Immunol. Methods 52:353, 1982; Thomas et al., J. Hematother. 2:297 (1993);
and U.S.
Patent No. 4,452,733, which are each incorporated herein by reference).
Magnetic particles
may also be obtained from commercial sources such as the microbeads available
from
Mitenyi Biotec.
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In accordance with a magnetic separation method of the invention, a sample
containing germ-line competent cells to be recovered, is reacted with one of
the above
described antibody reagents so that the antibody reagents bind to the targeted
germ-line
competent cells present in the sample to form cell conjugates of the targeted
cells and the
antibody reagents. The reaction conditions are selected to provide the desired
level of
binding between the targeted cells and the antibody reagents. The
concentration of the
antibody reagents is selected depending on the estimated concentration of the
targeted cells
in the sample. The magnetic particles are then added and the mixture is
incubated at the
selected temperature. The sample is then ready to be separated over a magnetic
filter
device. Preferably, the magnetic separation procedure is carried out using the
magnetic
filter and methods described in Miltenyi et al, 1990. Commercial magnetic
separation
systems such as the MiniMACS system available from Miltenyi Biotch may also be
used in
the magnetic separation methods of the present invention.
The sample containing the magnetically labelled cell conjugates is passed
through the magnetic filter in the presence of a magnetic field. The
magnetically labelled
conjugates are retained in the high gradient magnetic column and the materials
which are
not magnetically labelled flow through the column after washing with a buffer.
Antibodies to an epitope expressed by germ-line competent cells may also be
used to detect and quantify germ-line competent cells in an embryo. In
particular, the
antibodies may be used in immuno-histochemical analyses to localise germ-line
competent
cells to particular regions in the embryo.
Cytochemical techniques known in the art for localizing antigens using light
and electron microscopy may be used to detect germ-line competent cells.
Generally, an
antibody labelled with a detectable marker can be used to localize the germ-
line competent
cells in an embryo based upon the presence of the detectable marker.
Antibodies may also be
coupled to electron dense substances, such as ferritin or colloidal gold,
which are readily
visualised by electron microscopy.
The reagents suitable for applying the methods of the invention may be
packaged into convenient kits providing the necessary materials, packaged into
suitable
containers. Such kits may include all the reagents required to select a germ-
line competent
cell in a sample by means of the methods described herein, and optionally
suitable supports
useful in performing the methods of the invention.
II. Modification of Genm-line Competent Cells
Germ-line competent cells isolated from stage X (E-G&K) embryos in
accordance with the methods of the invention may be genetically modified by
random or
site-directed integration of DNA into the cells. Therefore, the present
invention relates to
the genetic modification of germ-line competent cells isolated by the methods
of the
invention comprising introducing a recombinant expression vector containing an
exogenous
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gene and the necessary elements for the transcription and translation of the
gene. Examples
of exogenous genes which can be introduced into the germ-line competent cells
include
vasoactive intestinal peptide, growth hormone, insulin-like growth factor i,
the IGF-I
receptor, the GH receptor, prolactin, the gonadotrophins, and gonadotrophin-
releasing
hormone (Etches et al, 1993 and references therein). Selection of the
necessary elements for
the transcription and translation of the genes may be readily accomplished by
one of
ordinary skill in the art. The necessary regulatory sequences may be supplied
by the native
genes and/or their flanking regions.
Transfer of DNA into the germ-line cells may be accomplished using
retroviral vectors (Shuman, Experientia, 47:897-904, 1991; Petropoulos et al.,
Journal of
Virology, 66:3391-3397, 1992; Salter et al., Manipulation of the Avian Genome,
pp. 135-150,
1993). Large DNA sequences (greater than 2000 base pairs), including those
designed to
promote specific integration within the genome via homologous recombination,
can be
introduced by liposome-mediated gene transfer.
Genetically modified germ-line competent cells can be cultured in vitro to
provide a population of genetically modified germ-Line competent cells.
Culture systems
which have been designed to support growth of unknown types of cells derived
from stage X
embryos (see Pain et al., 1996) can be used to support the growth of
transfected (and
untransfected} germ-line competent cells. In vitro culture techniques can also
be employed to
facilitate selection of transfected germ-line competent cells. For example,
the techniques
utilized to select transfected cells from non-transfected embryonic stem cells
in mice
(Mansour et al., Nature, 366:248-252, 1988) may be used in the present
invention.
Single germ-line competent cells may also be cloned using conventional
techniques to provide a homogenous population of genetically modified donor
cells.
III. Production of Germ-line Chimeras
The germ-line competent cells selected using the methods of the invention
may be used to produce chimeric chicken embryos. Chimeric chicken embryos are
produced
by isolating from a donor stage X (E-G&K) chicken embryo using the methods
described
herein, germ-line competent cells that have an epitope expressed by germ-line
competent
cells associated with their cell surface. In a preferred embodiment the
epitope is EMA-1 or
SSEA-1. The isolated germ-line competent cells may be transfected with a
recombinant
expression vector containing an exogenous gene and the necessary elements for
the
transcription and translation of the gene as described herein.
The germ-line competent cells are introduced into a recipient stage X (E-G&K)
chicken embryo from which a portion of the central disk of the embryo has been
removed.
The cells are introduced using conventional methods such as injection into the
subgerminal
cavity of the recipient embryo. A portion of the central disk of the recipient
embryo is
removed using physical techniques, or using antibodies to an epitope
conjugated to a toxin or
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to complement to kill recipient germ-line competent cells. Prior to removal of
the central
disk portion, recipient embryos may optionally be exposed to irradiation (e.g.
490-680 rads
of y irradiation from a 6~Co source). After introduction of the donor germ-
Line competent
cells into the recipient embryo, the recipient embryo is incubated to produce
a chimeric
embryo.
The recipient embryos containing the donor germ-line competent cells may be
transferred preferably on the fourth day of development, into surrogate
culture systems, to
provide the appropriate environment to support development of a chicken embryo
from
syngamy to hatching. Suitable surrogate culture systems are described in
Etches et al,
(1996b).
The methods described herein improve the rate of transmission of the donor
cell line to the recipient and provide larger numbers of chimeras. This
facilitates
commercial breeding programs to replace pure lines by genetically superior
lines.
It will be appreciated that the methods of the present invention may be
applied to other avian species including turkeys, ostriches, quail, pheasants,
ducks, and
geese.
IV. Identification of Germ-line Specific Molecules
The invention provides the isolation and identification of molecules
(including genes and proteins) that are associated with germ-line cells. Such
germ-line
specific molecules can be used as markers to identify and separate germ-line
cells from a
population of cells using the methods of the present invention.
The germ-line specific molecules can be isolated using a variety of techniques
known in the art. Methods to detect germ-line specific molecules include
digestion of the
germ-line cells with restriction endonucleases followed by analysis of the
resulting
fragments, differential hybridization of oligonucleotides, direct PCR
sequencing,
differential hybridization of oligonucleotides and denaturing gradient gel
electrophoresis.
In one example, the presence of a germ-line specific molecule can be detected
by the differential hybridization of oligonucleotides. In particular, germ-
line cells can be
isolated using the methods of the invention and the messenger RNA may be
obtained from
the cells. A cDNA library may be constructed from mRNA prepared from a pool of
control
cells. cDNA libraries may be synthetized using Oligo-dT primers and reverse
transcriptase
according to standard protocols. For example, cDNA may be cloned into plasmid
vectors
such as pSPORT R (Gibco BRL). Commercially available cDNA libraries may also
be used
such as phage display random libraries. The germ-line cell mRNA can be used to
probe a
library for sequences that specifically hybridize to the mRNA from the germ-
line cells but
not to mRNA isolated from control cells.
Hybridization conditions which may be used in the methods of the invention
are known in the art and are described for example in Sambrook J, Fritch EF,
Maruatis T. In:
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WO 98/38283 PCT/CA98/00145
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Molecular Cloning, A Laboratory Manual, 1989. (Nolan C, Ed.), Cold Spring
Harbor
Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by
reference. The
hybridization product may be assayed using techniques known in the art.
In another example, subtractive hybridization can also be used. Subtractive
hybridization can identify and enrich genes that are differentially expressed
in germ-line
competent cells. Subtractive hybridization can be carried out by hybridizing
between two
DNA (or RNA) populations that are closely related such as the germ-line
competent cells
of the invention and other cells present in the stage X embryos. By
subtractive
hybridization, the hybridized sequences common to both cell types can be
removed.
Subsequently, the unhybridized sequences can be preserved as a subtracted cDNA
library.
Subtractive hybridization techniques known in the art can be used for example
Christian E.
Gruber and Wu-Bo Li, "An Improved Subtractive Hybridization Method using
Phagemid
Vectors", Molecular Biology Current Innovations and Future Trends Part 1, A.M.
Griffen and
H.G. Griffen (eds), 1995, which is incorporated herein by reference.
In another example, germ-line specific molecules could be detected using
denaturing gradient gels. Restriction endonuclease fragments or PCR fragments
associated
with a selected nucleotide segment of germ-line specific cells can be resolved
on a
polyacrylamide gel containing gradients of denaturants, such as increasing
temperature,
increasing formamide, urea, and the like. The germ-line specific cell fragment
and control
fragments will denature at different positions in the gel leading to altered
migration
distances. The resolved fragments can the be detected by DNA hybridization for
restriction
fragments or direct DNA straining for PCR fragments.
Accordingly, the present invention provides a method of detecting germ-line
specific molecules using any of the above methods.
Regulatory sequences such as promoters that control expression of the germ-
line specific molecules may also be isolated.
Identification of germ-line specific molecules of the invention also permits
the identification and isolation, or synthesis of nucleotide sequences which
may be used as
primers to amplify a nucleic acid molecule of the invention, for example in
the polymerase
chain reaction (PCR).
Accordingly, the present invention includes a method of determining the
presence of a germ-line specific nucleic acid molecule of the invention is
provided
comprising treating the sample with primers which are capable of amplifying
the nucleic
acid molecule or a predetermined oligonucleotide fragment thereof in a
polymerase chain
reaction to form amplified sequences, under conditions which permit the
formation of
amplified sequences and, assaying for amplified sequences.
The polymerase chain reaction refers to a process for amplifying a target
nucleic acid sequence as generally described in Irmis et al, Academic Press,
1990 in Mullis el
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al., U.S. Pat. No. 4,863,195 and Mullis, U.S. Patent No. 4,683,202 which are
incorporated
herein by reference. Conditions for amplifying a nucleic acid template are
described in
M.A. Innis and D.H. Gelfand, PCR Protocols, A Guide to Methods and
Applications M.A.
Innis, D.H. Gelfand, J.J. Sninsky and T.J. White eds, pp3-12, Academic Press
1989, which is
also incorporated herein by reference.
The amplified products can be isolated and distinguished based on their
respective sizes using techniques known in the art. For example, after
amplification, the
DNA sample can be separated on an agarose gel and visualized, after staining
with
ethidium bromide, under ultra violet (UW) light. DNA may be amplified to a
desired
level and a further extension reaction may be performed to incorporate
nucleotide
derivatives having detectable markers such as radioactive labelled or biotin
labelled
nucleoside triphosphates. The primers may also be labelled with detectable
markers as
discussed above. The detectable markers may be analyzed by restriction and
electrophoretic separation or other techniques known in the art.
The conditions which may be employed in the methods of the invention using
PCR are those which permit hybridization and amplification reactions to
proceed in the
presence of DNA in a sample and appropriate complementary hybridization
primers.
Conditions suitable for the polymerase chain reaction are generally known in
the art. For
example, see M.A. Innis and D.H. Gelfand, PCR Protocols, A guide to Methods
and
Applications, M.A. Innis, D.H. Gelfand, J.J. Sninsky and T.J. White eds, pp3-
12, Academic
Press 1989, which is incorporated herein by reference. Preferably, the PCR
utilizes
polymerase obtained from the thermophilic bacterium Thermus aquatics (Taq
polymerase,
GeneAmp Kit, Perkin Elmer Cetus) or other thermostable polymerase may be used
to
amplify DNA template strands.
It will be appreciated that other techniques such as the Ligase Chain
Reaction (LCR) and NASBA may be used to amplify a nucleic acid molecule of the
invention
(Barney in "PCR Methods and Applications", August 1991, Vol.l(1), page 5, and
European
Published Application No. 0320308, published June 14, 1989, and U.S. Serial
No. 5,130,238
to Malek).
Hybridization and amplification techniques described herein may be used to
assay qualitative and quantitative aspects of the expression of the nucleic
acid molecules
encoding a germ-line specific molecule of the invention. For example, RNA may
be isolated
from a cell type or tissue known to express a nucleic acid of the invention
and tested utilizing
the hybridization (e.g. standard Northern analyses) or PCR techniques referred
to herein.
The techniques may be used to detect differences in transcript size which may
be due to
normal or abnormal alternative splicing.
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The isolated molecules may be used to prepare antibodies which can be used
in the methods of the present invention to select germ-line competent cells.
Conventional
methods can be used to prepare the antibodies as described in detail above.
The following non-limiting example is illustrative of the present invention:
Example
The following materials and methods were used in the studies described in
the Example:
~tiroation of germ-1'nP rnmpetent cells from ta;~P X cmhrypS
Donor embryos were obtained from Barred Plymouth Rocks that are
homozygous recessive (ii) at the I locus. Recipient embryos were obtained from
White
Leghorns that are homozygous dominant (II) at the I locus. The down colour of
the Barred
Plymouth Rocks and White Leghorns are black and yellow, respectively. These
phenotypes facilitate determination of chimerism by feather colour when chicks
hatch:
chicks with black down were designated as somatic chimeras whereas yellow
chicks were
designated as putative chimeras. The contribution of donor- and recipient-
derived cells to
the germ-line was estimated by mating chimeras to Barred Plymouth Rocks and
the extent
of germ-line chimerism was expressed as the ratio of the number of Barred Rock
to White
Leghom offspring.
Donor cells were obtained from Stage X blastoderms (Eyal-Giladi and
Kochav, 1976) isolated from freshly laid, unincubated eggs as described by
Carsience et al.
(1993). Briefly, the albumen was separated from the yolk, the embryo was
isolated, and
the blastodermal cells were dispersed by digesting the extracelluar matrix
with trypsin.
The cells were then washed and resuspended in DMEM (Dulbecco's Modified
Eagle's
Medium) containing 10% fetal bovine serum (FBS). Between 100 and 500 cells in
2-5 ~1 of
medium were injected into the subgerminal cavity of recipient embryos.
Recipient embryos were physically compromised by removing a portion of the
central disk or removing a portion of the lateral edge of the embryo, and
control embryos
were left intact. Approximately half of the embryos that were physically
compromised
and all of the control embryos were exposed to 490-680 rads of g irradiation
from a 6~C o
source within one hour after oviposition and within 2 h before they were
physically
compromised.
Donor cells were injected into all of the recipient embryos that were
irradiated but not physically compromised and approximately one half of the
recipient
embryos that were physically compromised with or without exposure to
irradiation. On
the fourth day after injection, the embryos were transferred to surrogate
shells and
incubated to term as described by Etches et al. (1996b).
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SNL 76/7 murine fibroblast feeder cells (a gift from A. Bradley, Baylor
College of Medicine, Houston, TX, USA) were seeded on 0.1% sterile bovine skin
gelatin
(Sigma, St. Louis, MO, USA) coated culture dishes {None, Denmark) at a density
of 1.25 x
105 cells/cm2. Feeder cells were maintained in Dulbecco's modified Eagle's
medium
(DMEM)(Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine
serum
(FBS) (Cansera, Rexdale, ON, Canada), 2mM L-glutamine (Gibco), 100 IU/ml
penicillin
and 100~.g/ml streptomycin (Gibco) at pH 7.2 for 24 hours at 37°C in 5%
C02.
Dissociation of Cells
Stage X blastoderms were isolated from Barred Plymouth Rock embryos into a
Petri dish in Dulbecco's phosphate buffered saline containing 1 g/L D-glucose
(PBS-G) using
sterile filter paper rings (Petitte et al., 1990). The entire blastoderm was
carefully cleaned
of excess yolk and gently transferred with a Pasteur pipette into a 15 ml
centrifuge tube
containing 2 to 3 ml of PBS-G plus 2% (v/v) chicken serum (ChS). Blastoderms
were pooled
at two per ml. The PBS-G + 2% ChS was removed and replaced with an equal
volume of
calcium and magnesium free phosphate buffered saline (CMF-PBS) + 2% ChS.
Blastoderms
were then incubated for 10 minutes on ice. The CMF-PBS was replaced with 0.05%
trypsin
(w/v) in 0.02% EDTA (w/v) (Gibco) and blastoderms were again incubated for 10
minutes on
ice. Trypsin was replaced with 1.0 ml of Opt-modified Eagle's medium I
(Optimem) (Gibco)
supplemented with 2% ChS, 4% (v/v) FBS, I00 IU/ml penicillin and 100 ~tg/ml
streptomycin at pH 7.2. The blastoderms were pooled and slowly dispersed by
aspiration
with a 10 ml pipette. A sample of cell suspension was used to determine cell
concentration
with a haemocytometer. Dissociated cells were centrifuged for 4 minutes at
1000 rpm. The
supernatant was removed and cells were resuspended in an appropriate volume of
Optimem.
Dispersed blastodermal cells were plated on SNL feeder cells at a
concentration of 1x105
cells/cm2 incubated for 1, 3, 7, or 18 hours at 37°C in 5% C02.
Determination of Immunohistochemistrv
Cultured blastodermal cells and whole mounts were fixed in 4%
paraformaldehyde (Sigma) for 10 minutes at 4°C. The preparations were
then immersed for
4 hours in PBS supplemented with 1 mg/ml bovine serum albumin (PBS-BSA)
(Boehringer
Mannheim, Laval, PQ, Canada), plus 5% (v/v) goat serum (Sigma) at 4°C
to saturate non-
specific binding sites. The blocking solution was replaced with supernatant
from SSEA-1
diluted 1:3 in PBS-BSA, EMA-1 diluted 1:10 in PBS-BSA or HNK-1 /NC-1 (a gift
from C.
Stem, Columbia University, NY, USA) diluted 1:2 at 4°C with gentle
rocking for at least 12
hours. SSEA-1 and EMA-1 antibody supernatants were obtained from the
Developmental
Studies Hybridoma Bank maintained by the Department of Pharmacology and
Molecular
Sciences, John Hopkins University School of Medicine, Baltimore, MD, and the
Department
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of Biological Sciences, University of Iowa, Iowa City, IA under contract NOi-
HD-6-2915
from the NICHD. The preparations were washed two times in PBS-BSA and
incubated for
2 hours at 4°C in the dark with affinity-purified goat anti-mouse IgM-
FITC conjugate
diluted 1:50 (Jackson ImmunoResearch, West Grove, PA, USA). After washing
twice with
PBS-BSA, the preparations were mounted with glycerol diluted 1:1 with PBS and
viewed
on a Nikon Diaphot-TMD inverted microscope equipped with epifluorescent
optics, excited
by a mercury lamp, using filters to allow emission at 470-490 nm.
Selection of cells a ine maeneti~c h~ar~c
Dissociated cell preparations were incubated with monoclonal antibodies
SSEA-1, EMA-1 or NC-1 (Immunotech, Westbrook, ME, USA) for 45 minutes at
4°C. Before
incubation, blastodermal cells were isolated as outlined above, except that
trypsin was
replaced with PBS-ChS. Subsequently, the cell preparation was washed in PBS-
BSA and
incubated with 100 ~tl of PBS-BSA containing rat anti-mouse IgM conjugated
microbeads
(Miltenyi Biotec, Sunnyvale, CA, USA) for 30 minutes at 4°C. The
mixture was then
washed again and resuspended in 500 ~tl of PBS-BSA. A steelwool separation
column (type
MS+: Miltenyi Biotech) was inserted into a MiniMACS magnetic system (Miltenyi
Biotech)
and rinsed with PBS-BSA. The cell suspension was run through the column.
Unbound
(negative) cells were flushed out with PBS-BSA. Cells labelled with the
superparamagnetic beads are magnetic in a magnetic field and bind to the
steelwool fibers
of the column, while unlabelled cells pass through the column (Miltenyi et
al., 1990).
Finally, the column was removed from the magnetic field and the bound
(positive) cells
eluted by rinsing with PBS-BSA. Immunohistochemistry was performed on the
cells before
and after magnetic cell sorting.
Results:
The reproductive capacity of birds derived from embryos in which the central
and lateral regions were extirpated with and without irradiation are shown in
Tables 1 and
2. It is evident that production of eggs and sperm and that the fertilizing
capacity of the
gametes were unaffected by the method of treating embryos. It can be
concluded, therefore,
that any residual germ-line competent cells remaining after extirpation of the
central
region had the capacity to proliferate and populate the germ-line with a
normal
complement of spermatogonia in males and oogonia in females.
The number of somatic and germ-line chimeras that were produced after
injection of Barred Plymouth Rock donor cells into White Leghorn recipient
embryos that
were compromised by irradiation and extirpation of approximately 500 cells
from the
central or lateral regions of the embryos are shown in Table 3-6. Somatic
chimeras hatch
with some black (donor-derived) pigmentation (see column 2 in Table 3-6).
Putative
chimeras have no black (donor-derived) pigmentation. Previous work from our
laboratory
has shown that putative chimeras usually are not germ-line chimeras. When
embryos were
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irradiated and approximately 500 cells were injected into the central regions
of the embryo
{Table 3), 5 of the 9 somatic chimeras produced donor-derived offspring, (i.e.
they were
germ-line chimeras). By comparison only 1 of 8 somatic chimeras produced by
injecting 500
cells into the lateral region of irradiated recipient embryos (Table 4)
produced donor-
s derived offspring. These data are consistent with and lend support to the
proposal that
germ-line competent cells are located in the center of the stage X embryo.
Injection of Barred Plymouth Rock donor cells into the central region of
recipient embryos that had been compromised by extirpating approximately 500
cells from
the center of the embryo yielded 3 germ-line chimeras from 9 somatic chimeras
(Table 5).
By comparison, injection of Barred Plymouth Rock cells into the lateral region
of recipients
from which cells had been removed from that lateral region yielded only 1 germ-
line
chimera among 10 somatic chimeras (Table 6). These data are also consistent
with the
hypothesis that germ-line competent cells reside in the central region of the
embryo. In
addition, the rate of germ-line chimerism produced following compromising
recipient
embryos by irradiation and by physically removing cells from the central
region are
similar. Irradiation of recipient embryos is believed to improve the rate of
germ-line
chimerism by slowing the rate of development of the recipient embryo. Since
the donor cells
have not been irradiated, they proliferate while cell division is inhibited in
the recipient
embryo and consequently, the ratio of donor recipient cells in the chimera is
increased
(Carsience et al., 1993). Removal of cells from the center of the embryo also
increases the
ratio of germ-line chimerism (from 0.6% to 11.3%, see Table 7). In this case,
the increase is
attributed to the removal of germ-line cells from the center of the recipient
and the
subsequent reintroduction of donor germ-line competent cells to the same area.
This
interpretation is supported by the very low rate of germ-line transmission
that follows
removal of cells from the lateral regions of the embryo (Table 7).
Identification of germ-line competent cells by immunofluorescence detection of
cells
exnr~ essing~enitopes
Identification of Cells Expressing Epitopes by ImmunofIuorescence
Labelling of cells expressing surface antigens recognized by EMA-1 and SSEA-
1 could be detected in stage X Barred Plymouth Rock whole embryos (Figures 2
and 3). A
mixture of non-, weakly and highly fluorescent staining cells were observed
after 1, 3, 7 and
18 hours in culture. Approximately 8%, 15% and 48% of cells cultured for 18
hours showed
intense staining for EMA-1 {Figures 4 and 5), SSEA-1 (Figures 6 and 7), and
HNK-1 /NC-1
(Figures 8 and 9), respectively. Cultures of SNL murine fibroblast cells were
used as
negative controls. Reagent controls consisted of replacing the primary
antibody with PBS-
BSA. None of the cells in the negative and reagent controls were unstained.
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PCT/CA98/00145 - -
Selection of cells expressing epitopes using magnetic beads
A three fold enrichment of Stage-X blastodermal cells expressing the SSEA-1
epitope was achieved using SSEA-1 coated microbeads. Immunofluorescence
verified that
the percentage of SSEA-1 staining cells increased from 14% to 45% after
separation.
Immunomagnetic isolation of EMA-1 positive cells resulted in a three fold
enrichment from
8% of the cells before purification to 28% after purification. Selection for
cells expressing
NC-1 epitopes resulted in a two fold increase. The percentage of NC-1 positive
cells
increased from 48% to 89% after separation. In all cases, viability of cells
after separation
was found to be greater that 94% as determined by trypan blue exclusion.
The rates of somatic and germ-line chimer~sm and the rate of germ-line
transmission of male and female chimeras made using the positive and negative
fractions
from MACS columns containing EMA-1, SSEA-1 and IsC-1 are presented in Table 8
and 9
respectmely. The overall rates of both somatic and germ-lme chimersim were low
in these
experiments, indicating that manipulation of the cells m the magnetic columns
reduced
their overall ability to contribute to recipient embryos. Preliminary evidence
from these
experiments indicates that the contribution to somatic and germ-line tissues
of the positive
and negative fractions selected using SSEA-1 and NC-1 were approximately
equal. Cells
that were selected by the EMA-1 antibody, however, yielded significant
contributions to
the germ-line in 2 of 3 chimeras (Table 8). These data indicate that germ-line
competent
cells can be selected using the EMA-1 epitope. Since the enrichment of EMA-1
positive cells
using MACS is from 8% to 28%, (see above), approximately 72% of the cells that
were
injected were not EMA-1 positive.
In summary, these data support the conclusion that a population of germ-line
competent cells exists in the central region of the stage X (E-G&K) embryo and
that the
germ-line competent cells can be isolated using a technique such as
magnetically activated
cell sorting using antibodies such as EMA-1 that recognize epitopes that are
specifically
expressed by germ-line competent cells.
While the present invention has been described with reference to what are
presently considered to be the preferred examples, it is to be understood that
the invention
is not limited to the disclosed examples. To the contrary, the invention is
intended to cover
various modifications and equivalent arrangements included within the spirit
and scope of
the appended claims.
All publications, patents and patent applications are herein incorporated by
reference in their entirety to the same extent as if each individual
publication, patent or
patent application was specifically and individually indicated to be
incorporated by
reference in its entirety.
Below full citations are set out for the references referred to in the
specification and detailed legends for the figures are provided.
i
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Table 1. Mean age and the range in age at the onset of egg production and
fertility of hens derived from
Stage X embryos that were irradiated or from which approximately 500 cells
were extirpated.
Irradiation Lateral Central NumberRange in Mean % Fertility
Mean age age ( 100
removal removal of hens at onsetat the x number of
of onset chicks
of
of cells of cells lay lay hatched/number
of
eggs set) and
range in
parentheses
+ + - 2 151 142-160 89(88-90)
+ - + 3 161 151-167 84(75-98)
- + - 6 I55 139-177 83(74-90)
- - + 4 139 117-158 80(77-82)
SUBSTITUTE SHEET (RULE 26)
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Table 2. Mean concentration of spermatozoa and fertility of males derived from
Stage X embryos
that were irradiated or from which approximately 500 cells were extirpated.
Irradiation Lateral CentralMean concentrationMean % Fertility
Number ( 109 ( 100 x
removal removal of males cells/ml) of number of chicks
spermatozoa
of cells of cells and range in hatched/number
parentheses of eggs
set) and range
in
parentheses
- 3 5.5 (4.9 - 5.9) 75 (66 - 91 )
2 6.6 (5.0 - 8.2) 67 (65 - 69)
- 3 5.2 (3.3 - 8.2) 71 (57 - 81 )
+ 4 3.6 (0.9 - 5.6) 66 (57 - 71 )
SUBSTITUTE SHEET (RULE 26)
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Table 3. The number of recipient- and donor-derived offspring from test mating
somatic and putative
chimeras to Barred Plymouth rocks. Group 1 - Recipient embryos compromised by
irradiation and
injected with -500 donor cells centrally.
DescriptionPercentage Number of Percentage
of of donor- of
Chimera black ~ ' Number donor-derived
pigmentationof offspring
~i derived
offspring
~ recipient-derived
offspring
I Somatic
female
2583-2584 90 0 ~ 135 0
2577-2578 1 0 I 100 0
2589-2590 75 10 I 116 0
2324-2325 95 ~ 43 38 i 53.1
2327-2328 99 ~ 3 14 17.6
i
Somatic
males
2575-2576 95 106 220 32.5
2579-2580 80 ~ 0 302 0
2581-2582 80 I 69 205 25.2
2329-2330 ~ 20 31 102 23.3
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Table 4. The number of recipient-and donor-derived offspring from test mating
somatic and putative
chimeras to Barred Plymouth rocks. Group 2 - Recipient embryos compromised by
irradiation and
injected with -500 cells laterally.
Description Percentage Number of Number of Percentage
of of donor- recipient-derivedof
Chimera black derived offspringoffspring donor-derived
pigmentation offspring
Somatic
female
2401-2402 99 0 0
2430-2431 2 29 154 ~ 15.8
I
2466-2467 30 I 0 139
I
2468-2469 95 0 127
2370-2471 95 0
Somatic males
2425-2526 75 0 312 0
2436-2437 20 0 297 0
!
2472-2473 40 ~ 0 I 240 p
~ I
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Table 5. The number of recipient-and donor-derived offspring from test mating
somatic and putative
chimeras to Barred Plymouth rocks. Group 3 - Recipient embryos compromised by
physically
removing -500 cells from the central area and injected with -50(? donor cells
centrally.
DescriptionPercentage Number of Number of Percentage
of of donor- recipient-derivedof
Chimera black derived offspring donor-derived
pigmentationoffspring offspring
Somatic
female
2499-2500 80 0 91 0
2531-2532 50 I O 57 I O
2533-2534 40 ~ 1 i 72 I 1.4
2591-2592 20 0 1 I 0
i
2593-2594 20 0 111 ~ 0
II
2347-2348 2 0 108 0
Somatic I
males
2505-2506 50 63 191 ( 24.8
2524-2525 45 24 270 8.1
2345-2346 1 0 ~ 159 I 0
i
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Table 6. The number of recipient - donor-derived offspring from test mating
somatic and putative
chimeras to Barred Plymouth rocks. Group 4 - Recipient embryos compromised by
physically
removing -500 cells form the lateral area and injected with -500 donor cells
laterally.
Description Percentage Number of Number of Percentage
of of donor- recipient-derivedof
Chimera black derived offspringoffspring donor-derived
pigmentation offspring
Somatic
female
2409-2410 20 I ~ 177 0.6
2415-2416 2 0 ~ 160 0
2305-2427 20 I 0 138 0
2444-2445 40 0 133 0
2448-2450 10 0 84 0
2460-2308 20 0 129 I 0
I
2474-2475 75 0 124 0
Somatic males
2417-2418 1 I 0 378 I 0
2419-2429 35 0 ~ 377 ~ 0
'
i 2456-2457 50 0 316 0
i
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Table 7. Rates of somatic chimerism expressed as the % of black pigmentation
in plumage and the
rate of germ-line transmission expressed as the number of donor-derived
offspring/total number of
offspring from chimeras derived from Stage X embryos that were irradiated or
from which
approximately 500 cells were extirpated.
Irradiation Lateral Central Number of Somatic Number Rate of germ-line
removal removal somatic chimerism (% of germ- transmission (number
of cells of cells chimeras black line of donor-derived
pigmentation) chimeras offspring/total
number of offspring x
100)
+ + - 8 57 1 15.8
+ - + 9 71 5 30.3
- + - 10 27 1 0.6
- - + 9 34 3 11.3
SUBSTITUTE SHEET (RULE 26)
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Table 8. Proportion of male somatic and germ-line chimeras made by injecting
cells selected using EMA-1,
SSEA-1 and NC-1 epitopes by magnetic activated cells sorting (MACS).
Ab Fraction % somatic chimerism Number of germ-line Rate of germ-line
chimeras/number of chimeras chimerism (%)
EMA-1 + 40 2/3 17
- I 0 0/ 1 _
SSEA-1 + 24 1/4 5.5
- 44 4/ 10 6. 9
NC-1 + 57 5/10 22.0
- 52 3/6
I 3.4
i
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Table 9. Proportion of female somatic and germ-line chimeras made by infecting
cells selected using EMA-1,
SSEA-1 and NC-1 epitopes by magnetic activated cells sorting (MACS).
Ab Fraction lo somatic chimerismNumber of germ-line Rate of germ-line
chimeras/number of chimerism (%)
chimeras
EMA-1 + 52 0/3 -
- 85 0/I -
SSEA-I + 45 2/7 3.2
- 54 3/1 I 4.5
NC-1 + 44 1/11 36
- 42 0/10 -
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FULL CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION
Brazolot, C.L., Petitte, J.N., Etches, R.J. and Verrinder Gibbins, A.M.
(1991). Efficient
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Carsience, R.S., Clark, M.E., Verrinder Gibbins A.M. and Etches, R.J. (1993)
Germ-line
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Kingdom.
Etches, R.J., Clark, M.E., Toner, A., Liu, G, and Verrinder Gibbins, A.M.
(1996a).
Contributions to somatic and germ-line lineages of chicken blastodermal cells
maintained in
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Etches, R.J., M.E. Clark, A.M. Verrinder Gibbins and M.B. Cochran (1996b).
Production of
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formation: a
complementary normal table and a new look at the first stage of the
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DETAILED FIGURE LEGENDS
Figure 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. At this time, the
embryo
contains 40,000-60,000 cells and is referred to as a Stage X embryo.
Figure 2. Phase contrast illumination micrograph of a microscopic field
illustrating the
expression of EMA-1 epitopes on stage X blastodermal cells cultured 18 hours
with SNL
feeder cells (magnification 300x).
Figure 3. Fluorescence micrograph of the same microscopic field as figure 2
illustrating the
expression of EMA-1 epitopes on stage X blastodermal cells cultured 18 hours
with SNL
feeder cells (magnification 300x).
Figure 4. Phase contrast illumination micrograph a microscopic field
illustrating the
expression of SSEA-1 epitopes after 18 hours in culture.
Figure 5. Fluorescence micrograph of the same microscopic field as figure 4
illustrating the
expression of SSEA-1 epitopes after 18 hours in culture. Note the variation in
fluorescence
intensity.
Figure 6. Phase contrast illumination micrograph of a microscopic field
illustrating the
SSEA-1 expression on stage X whole mount (magnification 300X).
Figure 7. Fluorescence micrographs of the same microscopic field as figure 1
illustrating the
SSEA-1 expression on stage X whole mount (magnification 300X).
Figure 8. Phase contrast illumination micrograph of a microscopic field
illustrating HNK-
1 /NC-1 expression on stage X blastodermal cells after 18 hours in culture.
Figure 9. Fluorescent micrograph of the same microscopic field as figure 8
illustrating
HNK-1/NC-1 expression on stage X blastodermal cells after 18 hours in culture.
