U.S. patent application number 11/045872 was filed with the patent office on 2006-01-19 for nuclear transfer embryo formation method.
This patent application is currently assigned to Trustees of Tufts College. Invention is credited to Eric W. Overstrom, Daniela Fischer Russell.
Application Number | 20060015950 11/045872 |
Document ID | / |
Family ID | 30770578 |
Filed Date | 2006-01-19 |
United States Patent
Application |
20060015950 |
Kind Code |
A1 |
Overstrom; Eric W. ; et
al. |
January 19, 2006 |
Nuclear transfer embryo formation method
Abstract
A nuclear transfer embryo is formed by destabilizing
microtubules of an oocyte, whereby essentially all endogenous
chromatin collects at a second polar body during meiosis of an
oocyte. The oocyte is fused with the nucleus of a donor somatic
cell of the same species of said oocyte prior to cessation of
extrusion of the second polar body from the oocyte, thereby forming
the nuclear transfer embryo. In one embodiment, the nuclear
transfer embryo is employed to impregnate an animal, such as a
mammal. In another embodiment, the donor nucleus is transgenic.
Inventors: |
Overstrom; Eric W.;
(Grafton, MA) ; Russell; Daniela Fischer; (Sio
Paulo, CA) |
Correspondence
Address: |
HAMILTON, BROOK, SMITH & REYNOLDS, P.C.
530 VIRGINIA ROAD
P.O. BOX 9133
CONCORD
MA
01742-9133
US
|
Assignee: |
Trustees of Tufts College
Medford
MA
|
Family ID: |
30770578 |
Appl. No.: |
11/045872 |
Filed: |
January 28, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US03/23464 |
Jul 29, 2003 |
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11045872 |
Jan 28, 2005 |
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10208653 |
Jul 29, 2002 |
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PCT/US03/23464 |
Jul 29, 2003 |
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Current U.S.
Class: |
800/6 ;
800/14 |
Current CPC
Class: |
C12N 15/873 20130101;
C12N 15/8771 20130101; A01K 2227/101 20130101 |
Class at
Publication: |
800/006 ;
800/014 |
International
Class: |
A01K 67/027 20060101
A01K067/027 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was supported, in whole or in part, by Grant
No. 2001-35205-09966 from the United States Department of
Agriculture. The Government has certain rights in the invention.
Claims
1-20. (canceled)
21. A method of cloning a mammal, comprising: a) destabilizing
microtubules of an oocyte, whereby essentially all endogenous
genetic material collects at a second polar body during meiosis of
said oocyte; and b) combining the oocyte with at least the nucleus
of a donor cell of the same species of said oocyte prior to
cessation of extrusion of the second polar body from said oocyte,
thereby forming a nuclear transfer embryo. c) impregnating a mammal
of the same species as the nuclear transfer embryo with the nuclear
transfer embryo under conditions suitable for gestation of the
cloned mammal; and d) gestating the embryo, thereby causing the
embryo to develop into the cloned mammal.
22. (canceled)
23. A method of producing a transgenic mammal, comprising the steps
of: a) destabilizing microtubules of an oocyte, whereby essentially
all endogenous genetic material collects at a second polar body
during meiosis of said oocyte; and b) combining the oocyte with at
least the nucleus of a donor cell of the same species of said
oocyte prior to cessation of extrusion of the second polar body for
said oocyte, thereby forming a nuclear transfer embryo. c)
impregnating a mammal of the same species as the nuclear transfer
embryo with the nuclear transfer embryo under conditions suitable
for gestation of the transgenic mammal; and d) gestating the
embryo, thereby causing the embryo to develop into the transgenic
mammal.
24. A method of cloning a mammalian fetus, comprising the steps of:
a) destabilizing microtubules of an oocyte, whereby essentially all
endogenous genetic material collects at a second polar body during
meiosis of said oocyte; and b) combining the oocyte with at least
the nucleus of a donor somatic cell of the same species of said
oocyte prior to cessation of extrusion of the second polar body
from said oocyte, thereby forming a nuclear transfer embryo. c)
impregnating a mammal of the same species as the nuclear transfer
embryo with the nuclear transfer embryo under conditions suitable
for gestation of the cloned mammalian fetus; and d) gestating the
embryo, thereby causing the embryo to develop into the cloned
mammalian fetus.
25. (canceled)
26. A method of cloning a non-human mammal, comprising the steps
of: a) destabilizing microtubules of an oocyte, whereby essentially
all endogenous genetic material collects at a second polar body
during meiosis of said oocyte; and b) combining the oocyte with at
least the nucleus of a donor somatic cell of the same species of
said oocyte prior to cessation of extrusion of the second polar
body from said oocyte, thereby forming a nuclear transfer embryo;
c) impregnating a non-human mammal of the same species as the
nuclear transfer embryo with the nuclear transfer embryo under
conditions suitable for gestation of the cloned non-human mammal;
and d) gestating the embryo, thereby causing the embryo to develop
into the cloned non-human mammal.
27. A method of producing a transgenic non-human mammal, comprising
the steps of: a) destabilizing microtubules of an oocyte, whereby
essentially all endogenous genetic material collects at a second
polar body during meiosis of said oocyte; and b) combining the
oocyte with at least the nucleus of a donor somatic cell of the
same species of said oocyte prior to cessation of extrusion of the
second polar body from said oocyte, thereby forming a nuclear
transfer embryo. c) impregnating a non-human mammal of the same
species as the nuclear transfer embryo with the nuclear transfer
embryo under conditions suitable for gestation of the transgenic
mammal; and d) gestating the embryo, thereby causing the embryo to
develop into the transgenic non-human mammal.
28. A method of cloning a non-human mammalian fetus, comprising the
steps of: a) destabilizing microtubules of an oocyte, whereby
essentially all endogenous genetic material collects at a second
polar body during meiosis of said oocyte; and b) combining the
oocyte with at least the nucleus of a donor somatic cell of the
same species of said oocyte prior to cessation of extrusion of the
second polar body from said oocyte, thereby forming a nuclear
transfer embryo. c) impregnating a non-human mammal of the same
species as the nuclear transfer embryo with the nuclear transfer
embryo under conditions suitable for gestation of the cloned
mammalian fetus; and d) gestating the embryo, thereby causing the
embryo to develop into the cloned non-human mammalian fetus.
29. A method of producing a protein of interest in an animal,
comprising the steps of: a) destabilizing microtubules of an
oocyte, whereby essentially all endogenous genetic material
collects at a second polar body during meiosis of said oocyte; and
b) combining the oocyte with at least the nucleus of a donor cell
of the same species of said oocyte prior to cessation of extrusion
of the second polar body from said oocyte, thereby forming a
nuclear transfer embryo. c) impregnating a mammal of the same
species as the nuclear transfer embryo with the nuclear transfer
embryo under conditions suitable for gestation of the cloned
mammal; d) gestating the embryo, thereby causing the embryo to
develop into the cloned mammal; and e) purifying the protein of
interest from the cloned animal.
30. The method of claim 29, wherein purification of the protein of
interest is expressed in tissue, cells or a bodily secretion of the
cloned animal.
31. The method of claim 29, wherein the tissue, cells or bodily
secretion is selected from the group consisting of: milk, blood,
urine, hair, mammary gland, muscle, viscera.
32. The method of claim 31, wherein said viscera is selected from
the group consisting of: brain, heart, lung, kidney, pancreas, gall
bladder, liver, stomach, eye, colon, small intestine, bladder,
uterus and testes.
33. A method of producing a heterologous protein in a transgenic
animal comprising the steps of: a) destabilizing microtubules of an
oocyte, whereby essentially all endogenous genetic material
collects at a second polar body during meiosis of said oocyte; and
b) combining the oocyte with at least the nucleus of a donor cell
of the same species of said oocyte prior to cessation of extrusion
of the second polar body from said oocyte, thereby forming a
nuclear transfer embryo. c) impregnating a mammal of the same
species as the nuclear transfer embryo with the nuclear transfer
embryo under conditions suitable for gestation of a transgenic
cloned mammal; d) gestating the embryo, thereby causing the embryo
to develop into the transgenic cloned mammal; and e) purifying the
protein of interest from the transgenic cloned animal.
34. The method of claim 33, wherein the genetically engineered
nucleus includes an operatively linked promoter.
35. The method of claim 34, wherein said promoter is selected from
the group consisting of: a host endogenous promoter, an exogenous
promoter and a tissue-specific promoter.
36. The method of claim 35, wherein said tissue-specific promoter
is selected from the group consisting of: mammary-specific
promoter, blood-specific promoter, muscle-specific promoter,
neural-specific promoter, skin-specific promoter, hair-specific
promoter and urinary-specific promoter.
37-40. (canceled)
Description
RELATED APPLICATION
[0001] This application is a continuation of International
Application No. PCT/US03/23464, which designated the United States
and was filed Jul. 29, 2003, published in English, which claims
priority to and is a continuation-in-part of U.S. application Ser.
No. 10/208,653, filed Jul. 29, 2002, the entire teachings of which
are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] Nuclear transfer methods have been developed and used
successfully to produce cloned sheep, cattle, mice, goats and pigs.
Two cell components are combined to produce a cloned embryo; the
donor nuclear genome (karyoplast) that is the target for clonal
replication, and the enucleated oocyte (cytoplast) whose
cytoplasmic constituency is sufficiently competent to facilitate
genome reprogramming and support embryonic development to term.
[0004] Mammalian oocyte cytoplasts have been prepared by physically
removing nuclear chromatin by micromanipulation techniques in
preparation to receive the donor genome. Enucleated oocytes
arrested at metaphase of meiosis II (MII) are subsequently
"reconstructed" by the addition of the donor karyoplast typically
using either electrofusion or microinjection techniques. However,
physical enucleation is generally technically demanding, time
consuming, inherently invasive and clearly damaging to cytoplast
spatial organization. Moreover, in certain instances, development
of reconstructed embryos is inherently inefficient.
[0005] One alternative strategy to physical enucleation has been to
treat oocytes with agents that modify the processes of karyokinesis
and cytokinesis and result in chemically enucleated oocytes at high
rates (>85%). However, certain studies have reported that
exposure of metaphase I and MII oocytes to etoposide, a
topoisomerase II inhibitor, and cycloheximide yields enucleated
cytoplasts with limited ability to support cleavage or blastocyst
development, and term development of reconstructed embryos has not
been reported.
[0006] Hence, a need exists for improved methods for developing
nuclear transfer embryos.
SUMMARY OF THE INVENTION
[0007] The present invention relates to methods of forming a
nuclear transfer embryo by destabilizing microtubules of an oocyte
(e.g., mammalian (human or non-human)), whereby essentially all
endogenous chromatin collects at (e.g. segregates into) a second
polar body during meiosis of the oocyte. The methods also include
combining the oocyte with at least the nucleus of a donor cell of
the same species of the oocyte prior to cessation of extrusion of
the second polar body from the oocyte, thereby forming a nuclear
transfer embryo. Certain conditions and/or compounds destabilize
the microtubules of the meiotic spindle of the oocyte. Examples of
chemicals that destabilize the microtubules of the oocyte include
demecolcine, paclitaxel, phalloidin, colchicine, and nocodozole.
Such conditions include alterations (e.g., increasing or
decreasing) in electromagnetic radiation (e.g., x-rays or heat), pH
or osmolality.
[0008] In one embodiment, the present invention further includes
activating the oocyte prior to exposing the oocyte to the chemical
that induces enucleation. Stages of an activated oocyte include
telophase II or anaphase II stage of meiosis. In another
embodiment, the oocyte is in a resting of meiosis (e.g., metaphase
II stage) during injection of the donor nucleus.
[0009] The present invent utilizes donor cells that are in various
stages of mitotic cell cycle, and can include several types of
cells. The present invention encompasses use of an active (e.g.,
G.sub.1, S or G.sub.2/M stage of a mitotic cell cycle) or inactive
(e.g., G.sub.0 stage of a mitotic cell cycle) donor cell. The donor
cell can be an active or inactive fibroblast cell, epithelial cell,
a somatic cell (e.g., adult or embryonic). The donor cell can also
be transgenic.
[0010] The present invention also encompasses methods for cloning a
mammal by forming the nuclear transfer embryo, as described herein,
and impregnating a mammal of the same species as the nuclear
transfer embryo with the nuclear transfer embryo under conditions
suitable for gestation of the cloned mammal; and gestating the
embryo, thereby causing the embryo to develop into the cloned
mammal.
[0011] The present invention further includes methods for producing
a transgenic mammals, by forming a nuclear transfer embryo using a
transgenic donor cell, as described herein, and impregnating a
mammal of the same species as the nuclear transfer embryo with the
nuclear transfer embryo under conditions suitable for gestation of
the transgenic mammal; and gestating the embryo, thereby causing
the embryo to develop into the transgenic mammal.
[0012] Yet another embodiment of the invention includes methods for
cloning a mammalian fetus, by forming a nuclear transfer embryo as
described herein, and impregnating a mammal of the same species as
the nuclear transfer embryo with the nuclear transfer embryo under
conditions suitable for gestation of the cloned mammalian fetus;
and gestating the embryo, thereby causing the embryo to develop
into the cloned mammalian fetus.
[0013] The present invention further pertains to methods of
producing a protein of interest in an animal, by forming a nuclear
transfer embryo as described herein, and impregnating a mammal of
the same species as the nuclear transfer embryo with the nuclear
transfer embryo under conditions suitable for gestation of the
cloned mammal; gestating the embryo, thereby causing the embryo to
develop into the cloned mammal; and purifying the protein of
interest (e.g., tissue, cells or a bodily secretion) from the
cloned animal. Examples of sources from which proteins that can be
purified from cloned animals include milk, blood, urine, hair,
mammary gland, muscle, viscera (e.g., brain, heart, lung, kidney,
pancreas, gall bladder, liver, stomach, eye, colon, small
intestine, bladder, uterus and testes).
[0014] The present invention also relates to methods of producing a
heterologous protein in a transgenic animal by forming a transgenic
nuclear transfer embryo, as described herein, and impregnating a
mammal of the same species as the nuclear transfer embryo with the
nuclear transfer embryo under conditions suitable for gestation of
a transgenic cloned mammal; gestating the embryo, thereby causing
the embryo to develop into the transgenic cloned mammal; and
purifying the protein of interest from the transgenic cloned
animal. In one embodiment, the genetically engineered nucleus
includes an operatively linked promoter (e.g., a host endogenous
promoter, an exogenous promoter and a tissue-specific promoter
(e.g., mammary-specific promoter, blood-specific promoter,
muscle-specific promoter, neural-specific promoter, skin-specific
promoter, hair-specific promoter and urinary-specific
promoter)).
[0015] In another embodiment, the present invention relates to
methods of forming a nuclear transfer embryo by performing the
steps in the following order: combining an oocyte with at least the
nucleus of a donor cell of the same species of the oocyte;
activating the oocyte; and destabilizing microtubules of the
oocyte, whereby essentially all endogenous chromatin collects at a
second polar body during meiosis of the oocyte, thereby forming a
nuclear transfer embryo. The oocyte can be in a metaphase II stage
of meiosis prior to activation, or in a telophase II or anaphase II
stage of meiosis after activation.
[0016] The present invention also includes methods of forming a
nuclear transfer embryo, by activating an oocyte; combining the
activated oocyte with at least the nucleus of a donor cell of the
same species of the oocyte in less than about 45 minutes (e.g., 40,
35, 30, 25, 20, 15, 10 or 5 minutes) after activation of the
oocyte; and destabilizing microtubules of the activated oocyte,
whereby essentially all endogenous chromatin collects at a second
polar body during meiosis of the oocyte, thereby forming a nuclear
transfer embryo.
[0017] The methods of the invention have advantages over methods of
nuclear transfer that employ mechanical enucleation (e.g.,
enucleation using a micropipette to remove the nucleus) of the
oocyte for several reasons. For example, using physical
enucleation, the nucleus along with other important spindle
associated elements are also removed. These spindle associated
elements include, for example, centrosome factors (.alpha.-tubulin,
pericentrin), kinases (e.g., C-mos, MPF, PAPK) and competence
factors (e.g., cdc25C, spindlin). Several of these elements are
important in forming a nuclear transfer embryo that is
developmentally competent (e.g., embryo's ability or likelihood to
develop successfully in vivo following an embryo transfer and
implantation to a recipient mammal). The method of the invention
removes the endogenous nucleus from the oocyte, while allowing
several of these spindle associated elements to remain in the
oocyte, thereby potentially significantly improving yield of
competent embryos.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] FIG. 1 is a schematic diagram showing the process of induced
enucleation and the introduction of the donor nucleus before
extrusion of the second polar body containing essentially all
endogenoses chromatin of the oocyte ceases.
[0019] FIG. 2 is a schematic diagram showing the process of induced
enucleation and the introduction of the donor nucleus before
cessation of extrusion of the second polar body containing
essentially all of the chromatin of a telophase II oocyte.
[0020] FIGS. 3A-3B are a series of histograms showing percent (%)
of complete second PB extrusion at several times post-activation
(45 min, 75 min, 105 min and 135 min) for untreated
ethanol-activated oocytes (EtOH) or demecolcine (Deme)-treated
activated oocytes at different times (0, 5, 10 and 15 min)
post-activation. FIG. 3A shows the time course for complete second
PB extrusion for B6D2F1 strain mouse oocytes, and FIG. 3B shows the
time course for complete second PB extrusion for CF-1 strain mouse
oocytes. Oocytes were analyzed by immunofluorescence at several
time-points after activation to assess the extent and completeness
of second PB extrusion. Different superscripts represent
significant differences (P<0.05) between different treatments
for each time-point and strain.
[0021] FIG. 4 is a series of histograms showing percent (%) of
complete second PB extrusion at several times post-activation (45
min, 75 min, 105 min and 135 min) for various types of B6D2F1 and
CF-1 strain oocytes (type C, type D, type E and type F) that were
treated with demecolcine (Deme) at different times (0, 5, 10 and 15
min) post-activation.
[0022] FIGS. 5A-5B are a set of histograms showing percent (%) of
enucleation rates in ethanol-activated B6D2F1 and CF-1 mouse
oocytes treated with demecolcine (Deme) at different times (0, 5,
10 and 15 min) post-activation (p.a.). FIG. 5A shows the percentage
of enucleated oocytes of only those activated oocytes that
completed second PB extrusion, and FIG. 5B shows the total of
activated oocytes. The value in each treatment represents the
combined results for all four time-points examined (45, 75, 105 and
135 min p.a.). Different superscripts represent significant
differences (P<0.05) between treatments for each strain of
oocytes. In both FIGS. 5A and 5 B, values for each treatment differ
significantly between strains.
[0023] FIG. 6 is a graphical representation showing the rates of
activation and the second PB extrusion in goat oocytes upon
treatment with demecolcine.
[0024] FIG. 7 is a schematic showing induced enucleation and the
introduction of the donor nucleus pre-activation and post
activation.
[0025] FIG. 8 is a histogram showing the meiotic status of bovine
oocytes fixed and stained at various timepoints (30 min. (n=63), 1
h (n=63), 1.5 h (n=67) and 17 h (n=51)) after activation.
[0026] FIG. 9 is a histogram showing the activation rates of
control and demecolcine treated oocytes fixed at 5 hours
(N=26-45/group) post activation.
[0027] FIG. 10 is a histogram showing induced enucleation rates of
demecolcine treated oocytes fixed at five hours (black bars,
n=26-45/group) and 17 hours (n=18-32/group) post-activation.
[0028] FIG. 11 is a histogram showing TCN, pycnic, interphase and
mitotic nuclei in day 8 blastocyts obtained by chemical enucelation
and partenogenetic activation followed or not by donor cell nuclear
transfer.
DETAILED DESCRIPTION OF THE INVENTION
[0029] The present invention relates to new methods of cloning
animals by enucleating an oocyte by induced enucleation and, prior
to the cessation of protrusion of a second polar body containing
essentially all of endogenous chromatin of the oocyte (e.g., the
completion of the enucleation), combining (e.g., injecting or
fusing) at least the nucleus from a somatic donor cell (e.g.,
karyoplast) with the oocyte. The nuclear material from the somatic
donor cell is combined with the oocyte (e.g., cytoplast) before the
protrusion of the second polar body containing essentially all of
the endogenous chromatin ceases.
[0030] The present invention utilizes "induced enucleation" which
refers to enucleation of the oocyte by disrupting the meiotic
spindle apparatus through the destabilization (e.g.,
depolymerization) of the microtubules of the meiotic spindle.
Destabilization of the microtubules prevents the chromatids from
separating (e.g., prevents successful karyokinesis), and induces
the oocyte genome (e.g., nuclear chromatin) to segregate unequally
(e.g., skew) during meiotic maturation, whereby essentially all
endogenous chromatin of the oocyte collects in the second polar
body.
[0031] Induced enucleation can be accomplished, for example, by
exposing an oocyte with compounds or conditions (e.g., at least one
compound and/or condition) that destabilize the microtubules, as
described above. Examples of compounds that destabilize the
microtubules include, but are not limited to, demecolcine,
Taxol.RTM. (e.g., paclitaxel), phalloidin, colchicine, and
nocodozole. Methods of chemically inactivating the DNA are known to
those of skill in the art. Completion of enucleation can be
determined by visually inspecting oocyte having stained
microfilaments e.g., with rhodamine pholloidin. In addition,
exposure of oocytes to certain conditions (e.g. increased or
decreased temperature, pH, osmolality) that induce destabilization
of the microtubules. In particular, oocytes are exposed to
temperatures, pH and/or osmolality that are above or below normal
body conditions for that species.
[0032] Before the enucleation process is completed, the nucleus
(e.g., genome) from the donor cell is introduced into the oocyte.
Completion of enucleation is signified by the cessation of the
second polar body extrusion (e.g., completion of cytokinesis or
effective cessation of active, or observable, continuing extrusion
of the second polar body). For example, a compound, such as
demecolcine, which destabilizes microtubules can, depending upon
the concentration employed, slow extrusion of a second polar body
to effectively terminate continuing extrusion and, thereby, prevent
completion of cytokinesis which, normally would mark the end point
of second polar body formation. The second polar body (PB), in the
case of induced enucleation, contains all of the endogenous nuclear
chromatin (e.g., the nucleus) of the oocyte. The introduction of
the donor nucleus can occur before, during or shortly after the
enucleation process begins (e.g., after exposure to the compound or
condition that induces enucleation), but before the cessation of
the second PB extrusion containing the nucleus of the oocyte.
Representative examples of complete second PB extrusion, induced
chemically with demecolcine, are described in the Exemplification.
Preferably, the second PB is fully extruded and contains the
nuclear chromatid of the oocyte. However, in some instances the
second PB is not fully extruded. Descriptions of this process in
the Exemplification demonstrate that in some instances, although
enucleation is completed and the second PB is formed, the second PB
is not fully extruded in all cases. Regardless of the extent to
which extrusion of the second PB actually occurs, the end point for
the completion of the enucleation process is cessation of extrusion
of a second PB containing essentially all endogenous nuclear
chromatin of the oocyte. Additionally, induced enucleation was
performed on mouse, goat and bovine embryos, and for each species
demecolcine caused the enucleation of the oocyte to form a second
PB containing essentially all of the endogenous chromatin of the
oocyte. See Exemplification.
[0033] When using a compound that destabilizes the microtubules to
enucleate an oocyte, the induced enucleation begins upon exposure
of the compound and continues until the protrusion of a second
polar body containing essentially all endogenous chromatin ceases,
such as by completing formation of the second polar body. The
length of time needed to complete enucleation depends on a variety
of factors including the specific compound or condition used for
the enucleation, and the species or strain within a species of
oocyte used. In general, induced enucleation for mammals using
demecolcine is about 5 hours post-activation. For example, when
using demecolcine to enucleate bovine oocytes, induced enucleation
(greater than 80%) generally requires about 5 hours
post-activation. Induced enucleation with demecolcine generally
takes about 1.5 hours for murine oocytes (greater than 50%), 4-6
hours for pig oocytes, 3-5 hours for human oocytes, and 5 hours for
goat oocytes (greater than 80%). One of skill in the art can
readily determine the length of time required to complete induced
enucleation.
[0034] Activated oocytes are those that are in a dividing stage of
meiotic cell division, and can include any meiotic phase except
metaphase II (e.g., metaphase I, anaphase I, telophase I, and
preferably, anaphase II and telophase II) stage. In particular,
activated oocytes refer to those metaphase II oocytes that have
been stimulated to resume meiosis naturally (e.g., fertilization)
or by artificial means (e.g., ethanol, ionomycin, electrical
change, or chemical activation). An activated oocyte is also
defined as one that has a protruding second polar body. Oocytes in
metaphase II are considered to be in a resting state and are
therefore arrested. The oocytes can be in the resting stage of
metaphase II (arrested), and then activated, using methods
described herein. The stage that the oocyte is in can be identified
by visual inspection of the oocyte under a sufficient
magnification. Methods for identifying the stage of meiotic cell
division are known in the art.
[0035] In another embodiment, the oocyte can be quiescent, in
metaphase II. In accordance with the present invention, the donor
nucleus is introduced to the metaphase II oocyte and then
activated. See FIG. 1. Once the metaphase II oocyte and the donor
nucleus are combined, the oocyte is activated and enucleation of
the endogenous nucleus is induced, as described herein, to thereby
form a nuclear transfer embryo. This nuclear transfer embryo is
ready to be implanted into an animal that is of the same species as
the embryo.
[0036] In one embodiment, the oocyte can be activated prior to
exposure to a microtubule destabilizing compound. For example, an
activated oocyte can be in the anaphase II or telophase II stage of
meiotic cell division, and then exposed to the microtubule
destabilizing compound. Before enucleation is complete, the donor
nucleus is introduced to the active, enucleated oocyte. See FIG. 2.
This process forms a nuclear transfer embryo which is ready to be
implanted into an animal that is of the same species as the
embryo.
[0037] Oocytes are activated by, for example, increasing their
exposure to calcium levels. Increasing levels of calcium, e.g., by
between about 10% and about 60% above the baseline levels, induces
activation or meiotic cell division of the oocyte. Baseline levels
are those levels of calcium found in an inactive oocyte. Rising
levels of calcium, coupled with decreasing levels of
phosphorylation further facilitates and sustains activation of the
oocyte. Several methods exist that allow for activation of the
oocyte. In particular, a calcium ionophore (e.g., ionomycin) is an
agent that increases the permeability of the oocyte's membrane and
allows calcium to enter into the oocyte. As the free calcium
concentration in the cell increases during exposure to the
ionophore, the oocyte is activated following reduction in MPF
(maturation promoting factor) activity. Such methods of activation
are described in U.S. Pat. No. 5,496,720. Ethanol has a similar
affect. Prior to or following enucleation, an oocyte in metaphase
II can be activated with ethanol according to the ethanol
activation treatment as described in Presicce and Yang, Mol.
Reprod. Dev., 37:61-68 (1994); and Bordignon & Smith, Mol.
Reprod. Dev., 49:29-36 (1998). Exposure of calcium to the oocyte
also occurs through electrical stimulation. The electrical
stimulation allows increasing levels of calcium to enter the
oocyte. At least one application or exposure to an activating agent
can be used for activating the oocyte. Also, more than one
application or exposures to activating agents can be used for
activating an oocyte. The time frame for a second activation can
be, for example, from about one minute after the first activation
to cessation of extrusion of the second polar body. The activating
agents for each activation can be different or the same. A second
activation can be used to ensure complete activation of the oocyte.
This second activation can be timed after a recovery is indicated
from the first activation.
[0038] In certain embodiments, the zona pellucida, can be removed
(e.g. mechanically) for optimizing the efficiency of the somatic
cell cloning. The removal prevents the potential possibility of
reintroducing the chromatin in the oocyte which can occur with an
incompletely extruded second polar body.
[0039] Oocytes can be obtained from a donor animal during that
animal's reproductive cycle. For example, oocytes can be aspirated
from follicles of ovaries at given times during the reproductive
cycle (exogenous hormone-stimulated or non-stimulated). Also at
given times following ovulation, a significant percentage of the
oocytes, for example, are in telophase. Additionally, oocytes can
be obtained and then induced to mature in vitro to arrested
metaphase II stage. Arrested metaphase II oocytes, produced in vivo
or in vitro, can then be induced in vitro to enter telophase. Thus,
oocytes in telophase can readily be obtained for use in the present
invention. In particular, oocytes can be collected from a female
animal following super ovulations. Oocytes can be recovered
surgically by flushing the oocytes from the oviduct of a female
donor. Methods of inducing super ovulations in, for example, goats
and the collection of the oocytes are described herein.
[0040] As described above, the enucleated oocyte is combined with
the nucleus of the donor cell. The donor cell can be active or
inactive. An activated (e.g., non-quiescent) donor cell is a cell
that is in actively dividing (e.g., not in a resting stage, G.sub.0
of mitosis). In particular, an activated donor cell is one that is
engaged in the mitotic cell cycle, such as G.sub.1 phase, S phase
or G.sub.2/M phase. The mitotic cell cycle has the following
phases, G.sub.1, S, G.sub.2 and M. The G.sub.2/M phase refers to
the transitional phase between the G.sub.2 phase and M phase. The
commitment event in the cell cycle, called START (or restriction
point), takes place during the G.sub.1 phase. "START" as used
herein refers to late G.sub.1 stage of the cell cycle prior to the
commitment of a cell proceeding through the cell cycle. The
decision as to whether the cell will undergo another cell cycle is
made at START. Once the cell has passed through START, it passes
through the remainder of the G.sub.1 phase (i.e., the pre-DNA
synthesis stage). The S phase is the DNA synthesis stage, which is
followed by the G.sub.2 phase, the stage between synthesis and
mitosis. Mitosis takes place during the M phase. If prior to START,
the cell does not undergo another cell cycle, the cell becomes
arrested. In addition, a cell can be induced to exit the cell cycle
and become quiescent or inactive. A "quiescent" or "inactive" cell,
is referred to as a cell in G.sub.0 phase.
[0041] In one embodiment, the donor cell and oocyte can be
"synchronous" with respect to the cell cycle. In this case,
synchronization refers to cells that are in the same stage of cell
division (e.g., in an active state). The meiotic cell stage of the
activated oocytes correlates to the mitotic stage of the cell cycle
of the activated donor cell. For example, an oocyte in telophase II
fused with the genome of a donor cell in the G.sub.1 stage before
completion of the enucleation process provides a synchronization
between the oocyte and the donor nuclei.
[0042] It is preferable that the donor cells also be in the same
stage of cell division. Using donor cells at certain phases of the
cell cycle, for example, G.sub.1 phase, allows for synchronization
of the donor cells. One can synchronize the donor cells and put
them in the same stage by depriving (e.g., reducing) the donor
cells of a sufficient amount of nutrients in the media that allows
them to divide. Once the donor cells have stopped dividing, then
the donor cells are exposed to media (serum) containing a
sufficient amount of nutrients to allow them to being dividing
(e.g., mitosis). The donor cells begin mitosis substantially at the
same time, and are therefore, synchronous. For example, the donor
cells are deprived of a sufficient concentration of serum by
placing the cells in 0.5% Fetal Bovine Serum (FBS) for about a
week. Thereafter, the cells are placed in about 10% FBS and they
will begin dividing at about the same time. They will enter the G1
phase about the same time, and are therefore, ready for the cloning
process.
[0043] Methods of determining which phase of the cell cycle a cell
is in are known to those skilled in the art, for example, U.S. Pat.
No. 5,843,705 to DiTullio et al., Campbell, K. H. S., et al.,
Embryo Transfer Newsletter, vol. 14(1):12-16 (1996), Campbell, K.
H. S., et al., Nature, 380:64-66 (1996), Cibelli, J. B., et al.,
Science, 280:1256-1258 (1998), Yong, Z. and L. Yuqiang, Biol. of
Reprod., 58:266-269 (1998) and Wilmut, I., et al., Nature,
385:810-813 (1997). For example, as described below in the
Examples, various markers are present at different stages of the
cell cycle. Such markers can include cyclines D 1, 2, 3 and
proliferating cell nuclear antigen (PCNA) for G.sub.1, and BrDu to
detect DNA synthetic activity. In addition, cells can be induced to
enter the G.sub.0 stage by culturing the cells on a serum-deprived
medium. Alternatively, cells in G.sub.0 stage can be induced to
enter into the cell cycle, that is, at G.sub.1 stage by serum
activation (e.g., exposing the cells to serum after the cells have
been deprived of a certain amount of serum).
[0044] The donor cell can be any type of cell that contains a
genome or genetic material (e.g., nucleic acid), such as a somatic
cell, germ cell or a stem cell. The term "somatic cell" as used
herein refers to a differentiated cell. The cell can be a somatic
cell or a cell that is committed to a somatic cell lineage.
Alternatively, any of the methods described herein can utilize a
diploid stem cell that gives rise to a germ cell in order to supply
the genome for producing a nuclear transfer embryo. The somatic
cell can originate from an animal or from a cell and/or tissue
culture system. If taken from an animal, the animal can be at any
stage of development, for example, an embryo, a fetus or an adult.
Additionally, the present invention can utilize embryonic somatic
cells. Embryonic cells can include embryonic stem cells as well as
embryonic cells committed to a somatic cell lineage. Such cells can
be obtained from the endoderm, mesoderm or ectoderm of the embryo.
Embryonic cells committed to a somatic cell lineage refer to cells
isolated on or after approximately day ten of embryogenesis.
However, cells can be obtained prior to day ten of embryogenesis.
If a cell line is used as a source for a chromosomal genome, then
primary cells are preferred. The term "primary cell line" as used
herein includes primary cells as well as primary derived cell
lines.
[0045] Suitable somatic cells include fibroblasts (for example,
primary fibroblasts), epithelial cells, muscle cells, cumulous
cells, neural cells, and mammary cells. Other suitable cells
include hepatocytes and pancreatic islets.
[0046] The genome of the somatic cell can be the naturally
occurring genome, for example, for the production of cloned
animals, or the genome can be genetically altered to comprise a
transgenic sequence, for example, for the production of transgenic
cloned animals, as further described herein.
[0047] Somatic cells can be obtained by, for example,
disassociation of tissue by mechanical (e.g., chopping, mincing) or
enzymatic means (e.g., trypsinization) to obtain a cell suspension
followed by culturing the cells until a confluent monolayer is
obtained. The somatic cells can then be harvested and prepared for
cryopreservation, or maintained as a stalk culture. The isolation
of somatic cells, for example, fibroblasts, is described
herein.
[0048] The nucleus of the donor cell is introduced before or upon
exposure to the chemical or condition used to induce enucleation,
or during any time prior cessation of protrusion of the second
polar body containing essentially all of the endogenous chromatin.
The donor nucleus and the enucleating oocyte can be combined in
variety of ways to form the nuclear transfer embryo. For example,
genome of a donor cell can be injected into the activated oocyte by
employing a microinjector (i.e., micropipette or needle). The
nuclear genome of the donor cell, for example, a somatic cell, is
extracted using a micropipette or needle. Once extracted, the
donor's nuclear genome can then be placed into the activated oocyte
by inserting the micropipette, or needle, into the oocyte and
releasing the nuclear genome of the donor's cell. See, for example,
McGrath, J. and D. Solter, Science, 226:1317-1319 (1984), the
teachings of which are incorporated by reference in their
entirety.
[0049] Alternatively, the genome of a donor cell can be combined
with an oocyte by fusion; e.g., electrofusion, viral fusion,
liposomal fusion, biochemical reagent fusion (e.g.,
phytohemaglutinin (PHA) protein), or chemical fusion (e.g.,
polyethylene glycol (PEG) or ethanol). The nucleus of the donor
cell can be deposited within the zona pelliduca which contains the
oocyte. The steps of fusing the nucleus with the oocyte can then be
performed by applying an electric field which will also result in a
second activation of the oocyte. Anaphase II and/or Telophase II
oocytes (e.g., oocyte having an extruding second polar body) used
are already activated, hence any activation subsequent to or
simultaneous with the introduction of genome from a somatic cell
would be considered a second activation event. With respect to
electrofusion, chambers, such as the BTX.RTM. 200
Embryomanipulation System for carrying out electrofusion are
commercially available from for example BTX.RTM., San Diego. The
combination of the genome of the donor cell with the oocyte results
in a nuclear transfer embryo.
[0050] A nuclear transfer embryo of the present invention is then
transferred into a recipient animal female and allowed to develop
or gestate into a cloned or transgenic animal. Conditions suitable
for gestation are those conditions that allow for the embryo to
develop and mature into a fetus, and eventually into a live animal.
Such conditions are known in the art. For example, the nuclear
transfer embryo can be transferred via the fimbria into the
oviductal lumen of each recipient animal female. In addition,
methods of transferring an embryo to a recipient known to those
skilled in the art and are described in Ebert et al,
Bio/Technology, 12:699 (1994). The nuclear transfer embryo can be
maintained in a culture system until at least first cleavage
(2-cell stage) up to the blastocyst stage, preferably the embryos
are transferred at the 2-cell or 4-cell stage. Various culture
media for embryo development are known to those skilled in the art.
For example, the nuclear transfer embryo can be co-cultured with
oviductal epithelial cell monolayer derived from the type of animal
to be provided by the practitioner.
[0051] The present invention encompasses the cloning of a variety
of animals. These animals include, for example, human or nonhuman
mammals, (e.g., canines, felines, murine species (e.g., mice,
rats), and ruminants (e.g., cows, sheep, goats, camels, pigs, oxen,
horses, llamas)). In particular, goats of Swiss origin, for
example, the Alpine, Saanen and Toggenburg bread goats can be used.
The donor cell and the oocyte are preferably from the same species,
and once combined, an animal of the same species is impregnated
with embryo.
[0052] "Cloning an animal" refers to producing an animal that
develops from an oocyte containing genetic information or the
nucleic acid sequence of another animal, the animal being cloned.
The cloned animal has substantially the same or identical genetic
information as that of the animal being cloned. "Cloning" also
refers to cloning a cell, which includes producing an oocyte
containing genetic information or the nucleic acid sequence of
another animal. The resulting oocyte having the donor genome is
referred to herein as a "nuclear transfer embryo."
[0053] The present invention also relates to methods for generating
transgenic animals. A transgenic animal is an animal that has been
produced from a genome from a donor cell that has been genetically
altered, for example, to produce a particular protein (a desired
protein). Methods for introducing DNA constructs into the germ line
of an animal to make a transgenic animal are known in the art. For
example, one or several copies of the construct can be incorporated
into the genome of a animal embryo by standard transgenic
techniques.
[0054] Embryonal target cells at various developmental stages can
be used to introduce transgenes. A transgene is a gene that
produces the desired protein and is eventually incorporated into
the genome of the activated oocyte. Different methods are used
depending upon the stage of development of the embryonal target
cell. The specific lines of any animal used to practice this
invention are selected for general good health, good embryo yields,
good pronuclear visibility in the embryo, and good reproductive
fitness. In addition, the haplotype is a significant factor.
[0055] Genetically engineered donor cells for use in the instant
invention can be obtained from a cell line into which a nucleic
acid of interest, for example, a nucleic acid which encodes a
protein, has been introduced.
[0056] A construct can be introduced into a cell via conventional
transformation or transfection techniques. As used herein, the
terms "transfection" and "transformation" include a variety of
techniques for introducing a transgenic sequence into a host cell,
including calcium phosphate or calcium chloride co-precipitation,
DEAE dextrane-mediated transfection, lipofection, or
electroporation. In addition, biological vectors, for example,
viral vectors can be used as described below. Samples of methods
for transforming or transfecting host cells can be found in
Sambrook et al., Molecular Cloning: A Laboratory Manual In Second
Edition, Cold Spring Harbor Laboratory, (Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. 1989). Two useful and
practical approaches for introducing genetic material into a cell
are electroporation and lipofection.
[0057] The DNA construct can be stably introduced into a donor cell
line by electroporation using the following protocol: donor cells,
for example, embryonic fibroblasts, are resuspended in phosphate
buffer saline (PBS) at about 4.times.10.sup.6 cells per mL. Fifty
micrograms of linearized DNA is added to the 0.5 mL cell
suspension, and the suspension is placed in a 0.4 cm electrode gap
cuvette. Electroporation is performed using a BioRad Gene Pulser
(Bio Rad) electroporator with a 330 volt pulse at 25 mA, 1000
microFarad and infinite resistance. If the DNA construct contains a
neomyocin resistance gene for selection, neomyocin resistant clones
are selected following incubation where 350 mg/mL of G418 (GIBCO
BRL) for fifteen days.
[0058] The DNA construct can be stably introduced into a donor
somatic cell line by lipofection using a protocol such as the
following: about 2.times.10.sup.5 cells are plated into a 3.5 cm
well and transfected with 2 mg of linearized DNA using
LipfectAMINE.RTM. (GIBCO BRL). Forty-eight hours after
transfection, the cells are split 1:1000 and 1:5000 and if the DNA
construct contains a neomyocin resistance gene for selection, G418
is added to a final concentration of 0.35 mg/mL. Neomyocin
resistant clones are isolated and expanded for cyropreservation as
well as nuclear transfer.
[0059] It is often desirable to express a protein, for example, a
heterologous protein, in a specific tissue or fluid, for example,
the milk of a transgenic animal. A heterologous protein is a
protein that is not naturally made by the cloned species (e.g., a
protein that is derived from a different species than the species
being cloned). The heterologous protein can be recovered from the
tissue or fluid in which it is expressed. For example, it is often
desirable to express the heterologous protein in milk. Methods for
producing a heterologous protein under the control of a
milk-specific promoter is described below. In addition, other
tissue-specific promoters, as well as, other regulatory elements,
for example, signal sequences and sequences which enhance secretion
of non-secreted proteins, are described below. The transgenic
product (e.g., a heterologous protein) can be expressed, and
therefore, recovered in various tissue, cells or bodily secretions
of the transgenic animals. Examples of such tissue, cells or
secretions are blood, urine, hair, skin, mammary gland, muscle, or
viscera (or a tissue component thereof) including, but not limited
to, brain, heart, lung, kidney, pancreas, gall bladder, liver,
stomach, eye, colon, small intestine, bladder, uterus and testes.
Recovery of a transgenic product from these tissues are well known
to those skilled in the art, see, for example, Ausubel, F. M., et
al., (eds), Current Protocols in Molecular Biology, vol. 2, ch. 10
(1991).
[0060] Useful transcriptional promoters are those promoters that
are preferentially activated in mammary epithelial cells, including
promoters that control the genes encoding protein such as caseins,
.beta.-lactoglobulin (Clark et al., Bio/Technology, 7:487-492
(1989)), whey acid protein (Gordon et al., Bio/Technology,
5:1183-1187 (1987)), and lactalbumin (Soulier et al., Febs Letts.,
297:13 (1992)). Casein promoters can be derived from the alpha,
beta, gamma, or kappa casein genes of any animal species; a
preferred promoter is derived from the goat .beta.-casein gene
(Ditullio, Bio/Technology, 10:74-77 (1992)). Milk specific protein
promoter or the promoters that are specifically activated in
mammary tissue can be derived from cDNA or genomic sequences.
[0061] DNA sequence information is available for the mammary
gland's specific genes listed above, in at least one, and often in
several organisms. See, for example, Richards et al., J. Biol.
Chem., 256:526-532 (1981) (.alpha.-Lactalbumin rat); Campbel et
al., Nucleic Acids Res., 12:8685-8697 (1984) (rat WAP); Jones et
al., J. Biol. Chem., 260:7042-7050 (1985) (rat .beta.-Casein);
Yu-Lee and Rosen, J. Biol. Chem., 258:10794-10804 (1983) (rat
.alpha.-Casein); Hall, Bio. Chem. J., 242:735-742 (1987);
(.alpha.-Lactalbumin human); Stewart, Nucleic Acids Res., 12:389
(1984) (Bovine .alpha. S1 and .kappa.1 Casein, cDNAs); Gorodetsky
et al., Gene, 66:87-96 (1988) (Bovine .beta.-Casein); Alexander et
al., Eur. J. Biochem., 178:395-401 (1988) (Bovine and
.kappa.-Casein); Brignon et al., Febs Let., 188:48-55 (1977)
(Bovine .alpha. S2 Casein); Gamieson et al., Gene, 61:85-90 (1987);
Ivanov et al., Biol. Chem. Hopp-Seylar, 369:425-429 (1988);
Alexander et al., Nucleic Acid Res., 17:6739 (1989) (Bovine
.beta.-Lactoglobulin); Vilotte et al., Biochimie, 69:609-620 (1987)
(Bovine .alpha.-Lactalbumin).
[0062] The structure and function of the various milk protein genes
are reviewed by Mercier & Vilotte, J. Dairy Sci., 76:3079-3098
(1993). If additional flanking sequences are useful in optimizing
expression of the heterologous protein, such sequences can be
cloned using the existing sequences as probes. Mammary gland
specific regulatory sequences from different organisms can be
obtained by screening libraries from such organisms using known
cognate nucleotide sequences, or antibodies to cognate proteins as
probes.
[0063] Useful signal sequences such as milk specific signal
sequences or other signal sequences which result in the secretion
of eukaryotic or prokaryotic proteins can be used. Preferably, the
signal sequence is selected from milk specific signal sequences,
that is, it is from a gene which encodes a product secreted into
milk. Most preferably, the milk specific signal sequence is related
to the milk specific promoter used in the construct. The size of
the signal sequence is not critical. All that is required is that
the sequence be of a sufficient size to effect secretion of the
desired recombinant protein, for example, in the mammary tissue.
For example, signal sequences from genes coding for caseins, for
example, .alpha., .beta., .gamma. or .kappa. caseins and the like
can be used. A preferred signal sequence is the goat .beta.-casein
signal sequence. Signal sequences from other secreted proteins, for
example, proteins secreted by kidney cells, pancreatic cells, or
liver cells, can also be used. Preferably, the signal sequence
results in the secretion of proteins into, for example, urine or
blood.
[0064] A non-secreted protein can also be modified in such a manner
that it is secreted such as by inclusion in the protein to be
secreted all or part of the coding sequence of a protein which is
normally secreted. Preferably, the entire sequence of the protein
which is normally secreted is not included in the sequence of the
protein but rather only a sufficient portion of the amino terminal
end of the protein which is normally secreted to result in
secretion of the protein. For example, a portion which is not
normally secreted is fused (usually at its amino terminal end) to
an amino terminal portion of the protein which is normally
secreted.
[0065] In one aspect, the protein which is normally secreted is a
protein which is normally secreted in milk. Such proteins include
proteins secreted by mammary epithelial cells, milk proteins such
as caseins, .beta.-lactoglobulin, whey acid protein, and
lactalbumin. Casein proteins including, alpha, beta, gamma or kappa
casein genes of any mammalian species. The preferred protein is
.beta.-casein, for example, goat .beta.-casein. Sequences which
encode the secreted protein can be derived from either cDNA or
genomic sequences. Preferably, they are of genomic origin, and
include one or more introns.
[0066] Other tissue specific promoters which provide expression in
a particular tissue can be used. Tissue specific promoters are
promoters which are expressed more strongly in a particular tissue
than in others. Tissue specific promoters are often expressed
exclusively in the specific tissue.
[0067] Tissue specific promoters which can be used include: a
neural-specific promoter, for example, nestin, Wnt-1, Pax-1,
Engrailed-1, Engrailed-2, Sonic-hedgehog: a liver specific
promoter, for example, albumin, alpha-1, antitrypsin; a
muscle-specific promoter, for example, myogenin, actin, MyoD,
myosin; an oocyte specific promoter, for example, ZP1, ZP2, ZP3; a
testus specific promoter, for example, protamine, fertilin,
synaptonemal complex protein-1; a blood specific promoter, for
example, globulin, GATA-1, porphobilinogen deaminase; a lung
specific promoter, for example, surfactin protein C; a skin or wool
specific promoter, for example, keratin, elastin;
endothelium-specific promoter, for example, TIE-1, TIE-2; and a
bone specific promoter, for example, BMP. In addition, general
promoters can be used for expression in several tissues. Examples
of general promoters, include .beta.-actin, ROSA-21, PGK, FOS,
c-myc, Jun-A, and Jun-B.
[0068] A cassette which encodes a heterologous protein can be
assembled as a construct which includes a promoter for a specific
tissue, for example, for mammary epithelial cells, a casein
promoter. The construct can also include a 3' untranslated region
downstream of the DNA sequence coding for the non-secreted
proteins. Such regions can stabilize the RNA transcript of the
expression system and thus increase the yield of desired protein
from the expression system. Among the 3' untranslated regions
useful in the constructs for use in the invention are sequences
that provide a polyA signal. Such sequences can be derived, for
example, from the SV40 small t antigen, the casein 3' untranslated
region or other 3' untranslated sequences well known in the art. In
one aspect, the 3' untranslated region is derived from a milk
specific protein. The length of the 3' untranslated region is not
critical but the stabilizing effect of its polyA transcript appears
imported in stabilizing the RNA of the expression sequence.
[0069] Optionally, the construct can include a 5' untranslated
region between the promoter and the DNA sequence encoding the
signal sequence. Such untranslated regions can be from the same
control region as that from which the promoter is taken or can be
from a different gene, for example, they can be derived from other
synthetic, semisynthetic or natural sources. Again, there specific
length is not critical, however, they appear to be useful in
improving the level of expression.
[0070] The construct can also include about 10%, 20%, 30% or more
of the N-terminal coding region of a gene preferentially expressed
in mammary epithelial cells. For example, the N-terminal coding
region can correspond to the promoter used, for example, a goat
.beta.-casein N-terminal coding region.
[0071] The construct can be prepared using methods known to those
skilled in the art. The construct can be prepared as part of a
larger plasmid. Such preparation allows the cloning and selection
of the correct constructions in an efficient manner. The construct
can be located between convenient restrictions sites on the plasmid
so that they can be easily isolated from the remaining plasmid
sequences for incorporation into the desired animal.
[0072] Transgenic sequences encoding heterologous proteins can be
introduced into the germ line of an animal or can be transfected
into a cell line to provide a source of genetically engineered
donor cells as described above. The protein can be a complex or
multimeric protein, for example, a homo- or hetromultimeric
proteins. The protein can be a protein which is processed by
removing the N-terminus, C-terminus or internal fragments. Even
complex proteins can be expressed in active form. Protein encoding
sequences which can be introduced into the genome of an animal, for
example, goats, include glycoproteins, neuropeptides,
immunoglobulins, enzymes, peptides and hormones. The protein can be
a naturally occurring protein or a recombinant protein for example,
a fragment or fusion protein, (e.g., an immunoglobulin fusion
protein or a mutien). The protein encoding nucleotide sequence can
be human or non-human in origin. The heterologous protein can be a
potential therapeutic or pharmaceutical agent such as, but not
limited to, alpha-1 proteinase inhibitor, alpha-1 antitrypsin,
alkaline phosphatase, angiogenin, antithrombin II, any of the blood
clotting factors including Factor VIII, Factor IX, and Factor X
chitinase, erythropoietin, extracellular superoxide dismutase,
fibrinogen, glucocerebrosidas, glutamate decarboxylase, human
growth factor, human serum albumin, immunoglobulin, insulin, myelin
basic protein, proinsulin, prolactin, soluble CD 4 or a component
or complex thereof, lactoferrin, lactoglobulin, lysozyme,
lactalbumin, tissue plasminogen activator or a variant thereof.
Immunoglobulin particularly preferred protein. Examples of
immunoglobulins include IgA, IgG, IgE, IgM, chimeric antibodies,
humanized antibodies, recombinant antibodies, single chain
antibodies and anti-body protein fusions.
[0073] Nucleotide sequence information is available for several of
the genes encoding the heterologous proteins listed above, in at
least one, and often in several organisms. See, for example, Long
et al., Biochem., 23(21):4828-4837 (1984) (Alpha-1 antitrypsin);
Mitchell et al., Prot. Natl. Acad. Sci. USA, 83:7182-7186 (1986)
(Alkaline phosphatase); Schneider et al., Embo J., 7(13): 4151-4156
(1988) (Angiogenin); Bock et al., Biochem., 27 (16):6171-6178
(1988) (Antithrombin); Olds et al., Br. J. Haematol., 78(3):
408-413 (1991) (Antithrombin III); Lyn et al., Proc. Natl. Acad.
Sci. USA, 82(22):7580-7584 (1985) (erythropoietin); U.S. Pat. No.
5,614,184 (erythropoietin) Horowtiz, et al., Genomics, 4(1):87-96
(1989) (Glucocerebrosidase); Kelly et al., Ann. Hum. Genet.,
56(3):255-265 (1992) (Glutamate decarboxylase); U.S. Pat. No.
5,707,828 (human serum albumin); U.S. Pat. No. 5,652,352 (human
serum albumin); Lawn et al., Nucleic Acid Res., 9(22):6103-6114
(1981) (human serum albumin); Kamholz et al., Prot. Matl. Acad.
Sci. USA, 83(13):4962-4966 (1986) (myelin basic protein); Hiraoka
et al., Mol. Cell Endocrinol., 75(1):71-80 (1991) (prolactin); U.S.
Pat. No. 5,571,896 (lactoferrin); Pennica et al., Nature,
301(5897):214-221 (1983) (tissue plasminogen activator); Sarafanov
et al., Mol. Biol., 29: 161-165 (1995).
[0074] A transgenic protein can be produced in the transgenic
cloned animal at relatively high concentrations and in large
volumes, for example in milk, providing continuous high level
output of normally processed protein that is easily harvested from
a renewable resource. There are several different methods known in
the art for isolation of proteins for milk.
[0075] Milk proteins usually are isolated by a combination of
processes. Raw milk first is fractionated to remove fats, for
example by skimming, centrifugation, sedimentation, (H. E.
Swaisgood, Development in Dairy Chemistry, I: Chemistry of Milk
Protein, Applied Science Publishers, NY 1982), acid precipitation
(U.S. Pat. No. 4,644,056) or enzymatic coagulation with rennin or
chymotrypsin (Swaisgood, ibid.). Next the major milk proteins can
be fractionated into either a clear solution or a bulk precipitate
from which this specific protein of interest can be readily
purified.
[0076] French Patent No. 2487642 describes the isolation of milk
proteins from skim milk or whey by performing ultra filtration in
combination with exclusion chromatography or ion exchange
chromatography. Whey is first produced by removing the casein by
coagulation with rennet or lactic acid. U.S. Pat. No. 4,485,040
describes the isolation of an .alpha.-lactoglobulin-enriched
product in the retentate from whey by two sequential ultra
filtration steps. U.S. Pat. No. 4,644,056 provides a method for
purifying immunoglobulin from milk or colostrum by acid
precipitation at pH 4.0-5.5, is sequential cross-flow filtration
first on a membrane with 0.1-1.2 mm pore size to clarify the
product pool and then on a membrane with a separation limit of 5-80
kD to concentrate it. Similarly, U.S. Pat. No. 4,897,465 teaches
the concentration and enrichment of a protein such as
immunoglobulin from blood serum, egg yolks or whey by sequential
ultra filtration on metallic oxide membranes with a pH shift.
Filtration is carried out first at a pH below the isoelectric point
(pI) of the selected protein to remove bulk contaminants from the
protein retentate, in next adding pH above the pI of the selected
protein to retain impurities and pass the selected protein to the
permeate. A different filtration concentration method is taught by
European Patent No. EP 467 482 B 1 in which defatted skim milk is
reduced to pH 3-4, below the pI of the milk proteins, to solubilize
both casein and whey proteins. Three successive rounds of ultra
filtration are diafiltration and concentrate the proteins to form a
retentate containing 15-20% solids of which 90% is protein.
Alternatively, British Patent Application No. 2179947 discloses the
isolation of lactoferrin from whey by ultra filtration to
concentrate the sample, fall by week cation exchange chromatography
at approximately a neutral pH. No measure of purity is reported in
PCT Publication No. WO 95/22258, a protein such as lactoferrin is
recovered from milk that has been adjusted to high ionic strength
by the addition of concentrated salt, followed by cation exchange
chromatography.
[0077] In all these methods, milk or a fraction thereof is first
treated to remove fats, lipids, and other particular matter that
would foul filtration membranes or chromatography medium. The
initial fractions thus produce can consist of casein, whey, or
total milk protein, from which the protein of interest is then
isolated.
[0078] PCT Patent Publication No. WO 94/19935 discloses a method of
isolating a biologically active protein from whole milk by
stabilizing the solubility of total milk proteins with a positively
charged agent such as arginine, imidazole or Bis-Tris. This
treatment forms a clarified solution from which the protein can be
isolated for example by filtration through membranes that otherwise
would become clogged by precipitated proteins.
[0079] Methods for isolating a soluble milk component such as a
peptide in its biologically active form from whole milk or a milk
fraction by tangential flow filtration are known. Unlike previous
isolation methods, this eliminates the need for a first
fractionation of whole milk to remove fat micelles, thereby
simplifying the process in avoiding losses of recovery of
bioactivity. This method can be used in combination with additional
purification steps to further remove contaminants and purify the
product (e.g., the protein of interest).
[0080] The following examples are intended to be illustrative and
not limiting in any way.
EXEMPLIFICATION
Example 1
Improved Method of Cloning Through the Introduction of Donor Cell
Nucleus Prior to Completion of Enucleation Process
[0081] Progress has continued with experiments designed to compare
in vitro development of control embryos (parthenotes) with that of
reconstructed NT embryos prepared by conventional NT, telophase NT,
IE and a novel simultaneous IE-NT paradigm (SIE/NT; see Table 1
below). As in all previous experiments, abattoir-sourced oocytes
were obtained having been submitted to 26 h of maturation in ACM
media @5% CO.sub.2 in air. Oocytes were denuded (enzyme/vortex) and
activated (5 .mu.M ionomyciin for 5 min, +/-CHX). See Example 2 for
detail on methods. IE was performed by treatment of oocytes with
demecolcine (0.4 .mu.g/ml) starting at 1.5 h and ending 5 h
post-activation (p.a.) and nuclei were injected 1.5-3 h p.a. In the
case of SIE/NT, donor karyoplasts (fibroblasts) were injected
immediately prior to or immediately after activation. In vitro
embryo development was evaluated over a period of 7 days.
[0082] Summary of Bovine NT In Vitro Development TABLE-US-00001
Total Frag- GROUP oocytes Cleavage Morula Blastocyst mented Control
67 58% 13% 21% 28% Parthnotes (39/67) (5/39) (8/39) Zona-intact
Control 30 86% NA 8% 41% Parthnotes (26/30) (2/26) Zona-free
Conventional 23 35% 17% 9% 22% MII (8/23) (4/23) (2/23) Enucleation
(CMII) Telophase 55 62% NA 12% 26% Enucleation (34/55) (4/34) (TE)
Induced 144 49% 17% 16% 20% Enucleation (IE) (71/144) (12/71)
(11/71) Simultaneous 145 54% NA 25% 18% IE-NT (79/145) (20/79)
(SIE/NT)
[0083] In summary, these data further confirm the comparative in
vitro development potential of NT embryos produced by conventional,
telophase and IE protocols. Moreover, the SE/NT protocol appears to
support the highest rate of blastocyst development when compared to
the other methods. This observation is further supports the
mechanism by which higher NT development rates can be obtained
using the IE method. This is founded on the concept that IE affords
spindle-associated enabling factors to compartmentalize within the
enucleating cytoplast in a manner and time frame so as to enhance
cytoplasmic-nuclear synchronization and chromatin remodeling.
Example 2
Demecolcine-Induced Oocyte Enucleation for Somatic Cell Cloning:
Coordination Between Cell Cycle Egress, Kinetics of Cortical
Cytoskeletal Interactions, and Second Polar Body Extrusions
[0084] Studies were designed to further explore the use of
pharmacological agents to create developmentally competent
enucleated mouse oocytes for animal cloning by somatic cell nuclear
transfer. Metaphase-II oocytes from CF-1 and B6D2F1 strains were
activated with ethanol and subsequently exposed to demecolcine at
various times post-activation. Chromosome segregation, spindle
dynamics and polar body (PB) extrusion were monitored by
fluorescence microscopy using DNA, microtubule and microfilament
selective probes. Exposure to demecolcine did not affect rates of
oocyte activation induced by ethanol but did disrupt the
coordination of cytokinesis and karyokinesis, suppressing the
extent and completion of spindle rotation and second PB extrusion
in a strain-dependent manner. Moreover, strain and treatment
specific variations in the rate of oocyte enucleation were also
detected. In particular, CF-1 oocytes were more efficiently
enucleated relative to B6D2F1 and demecolcine treatments initiated
early after activation resulted in higher enucleation rates than
when treatment was delayed. The observed strain differences are
possibly due to a combination of factors such as the time course of
meiotic cell cycle progression after ethanol-activation, the degree
of spindle rotation and the extent of second PB extrusion. These
results suggest that developmentally competent cytoplasts can be
produced by timely exposure of activated oocytes to agents that
disrupt spindle microtubules.
[0085] In the present study, the temporal consequences of
demecolcine-induced enucleation with reference to the cytoskeletal
remodeling that occurs during early phases of oocyte activation in
CF-1 and B6D2F1 mouse strains was investigated. In particular,
manifestations of demecolcine treatment on spindle
rotation/anchoring dynamics, and second PB formation and extrusion
were investigated.
Materials and Methods
Collection of Mature Oocytes
[0086] Hybrid B6D2F1 (C57BL/6.times.DBA/2) and outbred CF-1 female
mice, 8-12 weeks of age, were used as oocyte donors. Animal care
and procedures were conducted according to protocols approved by
the Tufts University Institutional Animal Care and Use Committee.
Females were induced to superovulate by intraperitoneal injection
of 5 IU of pregnant mare serum gonadotropin (PMSG, Calbiochem)
followed 48 h later by 5 IU of human chorionic gonadotropin (hCG,
Calbiochem). MII oocytes were collected from oviducts 16-17 h after
hCG administration in Hepes-buffered KSOM (H-KSOM, Specialty
Media). Cumulus cells were dispersed by incubation in 150 units/ml
of bovine testicular hyaluronidase (Sigma) in H-KSOM at 37.degree.
C. for 5 min. Cumulus-free oocytes were then washed three times in
fresh H-KSOM and immediately activated.
Oocyte Activation, Treatment and Culture
[0087] Oocytes were parthenogenetically activated by a 5-min
exposure to freshly prepared 7% (v/v) ethanol in H-KSOM at
37.degree. C. and then washed twice in H-KSOM. Removal of oocytes
from ethanol was considered as time zero post-activation (p.a.). To
monitor meiotic progression, activated control oocytes (EtOH
groups) were cultured for up to 2 h and 15 min and fixed at 30 min
intervals, starting 45 min p.a. Culture of activated oocytes was at
37.degree. C. under 5% COZ in air in KSOM medium containing 1 mg/ml
BSA and amino acids (16; Specialty Media).
[0088] Other activated oocytes were treated with the
microtubule-destabilizing drug demecolcine (Sigma) at a
concentration of 0.4 gg/ml in KSOM (Deme groups). These oocytes
were cultured in the continued presence of demecolcine starting
either immediately after activation (Deme 0 groups) or with a delay
of 5, 10 or 15 min after their removal from ethanol (Deme 5, Deme
10 and Deme 15 groups, respectively). Demecolcine-treated oocytes
were fixed at 30 min intervals, from 45 min to 135 min p.a.,
identical to control oocytes. To determine oocyte meiotic status at
the onset of demecolcine treatments, some control activated B6D2F1
and CF-1 oocytes were fixed at the same time drug exposure was
initiated in the treatment groups: 0, 5, 10 and 15 min p.a.
Fixation of Oocytes and Processing for Immunofluorescence
Analysis
[0089] At defined time-points after activation, control and
demecolcine-treated oocytes were fixed and extracted for 30 min at
37.degree. C. in a microtubule stabilizing buffer containing 0.1 M
PIPES, 5 mM MgCl.sub.2, 2.5 mM EGTA, 3.7% formaldehyde, 0.1% Triton
X-100, 1 g,M taxol, 0.01% aprotinin, 1 mM dithiothreitol (DTT) and
50% deuterium oxide. Fixed oocytes were stored until processing at
4.degree. C. in a PBS blocking solution containing 1% BSA, 0.2%
powdered milk, 2% normal goat serum, 0.1 M glycine, 0.2% sodium
azide and 0.01% Triton X-100. Wickramasinghe D, et al., Dev Biol
152:62-74 (1992).
[0090] A triple labeling protocol was used for the detection of
microtubules, microfilaments and chromatin by fluorescence
microscopy. Oocytes were first incubated for 1 h at 37.degree. C.
in a mixture of mouse monoclonal anti a-tubulin and anti (3-tubulin
antibodies (Sigma) at a 1:1000 final dilution. After several washes
in 0.1% polyvinylpyrrolidone (PVP)/PBS at room temperature, oocytes
were incubated at 37.degree. C. in PBS blocking solution for 30 min
and then in a 1:150 dilution of a donkey antimouse
fluorescein-conjugated IgG (Jackson ImmunoResearch) for 45 min at
37.degree. C. Oocytes were washed again several times in 0.1%
PVP/PBS and incubated at 37.degree. C. for 30 min in 10 units/ml of
Texas Red-conjugated phalloidin (Molecular Probes) to stain actin
filaments. Finally, after extensive washing in 0.1% PVP/PBS,
oocytes were incubated at room temperature for 15 min in 10
.mu.g/ml Hoechst 33258 (Molecular Probes) and mounted in 50%
glycerol/PBS containing 25 mg/ml sodium azide.
[0091] Labeled oocytes were examined using a Zeissu IM-35 inverted
epi-fluorescence microscope fitted with filters selective for
Hoechst, fluorescene and Texas Red and a 50 W mercury lamp.
Selected images were acquired using a Photometrics Cool Snap CCD
camera (Roper Scientific Inc., Trenton, N.J.) running on Metamorph
software (version 5.0, Universal Imaging Corp., Downington,
Pa.).
Statistical Analysis
[0092] All demecolcine treatments were repeated at least three
times and approximately 50 oocytes were examined per treatment at
each defined time-point. Data were analyzed by x.sup.2 test or
Fisher's exact test. A probability value of P<0.05 was
considered to be statistically significant.
Results
[0093] To monitor the effects of demecolcine on the meiotic cell
cycle progression after activation and determine its efficiency in
inducing oocyte enucleation, ethanol-activated oocytes of the
B6D2F1 and CF-1 mouse strains were cultured in the presence of drug
and analyzed at selected timepoints for microtubule (MT),
microfilaments and chromatin organization. Exposure to demecolcine
was continuous for 30-135 min, from 0, 5, 10 or 15 min p.a. Control
activated oocytes were cultured for the same period of time in the
absence of demecolcine.
Ethanol Activation Rates are not Affected by Demecolcine
Treatment:
[0094] The effect of demecolcine on the meiotic spindle was evident
15 min. after the onset of treatment, since spindles in treated
oocytes were smaller and displayed a lower MT density compared to
untreated control activated oocytes. Although MT density decreased
with extended exposure to drug, spindle MTs did not disappear
completely and even after 2 h. of treatment a few short MTs were
detected in the majority of the oocytes.
[0095] At 45 min p.a., 98% and 92.5% of the control activated
B6D2F1 and CF-1 oocytes, respectively, had resumed meiosis as
evidenced by chromatid segregation, spindle elongation and the
presence of a large, actin-rich cortical protrusion or, in a few
cases, a completely extruded second PB. These oocytes were
considered activated. Similar rates of activation at 45 min p.a.
(80-98%; Table 2) were observed in all groups of
demecolcine-treated oocytes according to the same criteria, except
that spindle elongation failed to occur. Although the extent of
chromosome separation was reduced, due to spindle disruption, two
distinct clusters of chromosomes were clearly visible in these
treated activated oocytes indicating that an effective anaphase had
occurred. The chromosomes were subcortical to the oocyte cortex,
and usually connected by a spindle remnant that resembled a
midbody. As in the control group, a small fraction of oocytes had
already extruded a second PB. Interestingly, a single group of
chromosomes and no detectable MTs were present in treated oocytes
that failed to activate. TABLE-US-00002 TABLE 2 Activation rates of
ethanol-activated oocytes (EtOH) and ethanol-activated oocytes
treated with demecolcine (Deme) at different times (0-15 min) post
activation activated oocytes (n) Strain Treatment 45 min* 75 min*
105 min* 135 min* B6D2F1 EtOH 98 (51) 98.1 (52) 100 (58) 100 (53)
Deme 0 96 (50) 98 (50) 96 (50) 96 (50) Deme 5 98 (50) 94 (50) 100
(55) 94.5 (55) Deme 10 94 (50) 98.1 (54) 100 (52) 100 (52) Deme 15
97.9 (47) 96 (50) 94 (50) 98 (51) CF-1 EtOH 92.5 (67) 97.sup.a (67)
97.3.sup.a (74) 100 (48) Deme 0 92 (50) 98.sup.a (50) 100.sup.a
(50) 95.9 (49) Deme 5 82 (50) 76.sup.b (50) 84.3.sup.b (51) 98 (51)
Deme 10 80 (50) 84.sup.b (50) 96.sup.a,b (50) 94.7 (57) Deme 15
94.2 (52) 100.sup.a (53) 96.1.sup.a,b (51) 95.9 (49) *Time
post-activation .sup.a,bValues with different superscripts within
the same column and strain differ significantly (P < 0.05)
[0096] In the B6D2F1 strain, activation rates of demecolcine
treated oocytes were equivalent to those of non-treated control
oocytes at all time-points examined (Table 2). While rates of
activation in some groups of CF-1 treated oocytes were lower than
in the control group at 75 min and 105 min p.a., this effect was
transitory and reversible since at 135 min after ethanol exposure
activation rates were again equivalent among all groups. Therefore,
normal rates of activation are obtained when activated oocytes are
cultured in the continuous presence of demecolcine.
Effect of Demecolcine on Spindle Rotation in Activated Oocytes:
[0097] As noted by others, two cortical protrusions formed adjacent
to each spindle pole shortly after activation in non-treated
control oocytes. One protrusion then regressed as the spindle
rotated towards the remaining protrusion, and assumed an
orientation perpendicular to the plasma membrane. Eventually this
structure was constricted at the oolemma and gave rise to the
second PB. Activated oocytes treated with demecolcine yielded two
classes that displayed either a single (type A oocyte) or double
(type B oocyte) cortical protrusions overlying the remnants of the
spindle. The two sets of chromosomes were closer to each other in
type A oocytes than in type B oocytes, suggesting that the
formation of one or two protrusions was probably dependent on the
extent of meiotic cell cycle progression before spindle disruption.
Consistent with this idea, type A oocytes were more frequently
observed when demecolcine treatment started immediately or 5 min
after activation (Deme 0 and Deme 5 groups, respectively) whereas
the incidence of type B oocytes predominated when treatment was
delayed for 10 or 15 min (Deme 10 and Deme 15 groups,
respectively).
[0098] Initiation of spindle rotation occurred in all groups of
demecolcine-treated oocytes, except for the group Deme 0 in the
CF-1 strain, but at lower rates than in control activated oocytes
(Table 3). Although only a few short spindle MTs were present in
treated oocytes, orientation of spindle remnants and the two
chromosomal sets relative to the plasma membrane was used as an
indicator of spindle rotation. CF-1 oocytes treated with
demecolcine consistently exhibited a comparatively low percentage
of activated oocytes undergoing a partial or complete spindle
rotation at all p.a. time points examined. Demecolcine also
impaired spindle rotation in B6D2F1 oocytes at 45 min and 75 min
p.a. when compared to the controls, but the effect was less
pronounced than in CF-1 oocytes. However, a dramatic decrease in
the percentage of B6D2F1 treated oocytes showing spindle rotation
occurred in all treatment groups at 105 min and 135 min p.a.,
suggesting that spindle rotation was reversed with prolonged drug
exposure. By 135 min p.a. oocytes showing complete spindle rotation
were observed in only 0-9.6% of the CF-1 and B6D2F1 treated
oocytes, compared to 100% in both control groups; and the lack of
differences between demecolcine treatments further attested to the
effectiveness of demecolcine on spindle rotation. In all, control
and treated oocytes showing a completely rotated spindle at 135 min
p.a. extrusion of the second PB had occurred. Together, these
results indicate that continued exposure to demecolcine after
oocyte activation inhibits spindle rotation independent of the time
of initiation of the treatment and the strain of oocyte, although
the kinetics of this inhibition varies between strains.
TABLE-US-00003 TABLE 3 Spindle rotation in ethanol-activated
oocytes (EtOH) and ethanol-activated oocytes treated with
demecolcine (Deme) at different times (0-15 min) post-activation
activated oocytes with a partially or completely rotated spindle
Strain Treatment 45 min 75 min* 105 min* 135 min* B6D2F1 EtOH
64.0.sup.a (50) 98.0.sup.a (51) 100.sup.a (58) 100.sup.a (53) Deme
0 37.5.sup.b (48) 30.6.sup.b (49) 0.sup.b (48) 8.3.sup.b (48) Deme
5 26.5.sup.b (49) 38.3.sup.b (47) 3.6.sup.b (55) 7.7.sup.b (52)
Deme 10 29.8.sup.b (47) 47.2.sup.b (53) 7.7.sup.b (52) 9.6.sup.b
(52) Deme 15 30.4.sup.b (46) 41.7.sup.b (48) 34.0.sup.c (47)
4.0.sup.b (50) CF-1 EtOH 58.1.sup.a (62) 84.6.sup.b (65) 94.4.sup.a
(72) 100.sup.a (48) Deme 0 0.sup.b (46) 0.sup.b (49) 0.sup.b (50)
0.sup.b (47) Deme 5 4.4.sup.b (45) 2.6.sup.b,c (38) 2.3.sup.b (43)
4.sup.b (50) Deme 10 2.5.sup.b (40) 2.4.sup.b,c (42) 4.2.sup.b (48)
1.8.sup.b (54) Deme 15 6.1.sup.b (49) 13.2.sup.c (53) 6.1.sup.b
(49) 2.1.sup.b (47) *Time post-activation .sup.a-cValues with
different superscripts within the same column and strain differ
significantly (P < 0.05)
Complete Extrusion of the Second Polar Body is Inhibited in the
Presence of Demecolcine:
[0099] While the onset of second PB formation was evident in all
treated activated oocytes, forming one or two cortical protrusions
overlying chromosomes, completion of second PB extrusion was
impaired in the presence of demecolcine. By 45 min p.a., a small
and similar percentage of activated control and treated oocytes
displayed a completely extruded second PB (FIGS. 3A & 3B).
Whereas the rates of PB extrusion in B6D2F1 and CF-1 control
oocytes increased progressively with time, reaching 100% at 135 min
p.a., complete PB extrusion in demecolcine-treated oocytes from
both strains was significantly decreased, with rates ranging from
23.1% to 70.2% at the various time-points p.a. examined. Even
though some differences were detected between treatments in both
strains, a correlation between rates of second PB extrusion and the
onset of demecolcine treatment could not be established. On the
other hand, comparison of second PB extrusion rates in
demecolcine-treated oocytes from both strains revealed significant
differences between groups Deme 10 at 75 min p.a., between all
treatment groups at 105 min p.a. and between groups Deme 0, Deme 10
and Deme 15 at 135 min p.a. These results suggest a
strain-dependent effect of demecolcine on the suppression of second
PB extrusion, being more pronounced in oocytes from the CF-1
strain.
[0100] In those demecolcine-treated oocytes that failed to extrude
a second PB, cortical protrusions enlarged over time and, in some
oocytes, showed signs of constriction at the oolemma. To determine
whether PB extrusion was merely delayed in these oocytes, they were
cultured for a longer period of time (4 h) before fixation and
analysis. In most of the oocytes the cortical protrusion/s were
reabsorbed while the formation of two pronuclei indicated that the
cell cycle progressed to early interphase. As described infra, FIG.
7C shows oocytes (Types A and B) that have been exposed to
demecolcine, and as a result completion of cytokinesis of the
second PB was prevented. FIG. 7C also depicts oocytes (Type D, E
and F) that have fully extruded second PB.
Characteristic Phenotypes are Observed in Activated Oocytes Treated
with Demecolcine that Complete Second Polar Body Extrusion:
[0101] All control activated oocytes extruded a second PB
containing half of the chromosomal complement and displayed a
midbody perpendicular to the plasma membrane. This phenotype was
classified as type C and was also observed in a fraction of
demecolcine-treated oocytes with completely extruded second PBs.
However, midbodies in type C treated oocytes were narrower and
shorter than in type C control activated oocytes, and were defined
as midbody-like structures. Other treated oocytes that completed
second B extrusion displayed characteristic phenotypes that were
never detected in control activated oocytes. Type D oocytes
deployed one set of chromosomes in the oocyte cytoplasm and one set
inside the extruded second PB, connected by a midbody-like
structure as in type C oocytes. However, the midbody-like structure
was oriented in parallel to the plasma membrane, indicating that
spindle rotation had not occurred. Moreover, a prominent
protuberance adjacent to the second PB was present in type D
oocytes, probably due to the subcortical position of the
chromosomal complement in the oocyte in the absence of spindle
rotation. Other demecolcine-treated oocytes displayed two (Type E)
or one (Type F) completely extruded second PBs that contained all
chromosomes. Therefore, type E and F represent totally enucleated
oocytes. Rotation of the spindle had not occurred in these oocytes
either, as was evidenced by the parallel orientation of the
remaining spindle MTs and the observation of two sets of
chromosomes inside the PB to the plasma membrane.
[0102] The frequency of each of these phenotypes varied according
to the onset of the demecolcine treatment with regards to
activation, the duration of the treatment, and the strain of the
oocyte, further indicating variability in the responsiveness of
demecolcine (FIG. 4). In B6D2F1 oocytes, type C was the most
frequent at 45 min and 75 min p.a. in all treatments, except for
Deme 0 group in which it was detected at similar rates as type D.
Prolonged exposure, independent of when treatment was initiated,
caused a shift to type D phenotype, as seen by the high percentage
of type D oocytes at 135 min p.a. in all treatments. CF-1 oocytes
exhibited a strikingly different response. For all treatments, type
F was the main phenotype in those oocytes that completed second PB
extrusion by 45 min p.a. An increase in the frequency of type D
oocytes was observed over time, and at 135 min p.a. type D and type
F oocytes appeared at a similar frequency, except for the group
Deme 5 in which most of the oocytes were still of type F.
Treatment of Activated Oocytes with Demecolcine Induces Enucleation
in a Strain-Dependent Manner:
[0103] According to the previous results, most CF-1 oocytes that
completed extrusion of the second PB were enucleated as a result of
demecolcine exposure, while the majority of B6D2F1 treated oocytes
retained half of the chromosomal complement. This result is
summarized in FIG. 10, which shows the combined results for all
time-points examined of the total oocytes enucleated as a result of
the various demecolcine treatments applied. Enucleation rates
ranging from 48.3% to 76.7% were obtained in those CF-1 oocytes
that completed second PB extrusion, and from 1% to 17.3% in the
B6D2F1 strain (FIG. 5A). In the four treatments applied, the rates
of enucleation were significantly higher in CF-1 than in B6D2F1
oocytes, suggesting that the efficiency of demecolcine in inducing
oocyte enucleation is strain-dependent. Moreover, enucleation
efficiency was also dependent on the time treatment was initiated
p.a., as indicated by the higher enucleation rates obtained in both
strains of oocytes when demecolcine treatment was initiated soon
after activation rather than later.
[0104] Because of the low rates of complete PB extrusion in
demecolcine-treated oocytes, when the total activated oocytes are
considered there is a dramatic decrease in the rates of enucleation
(FIG. 5B). Maximum enucleation rates of only 21% and 6.9% in CF-1
and in B6D2F1 oocytes, respectively, were obtained and again the
enucleation efficiency in all four treatments was higher in CF-1
than in B6D2F1 oocytes. Although enucleation rates of the total
activated oocytes were equivalent between treatments in CF-1
oocytes, some differences were detected between B6D2F1 oocytes
subjected to different treatments. These differences indicated,
again, that exposure to demecolcine early after activation results
in higher rates of enucleation than when the treatment is
delayed.
Meiotic Cell Cycle Progression after Activation is
Strain-Dependent:
[0105] In order to determine if the different efficiency of
demecolcine to induce enucleation in CF-1 and B6D2F1 oocytes could
be related to variations in the oocyte meiotic progression after
activation, some control activated oocytes were fixed at the same
time-points p.a. when the demecolcine treatments were initiated. As
demecolcine effects on the meiotic spindle are not immediate, the
meiotic progression at 45 mm p.a. was also recorded. Although the
time course of activation was similar in oocytes from the two
strains, the rate of cell cycle progression after the activation
stimulus was slightly different (Table 4). Release from MR arrest
and entry into anaphase followed a similar progression in the two
groups of oocytes after ethanol exposure, but the
anaphase-telophase transition proceeded faster in CF-1 oocytes.
Thus, 2.6% and 4.4% of activated CF-1 oocytes were at telophase II
10 min and 15 min p.a., respectively, while all activated B6D2F1
oocytes remained at anaphase II. By 45 min p.a. 87.1% of activated
CF-1 oocytes had entered telophase II, a value significantly higher
than the 60% observed for activated B6D2F1 oocytes. TABLE-US-00004
TABLE 4 Time course of activation rates and meiotic status of
B6D2F1 and CF-1 oocytes after ethanol activation % activated Strain
Time p.a.* n oocytes Meiotic status** B6D2F1 0 min 50 64.0 100% A 5
min 49 57.1 100% A 10 min 49 69.4 100% A 15 min 52 84.6 100% A 45
min 51 98.0 40% A, 60% T.sup.a CF-1 0 min 50 60.0 100% A 5 min 50
74.0 100% A 10 min 50 78.0 97.4% A, 2.6% T 15 min 50 90.0 95.6% A,
4.4% T 45 min 67 92.5 12.9% A, 87.1% T.sup.a *Removal from ethanol
is considered as t = 0 min post-activation (p.a.) **Only of
activated oocytes. A: anaphase; T: telophase .sup.aValues
significantly different between the two strains (P < 0.05)
Discussion
[0106] The microtubule-destabilizing drug, demecolcine, was used to
induce enucleation of pre-activated mouse oocytes of the B6D2F1
strain as a means to prepare competent cytoplasts for nuclear
transfer procedures. Additionally, the data described herein shows
the relationship between oocyte cell cycle control and the
cytoskeleton during exit from meiotic metaphase (M-phase).
[0107] Resumption of meiosis after fertilization or artificial
activation of M II-arrested oocytes is characterized by chromosome
segregation to the spindle poles, elongation and rotation of the
meiotic spindle, and extrusion of a second PB containing half of
the chromosomal complement of the oocyte. M-phase exit is triggered
by the inactivation of maturation-promoting factor (MPF) and it is
now well established that cyclin B degradation, and thus MPF
inactivation, requires an intact spindle. Consistent with this, MII
oocytes treated with demecolcine or nocodazole prior to in vitro
fertilization or parthenogenetic activation remain arrested in
M-phase, despite the occurrence of a normal pattern of calcium
oscillations. The exact mechanism by which the meiotic spindle
mediates the transition from meiotic M-phase to embryonic
interphase remains unclear. In the data described herein, oocytes
activated with ethanol prior to demecolcine treatment exhibited
activation rates comparable to activated control oocytes never
exposed to demecolcine. Ethanol exposure induces an immediate
increase in intracellular calcium and rapid progression into
anaphase, as evidenced by the rapidity of meiotic cell cycle
resumption in control activated oocytes from the two strains
analyzed in this work. Because a delay exists between the onset of
demecolcine application and detectable signs of spindle MT
disruption, the acute effects of ethanol on cell cycle resumption
are not impeded. In fact, as the results in control activated
oocytes show, most oocytes (>.sub.--60%) exited M-phase and
progressed to anaphase by the end of the 5 min ethanol exposure (0
min p.a.). Therefore, most of the oocytes were already at anaphase
II or at the anaphase-telophase transition when the demecolcine
treatment was applied. When demecolcine is applied after the
activation stimulus, activation proceeds in the presence of the
drug. However, at later stages there were clear consequences of
demecolcine exposure that altered the relationship between
karyokinesis and cytokinesis.
[0108] Also as shown herein, demecolcine binds tightly to tubulin
dimers and prevents MT polymerization, resulting in the loss of
dynamic spindle MTs in mitotic and meiotic cells.
Immunofluorescence staining with antitubulin antibodies confirmed
the time course and extent of spindle disruption by demecolcine and
showed further that few short MTs remain in the majority of oocytes
even after prolonged (2 h) drug exposure. The presence of these
spindle remnants reflects differential stability of some MTs in the
spindle, and likely correspond to interpolar MTs. Spindle
disruption impaired the extent of chromatid segregation under these
conditions. However, because oocytes were activated prior to
demecolcine treatment, the observed variable degrees of chromosome
segregation most likely result from the time of demecolcine
administration, its uptake kinetics and variations in anaphase
onset or duration. These results establish that cell cycle
activation occurs prior to gross disruptions of spindle
stability.
[0109] In early telophase, the meiotic spindle rotates from a
parallel to a perpendicular orientation relative to the plasma
membrane coincident with the initiation of second PB formation.
Although the mechanism of spindle rotation is unclear, the presence
of an actin-rich cortical domain overlying the spindle coupled with
the inhibition of spindle rotation in both mouse and Xenopus
oocytes treated with cytochalasin suggests that the interaction of
spindle MTs with actin filaments of the cell cortex mediates
spindle rotation and serves to coordinate karyokinesis and
cytokinesis. Consistent with this, disruption of the spindle should
also inhibit its rotation, as the results with demecolcine
demonstrate. In fact, some demecolcine treated oocytes undergo some
degree of spindle rotation, especially in the case of B6D2F1 eggs,
but the process is completed in less than 10% of the oocytes. These
observations suggest that spindle rotation is initiated before
demecolcine induces depolymerization of the spindle MTs, which as a
result perturbs interactions between MTs and cortical
microfilaments and impairs further rotation of the spindle.
Although suppression of spindle rotation occurred in both strains
of oocytes examined, the percentage of oocytes with partially or
completely rotated spindles at 45 and 75 min p.a. was higher in the
B6D2F1 than in the CF-1 strain. This result indicates
strain-dependent variation in the kinetics of inhibition of spindle
rotation induced by demecolcine, that can not be related to
interstrain differences in (a) the initiation and progression of
spindle rotation and in (b) the rate of cell cycle progression (as
detected in control activated oocytes from the two strains). Thus,
these strain-dependent variations could be due to other factors
associated with elongation and anchoring of the spindle such as
centrosome positioning.
[0110] An additional effect of demecolcine was inhibition of second
PB extrusion. The initial phase of PB formation, described as a
"furrowing" of the plasma membrane in the region overlying the
spindle, occurred in most treated oocytes, as evidenced by the
formation of one or two actin-rich cortical protrusions. However,
later "furrow constriction" and abscission was generally impaired
in oocytes activated in the presence of demecolcine. Nocodazole or
demecolcine treatment prior to furrowing and cleavage in sea urchin
eggs has shown that MTs are required for furrow stimulation and the
formation of the actomyosin contractile ring. But once furrowing
has been stimulated, MTs are unnecessary. MTs are important for
abscission, as depolymerization of the central spindle in late
anaphase blocks the completion of cytokinesis. Several proteins
necessary for cytokinesis have been localized to the central
spindle and it has been suggested that MTs could serve as tracks
along which these proteins and other components of the cell move
into the cleavage furrow. Specifically, the presence of a
functional midbody is required in mammalian cells to complete
division. Formation of the midbody begins in anaphase, when MT
bundles assemble in the central spindle, but functional midbody
assembly also requires formation of new MTs nucleated by y tubulin
centers during telophase. In view of this, suppression of new MT
polymerization would be expected in demecolcine-treated oocytes and
could underlie the inhibition of second PB extrusion.
Interestingly, midbody-like structures were detected in some of the
treated oocytes that completed second PB extrusion, and
specifically in all type C and type D oocytes. As MTs that form the
central spindle and the midbody are extremely stable, it is
possible that some MT bundles could assemble in these oocytes
before extensive MT depolymerization, forming a midbody-like
structure that persisted. However, detection of these midbody-like
structures in oocytes with a completely extruded second PB argues
against the need of newly nucleated MTs for the completion of
cytokinesis, unless this is not required for PB abscission or a
different mechanism was used in these oocytes to complete division.
In fact, second PB extrusion in type E and type F oocytes was
completed in the absence of a midbody or a midbody-like structure.
Spindle rotation had not occurred in these oocytes and the spindle
remnants together with all chromosomes were extruded inside the
second PB, leaving an enucleated oocyte. Interestingly, chemically
enucleated mouse oocytes produced by a combined treatment with
etoposide and cycloheximide also extrude PBs containing all oocyte
chromosomes without involvement of the spindle. Completion of
cytokinesis in the absence of MTs has also been reported in other
studies and a midbody independent mechanism for cytokinesis has
been proposed to exist in mammalian cells. Thus, it is also
possible that in all or some of the demecolcine-treated oocytes
that completed second PB extrusion this alternative mechanism was
used due to the absence of a midbody or the presence of a
non-functional midbody-like structure. The mechanism of PB
extrusion and its dependence on midbody integrity will require
further study.
[0111] Suppression of second PB extrusion in the presence of
demecolcine was independent of the time of treatment but was
dependent on the strain of the oocytes tested. In general, the
incidence of second PB extrusion was lower in CF-1 than in B6D2F1
treated oocytes. Almost all B6D2F1 treated oocytes with an extruded
second PB were of type C or type D and exhibited a midbodylike
structure. On the other hand, as the results in control activated
oocytes show, extrusion of the second PB seems to proceed somewhat
faster in the B6D2F1 strain. Therefore, strain specific variations
in the time course of midbody formation and second PB extrusion can
explain the observed differences between CF-1 and B6D2F1 treated
oocytes.
[0112] Timely perturbation in spindle function during second PB
extrusion also resulted in oocyte enucleation. Inhibition of
spindle rotation and the extent of chromosome migration in the
presence of demecolcine probably contributed to the expulsion of
the entire chromosome complement inside one, or occasionally two,
second PBs. As the results described herein, the onset of the
demecolcine treatment in relation to activation is key to achieving
enucleation. Application of demecolcine immediately or a few
minutes after ethanol exposure results in higher enucleation rates
than application of the drug 15 min after activation, and this
finding suggests that the extent of chromatid segregation is a key
determinant of enucleation. In addition, a strain effect was also
observed for enucleation efficiency, but the reasons for this are
unclear. If the proximity of the two groups of chromosomes were
decisive for enucleation, slower progression into telophase after
activation would favor enucleation. However, a faster
anaphase/telophase transition was observed in control activated
oocytes of the CF-1 strain, with higher rates of enucleation in all
demecolcine treatments, than of the B6D2F1 strain, with lower rates
of enucleation. Thus, other parameters account for the
strain-dependent efficiency of enucleation.
[0113] The majority of CF-1 oocytes treated with demecolcine that
completed second PB extrusion were enucleated, and enucleation
rates close to 80% were obtained. However, since many activated
oocytes failed to complete second PB extrusion, the overall
enucleation efficiency approximated 20%. Therefore, at least in the
CF-1 strain, impairment of PB extrusion is a limitation to
enucleation. Shorter treatments with demecolcine, that would allow
MT regeneration by late telophase, can promote the completion of
second PB extrusion. In fact, preliminary studies with oocytes
exposed to demecolcine for only 15, 30 or 45 min resulted in
slightly higher rates of second PB extrusion but the rates of
oocyte enucleation were also reduced (unpublished results). As the
effects of demecolcine on MT depolymerization and regeneration are
not immediate with respect to time of application and removal,
synchronization of treatment with oocyte cell cycle stage can be
difficult to achieve. Possibly the use of other MT-disrupting drugs
such as nocodazole, which have more rapid and reversible effects,
provide better control over the integration of cytokinesis and
karyokinesis.
[0114] Culture of activated mouse oocytes in the presence of
demecolcine results in normal rates of oocyte activation and
progressive cytoskeletal changes after activation. Disruption of
spindle MTs by demecolcine impairs chromosome migration, suppresses
spindle rotation, inhibits second PB extrusion, alters chromosome
partitioning and thereby results in the generation of enucleated
oocytes. Enucleation efficiency depends both on the onset of the
demecolcine treatment in relation to oocyte activation and on the
genetic background of the oocyte. This protocol can then be applied
to prepare recipient cytoplasts in nuclear transfer procedures.
Example 3
Activated Bovine Cytoplasts Produced by Induced Enucleation Support
Development of Nuclear Transfer Embryos In Vitro
[0115] Poor efficiency of somatic cell NT has been associated with
the preparation of developmentally competent enucleated cytoplasts.
Induced enucleation (IE) of mouse oocytes has been shown to support
enhanced term development of cloned mice. This study characterized
the kinetics and phenotypic progression of bovine oocytes subjected
to IE, and evaluated their developmental competence to support NT
embryo development in vitro. In vitro matured (26 h) oocytes were
denuded, activated (5 pM ionomycin, 5 min, then 10 .mu.g/mL
cycloheximide, 5 h) and cultured for up to 5 h post-activation
(pa). Oocyte enucleation was induced by demecolcine (0.4 .mu.g/ml,
DM) exposure at 30, 60, 90 and 120 min post activation for various
time periods (1 to 4.5 h). Activation rates and meiotic progression
of control and DM treated oocytes (n=31-49/gp) was evaluated at 5
hpa by immunofluorescence microscopy (microtubule-Mab-FITC,
microfilament-Texas red-phalloidin and chromatin-H33258). DM
treatment at 30 min pa resulted in low activation rates (10-16%)
whereas DM exposure at 1, 1.5 or 2 hpa resulted in higher (79-100%)
oocytes activation rates. Onset and duration of DM treatment
significantly altered IE rates, which varied from 60-91% at 5 hpa.
Maximum rates of IE were obtained when oocytes were exposed to DM
between 1.5 and 5 hpa (91% IE at 5 hpa). DM treatments elicited a
range of distinct oocyte spindle, chromatin, microfilament and PB
phenotypes. Development of reconstructed IE embryos was evaluated
by culture in vitro for 7 days. Mechanically isolated adult
fibroblast nuclei were injected into IE cytoplasts between 1.5-3
hpa (n=106). Parthenogenetically activated and DM treated oocytes
were cultured simultaneously for 7 days and served as controls.
Control group cleavage and morula/blastocyst rates were 49% (
23/47) and 30% ( 7/23) respectively, whereas IE group rates were
48% ( 51/106) and 27% ( 14/51) respectively. These results
demonstrate that the IE method can be used to produce enucleated
bovine cytoplasts and that IE cytoplasts are competent to support
in vitro development. This technically simple approach provides a
more efficient method to prepare competent cytoplasts for use in
nuclear transfer procedures.
[0116] Another set of experiments used bovine oocytes that have
been subjected to induced enucleation by demecolcine and control
Telophase II bovine oocytes. In these experiments, the extrusion of
the second PB in bovine oocytes had ceased. Prior to cessation of
the formation of the second PB, the nucleus of a donor cell can be
introduced into the oocyte to obtain a nuclear transfer embryo.
Example 4
Induced Enucleation of Mouse and Goat Oocytes: Kinetic and
Phenotypic Characterizations
[0117] In general, the rates of successful somatic cell cloning of
animals are poor, in part due to low efficiency in the production
of competent cytoplasts when prepared by mechanical enucleation of
MII oocytes. A method to induce enucleation of activated oocytes
has been developed that supports enhanced rates of development of
cloned mice to term. This study was designed to characterize the
kinetics and progressive phenotypes observed during induced
enucleation (IE) of activated mouse and goat oocytes. In vivo
ovulated mouse oocytes (B6D2F1, n-959; CF 1, n=999) and in vitro
matured goat oocytes (n-163) were denuded. activated (7% ethanol)
and incubated for up to 3.5 hr in KSOM. Enucleation was induced by
continuous exposure to demecolcine (0.4 g/ml) commencing at 0-30
min post-activation. Non-demecolcine treated activated oocytes
served as controls (n=50/rep). At selected time points oocytes
(n=11.50) were fixed/extracted and processed for immunofluorescence
microscopy to document activation and meiotic progression based on
spindle (microtubules), chromatin (H33258) and polar body (PB.
microfilaments) phenotypes. In mouse oocytes, activation rates were
high and similar in both strains (89-10096). In contrast, the pace
of meiotic progression and PB extrusion was strain dependent.
Maximum IE rates (23-100%) were observed when oocytes were exposed
to demecolcine immediately after activation, and the efficiency was
also strain-dependent. IE rates diminished progressively when
demecolcine treatment was delayed with respect to the time of
activation. A range of distinct spindle, chromatin, PB and oocyte
phenotypes were observed in response to demecolcine. In goat
oocytes, activation was evident by anaphase onset at 30 min and
activation rates of 69-94% were observed. When treated continuously
with demecolcine from 30 thin post-activation, 63% of activated
oocytes displayed all nuclear chromatin within the extruding second
PB. Unlike the mouse, spindle, chromatin, PB and oocyte phenotypes
displayed little variation. These results demonstrate that the IE
method can be successfully employed to produce enucleated mouse and
goat cytoplasts and therefore has broad utility for somatic cell
cloning in many animal species.
[0118] Also studied were goat oocytes subjected to induced
enucleation by demecolcine, and control Telophase II bovine
oocytes. In these experiments, the extrusion of the second PB in
goat oocytes had ceased. Prior to cessation of the formation of the
second PB, the nucleus of a donor cell can be introduced into the
oocyte prior to obtain a nuclear transfer embryo, as described
herein.
Example 5
Activated Bovin Cytoplasts Prepared by Induced Enucleation Support
Development of Nuclear Transfer Embryos In Vitro
[0119] The kinetics and phenotypic progression of boving oocytes
subjected to demecolcine induced IE and the evaluation of their
ability to support NT embryo development to the blastocyte stage in
vitro was studied. Democeocine treatments elicited a range of
distict changes involving oocyte spindle, chromatin, microfilament
and polar body phenotypes as observed ty immunofluoresnce
microscopy. Onset and duration of demecolcine treatment
significantly altered the activation and IE rates, which varied
from 10-100% and 60-91%, respectively, at 5 h post-activation.
Maximum rates of induced enucleation (IE) were obtained when
oocytes were exposed to demecolcine between 1.5 and 5 hpa (91% IE
at 5 h post-activation). Development of reconstructed demecolcine
treated embryos was evaluated by culture in vitro. After mechanical
isolation, adult fibroblast nuclei were injected into IE cytoplasts
following two different protocols. These results demonstrate that
the IE method can be used to produce enucleated bovine cytoplasts
and that IE cytoplasts are competent to support development at
least to the blastocyst stage in vitro. This technically simple
approach may provide a more efficient method to enhance the success
rate of nuclear transfer procedures.
Materials and Methods
Oocyte Recovery, Maturation and Activation
[0120] Bovine cumulus-oocyte complexes (COCs) were recovered by
follicular aspiration from slaughterhouse-derived ovaries and
shipped to the laboratory in maturation medium at 38.5.degree. C.
After 26 h of in vitro maturation, cumulus cells were removed from
COCs by a 10 min treatment with 1200 UI/ml of hyaluronidase (Sigma)
followed by vortexing. Activation of denuded oocytes was achieved
by treatment with 5:M ionomycin (Sigma) for 5 minutes at 375 C
followed by a 5-h culture (38.5.degree. C., 5% CO.sub.2 in air) in
maturation medium supplemented with 10 .mu.g/ml cycloheximide (CHX,
Sigma). The beginning of ionomycin treatment was considered as time
zero post-activation. To monitor normal meiotic cell cycle
progression under our experimental conditions, activated oocytes
(control oocytes) were fixed at 30 min (n=63), 1 h (n=63), 1.5 h
(n=58), 5 h (n=67) and 17 h (n=51) post-activation and processed
for immunofluorescence microscopy, as described below.
Demecolcine Treatment of Activated Oocytes
[0121] To define the optimal protocol for induced enucleation,
activated oocytes (n=652) were submitted to several treatments (2
replicates each) with demecolcine (0.4 .mu.g/ml, Sigma) starting at
30, 60, 90, and 120 min post-activation. Demecolcine was removed at
30 min intervals, from 60 min to 270 min (4.5 h) post-activation.
After demecolcine treatment each group of oocytes was washed 3
times in demecolcine free media and transferred to fresh culture
media (Cooks, cleavage) until fixed. Half of the oocytes in each
demecolcine-treated group was fixed after 5 hours of activation,
while the other half was fixed at 17 hrs post-activation. Oocytes
were fixed with 2.5% paraformaldehyde supplemented with 10% triton
X-100 (Sigma, 5 min at 37.degree. C.) followed by Microtubule
Stabilization buffer-extraction fixative (MTSB-EX, 10% Triton-X)
incubation for 25 min at 37.degree. C.
Oocyte Fixation and Immunofluorescence Processing
[0122] Activated control and demecolcine-treated oocytes were fixed
by incubation for 5 min at 37.degree. C. in 2.5% paraformaoldehyde
supplemented with 10% Triton X-100 (Sigma) followed by 25 min at
37.degree. C. in a Microtubule Stabilization Buffer-Extraction
Fixative (MTSB-EX, 3) containing 10% Triton X. Unspecific binding
sites of fixed oocytes were blocked by overnight treatment at
4.degree. C. with a blocking solution composed of 0.2% sodium
azide, 1% BSA, 0.2% powdered milk, 2% normal goat serum 0.1M
glycine and 0.01% Triton-X 100 in phosphate-buffered saline (PBS).
Spindle structure was assessed after primary labeling of the
oocytes with a mixture of mouse monclonal anti .alpha.-tubulin and
anti .beta.-tubulin antibodies (Sigma) at a 1:500 dilution for 8 h
at 4.degree. C. Secondary labeling was performed with a 1:100
dilution of donkey anti-mouse fluorescein-conjugated IgG (Jackson
ImmunoResearch) for 3 h at 37.degree. C. Texas red-conjugated
phalloidin (5 U/ml, 30 min at 37.degree. C.) was used for the
visualization of cell surface actin filaments. Oocytes were then
stained with a DNA-specific fluorochrome dye (10 .mu.g/ml of
Hoechst 33258, Sigma), mounted onto glass slides using a 50%
glycerol/PBS mounting solution, and examined by epifluorescence
microscopy.
Scoring Criteria
[0123] Oocytes were considered to be activated when the second PB
was present at 5 h post-activation or when at least one pronucleus
could be observed at 17 h post-activation. The normal pattern of
meiotic progression was determined in ionomycin and cycloheximide
(CHX) activated oocytes (controls) subjected to the same fixation
and labeling procedures.
[0124] The meiotic status was scored as follows: "Immature (MI-TI)"
when a large meiotic spindle was detected with chromosomes aligned
between the two spindle poles (MI) or with two sets of chromosomes
each migrating towards the opposite spindle poles (AI-TI) and no
PBs could be observed; "MII stage", when chromosomes were located
along the midplane between the two spindle poles and the first PB
was present in the periviteline space; "AII stage" when two sets of
chromosome migrating towards opposite poles and, "TII stage", when
two chromosome groups were intercalated by the characteristic
remainder of telophase spindle and one or two PBs were present in
the periviteline space. The oocytes in "pronuclear stage" were
scored according to the number of pronuclei (PN) and PBs
detected.
[0125] Oocytes were categorized as enucleated when all the DNA
content was located in the PBs, as determined by Hoechst staining,
and PB extrusion seemed to be complete, as assessed by actin
labeling.
Nuclear Transfer and In Vitro Culture of Reconstituted Embryos
[0126] Recipient cytoplasts for NT experiments were produced by
demecolcine treatment between 1.5 and 5 h post-activation, as
described previously. Embryos were reconstituted by direct
microinjection of mechanically isolated proliferating fibroblast
nuclei (line ACT 00448 BFF056H, passage 6). Prior to NT, monolayers
of fetal fibroblasts were trypsinized and resuspended in
manipulation media. Cytoplasmic membranes and content were removed
by repetitive pipetting of individual cells using a 12-14 mm
diameter glass micropipette in 12% polyvinylpyrrolidone (PVP-360,
Sigma) microdrops.
[0127] Intracytoplasmic NT was performed in the first group
(Treatment 1, n=107), 5 to 10 minutes prior to the initiation of
the activation-demecolcine treatment while in the second group
(Treatment 2, n=106) the embryos were reconstructed between 1.5 and
2.5 h post activation (FIG. 7). Micromanipulation of the second
group was performed in the presence of CHX and demecolcine. All the
oocytes were exposed to cytochalasin B (5 mg/ml) treatment for 20
min prior to micromanipulation. Activated oocytes treated with
demecolcine but not subjected to micromanipulation served as the
control group (IE oocytes, n=47).
[0128] Reconstituted and IE embryos were transferred to culture
media (Cooks, Cleavage.RTM.) supplemented with 10% heat-inactivated
fetal bovine serum (FBS) on mouse fibroblast feeder layers under
oil. After 3 days in culture, cleaved embryos were transferred to
blastocysts culture media (Cooks, Blast.RTM.) supplemented as
described for cleavage. Cleavage rates were determined 2 days post
activation and blastocyst rates were assessed at day 7 of in vitro
culture.
Karyotype Analysis of Reconstructed Embryos
[0129] Day 8 blastocysts obtained after NT (n=8 Treatment 1, n=6
Treatment 2), or IE (n=4) were incubated with 0.4 .mu.g/ml
demecolcine for 16 h at 38.5.degree. C. for the induction of
mitotic arrest. Each embryo was then transferred to a 72 well dish
containing 15:1 of 0.65% KCl solution and incubated for 15 min.
Embryos were then placed separately on methanol chilled slides and
fixed with 3 drops of chilled methanol-acetic acid mixture (3:1).
Slides were air dried overnight and stained with 4% Giemsa for 10
min, washed with tap water and kept at -20.degree. C. until
analyzed. All analyzable cells were counted, categorized
accordingly to morphology, and recorded. The total cell number
(TCN) was determined by counting all the cells including pycnotic,
interphase and mitotic cells with distinguishable chromosomes. A
nucleus was considered pycnotic and thus inactive, when the
chromatin was extremely condensed and the nuclear periphery was
defined. All nuclei containing distinguishable chromosomes were
categorized as mitotic and further classified as near haploid, near
diploid and near tetraploid when around 30, 60 or 120 chromosomes,
respectively, were encountered in the spreads.
Results
Meiotic Progression of Control Activated Oocytes
[0130] The meiotic cell cycle progression of control
parthenogenetically activated bovine oocytes matured in vitro for
26 h was examined at 5 intervals, from 30 min to 17 h
post-activation. The group of oocytes fixed at 30 min
post-activation provided information as to the resumption of
meiosis I, as an indicator of nuclear maturation status at the time
artificial activation (26 hrs post maturation). Those fixed at the
last interval indicated whether the parthenogenetically activated
oocytes were capable of progressing to pronuclear stage within 17
hrs post-activation. FIG. 8 shows the meiotic status of activated
oocytes observed at 30 min, 1 h, 1.5 h, 5 h and 17 h
post-activation. At 30 min post-activation the meiotic stage of
oocytes ranged from MI to early TII, with most of the oocytes in
MII (71.4%). Progression to AII and then to TII was observed in
oocytes fixed at 1 and 1.5 h post-activation. At 5 h
post-activation the majority of oocytes (80.6%) were in TII, and at
least one PN could be observed in 80.7% of the activated oocytes at
17 h post-activation. The rate of immature oocytes (MI-TI) ranged
from 6.9 to 12.7% between groups.
Meiotic Progression and Induced Enucleation of Demecolcine Treated
Activated Oocytes
[0131] Next, the effect of demecolcine on the meiotic progression
of bovine oocytes after activation was evaluated. Several
treatments were performed to establish an efficient protocol for
demecolcine-induced enucleation of oocytes as described in the
Materials and Methods section above. The results obtained after
demecolcine exposure are shown in FIG. 9 for oocytes fixed at 5 and
17 h post-activation. When demecolcine treatment was initiated 30
min post-activation and lasted for 1 h, 60% of the oocytes resumed
meiosis, a value significantly lower than the estimated 100%
observed in the control group (FIG. 9). Moreover, demecolcine
treatment of oocytes starting at 30 min post-activation and lasting
for 2 or 4.5 h seemed to arrest meiotic progression as low rates of
activation were observed in these oocytes (10.3-16%, FIG. 9). The
remaining groups presented activation rates ranging from 79 to 100%
(FIG. 3), equivalent to the control group.
[0132] Maximal enucleation rates, determined as the number of
activated oocytes that were enucleated at 5 h post-activation, were
observed when oocyte exposure to demecolcine was initiated at 1.5 h
post-activation and lasted for 3.5 h (91%, FIG. 10). Further
culture of oocytes after the withdrawal of the drug induced a
decrease in IE rates as seen when oocytes were fixed at 17 h
post-activation (45 to 65% decrease depending on treatment, FIG.
10). A positive correlation of enucleation rates was observed
between the results obtained from oocytes fixed at 5 and 17 h
post-activation. Thus, demecolcine treatment between 1.5 and 5 h
post-activation also resulted in maximal IE rates when oocytes were
fixed at 17 h post-activation.
[0133] Immunofluorescence analysis of demecolcine treated oocytes
revealed that the microtubule inhibitor was able to disrupt the
meiotic spindle and to enable PB extrusion with all the oocyte's
DNA content. A more intense background was also observed in
demecolcine treated oocytes indicating depolimerization of tubulin,
which could be seen dispersed in the cytoplast. Several ooplasmic
membrane and spindle phenotypes were observed in demecolcine
treated oocytes. Multiple PB extrusion and a variety of different
PB sizes were also observed in the oocytes treated with
demecolcine.
[0134] At 5 h post-activation demecolcine treated oocytes often
showed an acentric localization of the chromosomal plate, which
approached the oocytes plasma membrane. The region of the plasma
membrane right above the chromosomes displayed, in most cases a
"cauliflower" like protrusion, which was repeatedly localized
adjacent to the PB. The reason behind this difference in the
membrane conformation near the chromosomal plate remains unclear
however, we believe it could represent the initiation site for the
oocyte induced enucleation.
[0135] Abnormal spindle shapes, such as multipolar spindles or
aster-like spindle were observed in a minority (less than 1%) of
oocytes treated with the microtubule inhibitor within 5 h
post-activation. The majority of oocytes however did not display a
complete spindle like structure, instead tubulin could be seen
dispersed in the cytoplast and aggregated around the chromosomes,
with the remainder of the spindle contained within the PBs.
In Vitro Development of Reconstructed Chemically Enucleated
Embryos
[0136] Also, the in vitro developmental competence of cytoplasts
prepared by chemical enucleation was evaluated. Nuclear transfer
was performed before (treatment 1) or during (treatment 2) oocytes
exposure to demecolcine, thus confirmation of enucleation prior to
nuclear transfer was not possible. However, in order to access
enucleation of demecolcine treated recipient oocytes, 9 oocytes
from treatment 1 and 10 oocytes from treatment 2 were randomly
chosen after NT, fixed at 7 h post-activation and labeled as
described above. Control IE oocytes that did not receive donor
nuclei were also fixed and monitored. Of these 20% (1/5) were
completely enucleated whereas 60% (3/5) were in pronuclear stage,
indicating incomplete chromosomal extrusion. The remaining oocyte
did not undergo maturation. Complete enucleation and incorporation
of donor nuclei took place in 22% ( 2/9) and 50% ( 5/10) of
treatment 1 and 2 micromanipulated oocytes. Incomplete enucleation
was observed in 67% ( 6/9) and 40% ( 4/10) of treatment 1 and 2
oocytes, respectively. One oocyte of each group did not undergo
activation. Intracytoplasmic injection of the donor nucleus prior
to oocyte activation and treatment with demecolcine yielded 54%
cleavage rates and 19% of blastocyts (Table 5). Lower cleavage and
blastocyts rates (48 and 16%, respectively) were observed when
donor cell nuclei were transferred into the recipient cytoplasts
between 1.5 and 3 h post-activation. Moreover similar in vitro
development was observed in IE activated oocytes (Table 5). High
fragmentation rates were observed in control IE embryos (35%) and
in NTs produced by nuclear injection after activation (treatment 2,
27%). Whereas the embryos produced by NT before activation
displayed lower fragmentation (19%), indicating a possible effect
of timing of nuclear injection on the fate of early embryo
development.
[0137] Table 5. Embryo development after intracytoplasmic injection
of donor bovine fetal fibroblast into recipient oocytes prepared by
chemical enucleation. Donor nuclei were injected 5 to 10 min prior
to activation (treatment 1) or 1.5 to 2 h post-activation
(treatment 2). Control oocytes were parthenogenetically activated
and enucleated by demecolcine treatment (IE). Chromosome make up of
reconstructed (chemically enucleated) embryos. TABLE-US-00005 TABLE
5 Oocytes Embryo development Nuclear Manipulated Cleaved Morula
Blastocyst Fragmented transfer (n) % (n) % (n) % (n) % (n) Control
49 4 26 35 (IE) 47 (23/47) (1/23) (6/23) (8/23) 54 5 19 19
Treatment 107 (58/107) (3/58) (11/58) (11/58) 1 48 12 16 27
Treatment 106 (51/106) (6/51) (8/51) (14/51) 2
[0138] Nuclear transferred and IE control blastocysts were examined
to enumerate the cells according to their ploidy. The ratio of
cells in mitosis to total cell number (TCN) was not significantly
different between the control (43.2%, n=3), treatment 1 (48.8%,
n=8) or treatment 2 (36.3%, n=5) groups. Ninety percent of the
analysed NT embryos showed a majority of diploid (.+-.60)
chromosomal makeup, indicating successful enucleation by the
microtubule inhibitor. Heteroploidy was observed in 100% of the
control embryos (n=4), 62.5% of treatment 1 derived embryos (n=5)
and 16.7% of blastocyst in treatment 2 (n=6). Additionally, 37.5%
and 66.7% of embryos produced by treatment 1 and 2, respectively,
were diploid in all cells analyzed. One embryo derived from
treatment 2 displayed a tetraploid chromosomal make up.
[0139] None of the embryos in treatment 2 had haploid (30
chromosomes), or nearly haploid (.+-.30) cells, whereas 50% ( 4/8)
of the blastocysts in treatment 1 were heteroploid showing a range
from haploid to diploid cells
[0140] No major differences were noted in the mean chromosome make
up between the treatment groups. All blastocysts in the control
group presented an heteroploid nuclear composition.
[0141] FIG. 11 shows the mean distribution pattern with reference
to the type of nuclei (pycnotic, interphase or nuclei with visible
chromosomes) observed in day 8 blastocysts subjected to chemical
enucleation and nuclear transfer (treatment 1 and 2) or chemical
enucleation alone (controls). No significant difference was
observed in any of these parameters between the different
groups.
Discussion
[0142] Microtubules and microfilaments are major cytoskeletal
components that modulate the movement of chromosomes during meiosis
and cell division. A non-invasive method for the preparation of
enucleated oocytes for the production of NT embryos, based on the
chemical disruption of the oocyte's microtubules during the second
meiotic division is described.
[0143] Demecolcine is a specific microtubule inhibitor shown to be
capable of inducing the expulsion of the entire chromosome
complement of an oocyte within the PBs, resulting in enucleated
cytoplasts. This technique causes minimal harm to recipient oocytes
during enucleation, as opposed to the mechanical preparation of
enucleated cytoplast in which 10 to 30% of the oocyte's content is
removed. The oocyte's cytoplasm surrounding the nucleus is believed
to contain important organelles and proteins which are essential
for the maintenance of embryonic developmental competence.
[0144] Demecolcine is a reliable and potent reversible microtubule
inhibitor, which enabled enucleation of up to 91% of the activated
bovine oocytes. Exposure of bovine oocytes to this drug starting at
1 h post-activation or later did not seem to affect the meiotic
progression since the majority of the oocytes (>79%) reached
telophase II stage before 5 h post-activation, and pronuclear stage
before 17 h post-activation. Interestingly a significant decline in
enucleation rates was observed at 17 h after activation, i.e. after
12 h of culture in demecolcine-free media. This decrease could have
been caused by the reintegration of chromosomes into the oocyte
after incomplete extrusion of PBs or by re-fusion of PBs to
enucleated oocytes. Similar results were reported by Elsheikh et
al. (J. Vet Med Sci 59(2):107-13 (1997)) who noted 23% of
spontaneous PB re-fusion in chemically enucleated mouse oocytes.
Moreover, karyotype analysis of parthernogenetically activated
blastocysts treated with demecolcine showed a majority of embryos
with diploid chromosomal make up confirming incomplete PB extrusion
and thus enucleation of treated oocytes. This difficulty could
certainly be overcome by the removal of the second PB after 5 h of
activation, when the chromosomal content of 91% of the treated
oocytes was present in the PBs, minimizing the invasiveness of the
current mechanical enucleation procedure and enabling the removal
of minimal amounts of cytoplast.
[0145] This study shows that the overall developmental performance
of in vitro cultured NT embryos is not affected by the chemical
treatment of the oocytes with demecolcine as evidenced by our in
vitro developmental rates, in which 24-28% of artificially
enucleated oocytes able to cleave and reach the blastocyst stage
following activation and/or nuclear transfer.
[0146] Interestingly, lower rates of embryonic fragmentation were
observed when nuclei were transferred into enucleated cytoplasts
before activation (19 vs 27%). Similar observations were published
by Wakayama and Yanagimachi (Reproduction, 122:49-60 (2001)) based
on nuclear transfer of mouse oocytes before and after activation
using mechanical enucleation.
[0147] As shown above induced enucleation can be accomplished in
bovine oocytes using demecolcine. It is likely that many technical
factors such as oocyte activation, chemical treatments and in vitro
manipulation may affect development of cloned embryos. The low
number of haploid nuclei may indicate a loss of developmentally
incompetent nuclei (haploid) moreover this loss does not seem to
significantly influence development of embryos to blastocyst
stage.
Example 6
Induced Enucleation for SCNT in Bovine Without
Micromanipulation
[0148] By producing competent enucleated cytoplasts without
micromanipulation, IE has the potential for application to large
scale production of viable cloned embryos for basic research and
commercial animal production. Bovine oocytes were first activated
(Act I, 10 .mu.M ionomycin in Tyrodes-Hepes, 5 min) 22-25 hr post
IVM and placed into M199 containing 5% FBS, 10 .mu.g/ml
cycloheximide (CHX), 2 .mu.g/ml cytochalasin D (CD), 0.4 .mu.g/ml
demecolcine (DEME) for 20 min, followed by a second activation (Act
II) and subsequent culture (M199-5% FBS, 10 .mu.g/ml CHX) for 20
min. ZPs were removed by acidified Tyrodes solution, washed and
transferred into M199-5% FBS, 10 .mu.g/ml CHX, 0.4 .mu.g/ml DEME,
2% DMSO and 8.5 mM CaCl.sub.22H.sub.2O. After 1 hour, the extruding
second polar body (PB2) containing all nuclear chromatin is evident
and removed using a hand-held glass pipette (15-20 .mu.m I.D.)
under a stereoscope. Enucleation was assured by Hoechst 33342
staining and UV light. IE cytoplasts were treated with PHA (200
.mu.g/ml) to facilitate adhesion of a donor somatic cell
(fibroblast, passage 3-4). Nuclear transfer (NT) embryos were
reconstructed by electric fusion (150 V/mm, 10 .mu.sec, 1 pulse).
NT embryos were packaged in artificial ZPs (0.4% alginate and 0.1%
CaCl.sub.2.2H.sub.2O) and cultured in BARC medium for 7 days. At
1.6 hr post Act I, 80% of oocytes respond to the double
activation-IE protocol. Of the responding oocytes, an enucleation
rate of 75-90% was achieved. PB removal without manipulation is
rapid taking 2-5 sec to enucleate a single IE oocyte. Culture of IE
NT embryos resulted in 6-23% development to the blastocyst stage,
which was similar to development rates of parthenogenetic control
embryos. The results of this study demonstrate that the modified IE
protocol is an effective method to produce cloned, blastocyst-stage
bovine embryos. This method represents a novel strategy for more
efficient somatic cell cloning without the need for
micromanipulations. Further investigation of the cellular
mechanisms and comparison with conventional cloning methods to
produce healthy offspring is warranted, and will reveal the utility
of this novel method.
[0149] This method, by preparing activated zona-free cytoplasts by
IE results in the extrusion of oocyte nuclearchromatin in PB2 and
simple agglutinin-facilitated reconstruction, represents a novel
strategy for more efficient somatic cell cloning without the need
for micromanipulation. Further investigation of the cellular
mechanisms involved, and validation of this approach by comparison
with conventional cloning methods to produce healthy offspring is
ongoing, and will reveal the utility of this novel methodology.
Materials and Methods
[0150] The modified IE method employs double activation
(ionomycin), zona pellucida removal, agglutin-facilitated
reconstruction and in vitro development using an artificial zona
pellucida (ZP). Slaughterhouse-sourced bovine oocytes were first
activated (Act I, 10 mM ionomycin in Tyrodes-Hepes, 5 min) 22-25
hour post IVM and placed into M199 containing 5% FBS, 10 mg/ml
cycloheximide (CHX), 2 mg/ml cytochalasin D (CD), 0.4 mg/ml
demecolcine (DEME) for 20 min, followed by a second activation (Act
II, 10 mM ionomycin) and subsequent culture (M199-5% FBS, 10 mg/ml
CHX) for 20 min. ZPs were removed (acidified Tyrodes solution, pH
1.79), washed and transferred into M199-5% FBS, 10 mg/ml CHX, 0.4
mg/ml DEME, 2% DMSO and 8.5 mM CaCl.sub.2. After 1 hour, the
extruding second polar body (PB2) containing all nuclear chromatin
is evident and removed using a hand-held glass pipette (15-20 mm
I.D, 100 mm O.D.) under a stereoscope. Enucleation was assured by
Hoechst 33342 staining and UV illumination. IE cytoplasts were
immediately treated with PHA (200 mg/ml) to facilitate adhesion of
a donor somatic cell (primary fibroblast, passage 3-4). Nuclear
transfer (NT) embryos were reconstructed by electric fusion of
couplet units (150 V/mm, 10 msec, 1 pulse) in fusion medium (0.253
M D-Sorbitol, 0.5 mM MgAc, 0.05% PVA). NT embryos were packaged in
artificial ZPs, which were comprised of 0.4% alginate and 0.1%
CaCl2?2H2O, and NT embryos were cultured in BARC medium for 7
days.
Results
[0151] At 1.6 hr post Act I, 80% of oocytes respond to the double
activation-IE protocol. Of the responding oocytes, an enucleation
rate of 75-90% was achieved. PB removal without manipulation is
rapid taking 2-5 sec to enucleate a single IE oocyte. The Culture
of zone-free reconstructed IE NT embryos in artificial zona
resulted in 6-23% development to the blastocyst stage, which was
similar to development rates of parthenogenetic control embryos.
shown (Table 6) demonstrate that double activation can
significantly increase the rate of development to the blastocyst
stage when compared with a single activation. TABLE-US-00006 Stages
of Development to (%) No. No. Cleavage 16 cell to Groups Replicates
Oocytes (2-8) morula Blastocyst control ACT I 11 271 182(67).sup.a
57(21).sup.a 35(13).sup.a control ACT II 8 207 152(73).sup.a
35(17).sup.a 27(13).sup.a IE NT 8 238 152(64).sup.b 37(16).sup.a
36(15).sup.a
[0152] Significant difference b vs. a, p<0.05
[0153] The teachings of all the patents, patent applications and
publications cited herein are incorporated by reference in their
entirety. In particular, U.S. patent application Ser. No.
09/432,906, filed Nov. 2, 1999, entitled, "Methods for Cloning
Animals," by Baguisi et al. is incorporated herein by reference in
its entirety.
[0154] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
* * * * *