Note: Descriptions are shown in the official language in which they were submitted.
3 ~
--2--
FIELD OF THE INVENTION
The present invention is generally directed to
an improved process for cloning or multiplying mammalian
embryos and to an improved process for transferring the
nuclei of donor embryos into enucleated recipient
oocytes, that is, oocytes from which the nuclei have been
removed. The present invention is specifically directed
to enhancing mammalian embryo development after nuclear
transfer by including a maintenance period prior to
fusion which promoted increased intercellular surface
area contact.
DESCRIPTION OF THE PRIOR ART
Advanced genetic improvement and selection
techniques continue to be sought in the field of animal
husbandry. With specific reference to dairy cattle, for
example, significant increases in milk production have
been made with the wide scale use of genetieally superior
sires and artificial insemination. Dairy COW6 today
produce nearly twice as much milk as they did 30 years
ago. Further genetic improvement can be accomplished by
the multiplication of superior or genetically manipulated
embryos by cloning.
It has now becom~ an accepted practice to
transplant embryos in cattle to aid in the production of
genetically superior stock. The cloning of embryos
together with the ability to transplant the cloned
embryos makes it possible to produce multiple genetically
identical animals. Embryo cloning is the proeess of
transferring the nucleus of an embryonic donor cell to an
enucleated recipient ovum or oocyte. The clone then
-3~ 3ti~
develops into a genetically identical offspring to the
donor embryo.
Nuclear transfer was first accomplished in
Amoeba sphaeronucleus in 1939 by Comandon and de Fonbrune
("Greffe Nucleaire Totale, Simple ou Multiple, Chez une
Amibe," Soc. Biol. 130: 744, 1939). This was followed in
1952 by successful nuclear transfer in Rana PiPiens by
Briggs and King ("Transplantation of Living Nuclei from
Blastula Cells into Enucleated Frogsl Eggs," Zooloqy 38:
455-463, 1952). The procedure for successful nuclear
transfers, according to Briggs and King (supra), included
the following:
l) activation of a recipient oocyte:
2) enucleation, i.e., the process of removing or
inactivating the chromosomes from the recipient oocyte;
and
3) transfer of a whole blastomere (a cell resulting
from embryo cleavage prior to gastrulation) with a
nucleus, from a blastula or early gastrula stage embryo
back to the enucleated oocyte.
Embryo cloning has now been successfully
performed in cattle, sheep, rabbits, pigs and mice
(Prather, R.S. et al., 1987, Biol. ReProd., 37: 859-866;
~'illadsen, S.M., 1986, Nature, 320: 63-65; Stice, S.L.,
and J.M. Robl, 1989, Biol. Reprod., 39: 657-664; Prather,
R.S. et al. r 1989, Biol. Reprod., 41: 414-418; Tsunoda,
Y. et al., :L987~ J. Ex~. Zool., 242: 147-151).
Elsdale et al. ("A Description of the Technique
for Nuclear Transplantation in XenoPus laevis," J.
Embrvol. EXP. MorPh., 8(4): 437-444, 1960), utilized
ultraviolet irradiation to, in one step, inactivate thP
egg pronucleus and activate the unfertilized oocyte. In
the axolotl, activation was reported by electrical shock
with chromosomes of the egg nucleus being eliminated by
ultraviolet irradiation, (Briggs, R., et al. r
"Transplantation of Nuclei of Various Cell Types from
Neurulae of the Mexican Axolotl (Ambvstoma meXlCanUm),"
J . ~ C .~
Develop. Biol. 10: 233, 1964). Transfer of a whole
blastomere containing a nucleus into the enucleated
oocyte via a small bore micropipette was the common
method of nuclear transfer for all these techniques.
Two techniques have been used for nuclear
transfer in the mouse. Illmensee and Hoppe used a
totally surgical method in which a micropipette was
inserted through the plasma membrane and into the
cytoplasm of a pronuclear stage embryo for pronuclear
removal and subsequent insertion of donor nuclei
(Illmensee, K~ and Hoppe, P.C., "Nuclear Transplantation
in Mus musculus: Development Potential of Nuclei from
Preimplantation Embryos," Cell 23: 9, 1981). McGrath and
Solter reported a nondisruptive method of transplanting
nuclei (McGrath, J. and Solter D., "Nuclear
Transplantation in the Mouse Embryo by Microsurgery and
Cell Fusion," Science, 220: 1300, 1983)~ Nuclei were
removed as membrane bounded pronuclear karyoplasts
without penetrating the plasma membrane of the embryo.
The nucleus was inserted into a recipient cell by cell
fusion, using Sendai virus as the fusigenic agent. A
small volume of Sendai virus suspension was aspirated
after removal of the donor nucleus and the virus
suspension and the pronuclear karyoplasts were injected
sequentiall~y into the perivitelline space of the
recipient embryo. At best, the microsurgical method of
Illmensee and Hoppe (supra) was about 30-40% e~icient,
whereas the nondisruptive method of McGrath and Solter
(su~ra) was greater than 90% efficient. These techniques
have been successful in producing blastocyst stage
embryos which do not continue development to term.
Reports that Illmensee and Hoppe produced three live mice
have been questioned.
It was later reported that blastocyst stage
embryos and mice were produced by transferring nuclei
into enucleated pronuclear zygotes only when the donor
cell stage was also pronuclear or at a very early two-
2~3~8
--5--
cell stage (McGrath, J. and Solter, D., "Inability of
Mouse ~lastomere Nuclei Transferred to Enucleated Zygotes
to Support Development In Vitro," Science, 226: 1317-
1319, 1984; Surani, M.A.H. et al., "Nuclear
Transplantation in the Mouse: Heritable Differences
Between Paternal Genomes after Activation of the
Embryonic Genome." Cell, 45: 127-136; 1986; and Robl,
J.M. et al., "Nuclear Transplantation in Mouse Embryos:
Assessment of Recipient Cell Stage," Biol. Reprod., 34:
733-739, 1986).
While cloning procedures have been successful
for a variety of species, the embryo development after
nuclear transfer is lower than non-manipulated
contemporaries (Bondioli, K.R. et al. 1990, "Production
of Identical Bovine Offspring by Nuclear Transfer,"
Therioaenoloay, Vol. 13, No. 1, pgs. 165-174).
Embryo development after nuclear transfer has
been improved by various procedures invoIving arresting
the embryo cytoskeleton with a cytochalasin B culture
during cell fusion. Cytochalasin causes the embryo
cytoskeleton to become more elastic, making the embryo
more conducive to cell fusion (McGrath, J. and Solter,
D., 1983, supra.). It is believed that periods of
cytoskeletal repair before cell fusion may increase
subsequent embryo development.
SUMMARY OF THE INVENTION
The present invention is directed to a
technique which improves known methods of producing
cloned mammalian embryos by transferring a nucleus from a
donor embryo to a recipient oocyte. The improved cloning
method includes isolating a donor membrane-bounded
nucleus from a cell of a donor embryo, removinq the
nuclear chromosomal material from an oocyte to create an
enucleated recipient oocyte, maintaining the donor
membrane-bounded nucleus and the enucleated recipient
oocyte for a period of time sufficient to increase
~ - .
-6- ~ 8
intercellular surface area contact between the donor
membrane-bounded nucleus and the enucleated recipient
oocyte, and fusing the membranes of the donor membrane-
bounded nucleus and the enucleated recipient oocyte
together to form an embryonic single cell with a nucleus
from the donor embryo, referred to herein as a "nuclear
transfer embryo" or an "NT". The improvement of this
invention lies in the maintenance period prior to fusion.
By maintaining the nuclells of the membrane-
bounded donor embryo and the enucleated recipient oocyte
in a maintenance medium for an elongated period of time,
typically 24 - 52 hours after oocyte aspiration from
ovarian follicles, the nucleus and oocyte undergo a
physiological change which enhances the ability of each
of the membranes to fuse. This results in increased
fusion and developmental rates.
Further objects, features and advantages of the
present invention will be apparent from the following
detailed description and appended claims.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to an
improvement in the series of steps which collectively
result in the cloning of mammalian embryos by nuclear
transplantation. Although it is contemplated that the
embryo cloning procedure of the present invention may be
utilized on a variety of mammals, the procedure will be
described with reference to the bovine species. However,
the present invention does not restrict the cloning
procedure to bovine embryos.
The cloning procedure includes a non-disruptive
method of removing the nucleus from a mature recipient
oocyte and isolating a nucleus from a donor embryo,
bounded by a membrane, either by removal of the nucleus
3~ from the donor embryo or by isolating a blastomere
itself. The nucleus is then positioned so it is adjacent
-7-- ~ é~ 8
to the recipient oocyte and the nucleus is fused with the
recipient cell to form an embryonic single cell.
The standard embryo cloning process follows a
basic six step procedure:
1) selecting a proper recipient oocyte and a
multicellular embryo donor for nuclear transfer
2) isolating a blastomere from the donor;
3) enucleating, i.e., removing the nuclear material
from the recipient oocyte;
4) introducing the membrane-bounded nucleus of the
blastomere adjacent to the enucleated recipient oocyte;
5) orienting the nucleus and the recipient oocyte
for cell fusion; and
6) fusing the membrane surrounding the nucleus to
the membrane of the recipient oocyte and activatiny the
recipient oocyte by dielectrophoresis.
The overall procedure disrlosed herein may be
described as cloning or as multiplication of embryos by
nuclear transfer followed by a prolonged maintenance
period to increase fusion and developmental rates of
multiple genetically identical embryos, and ultimately,
animals.
Donor Cell EmbrYos
The donor embryos may be obtained by flushing
from surgically recovered oviducts or may be
nonsurgically flushed from the uterus in manners known to
the art. The donor embryo is selected prior to any
significant cellular differentiation. In other words,
totipotent embryonic stem cells are used, which are
defined as those cells still capable of differentiation
to eventually form all the required embryonic cell typesO
While not wishing to be limited to a particular cell
division stage, a preferred donor embryo would have a
development of from 2-64 cells. It is, however, within
the scope of the present invention to include donor
embryos at later stages of development as long as
~3~g8
--8--
significant cell differentiation has not occurred in the
donor embryo being used. For example, the inner cell
mass of a later stage embryo may remain totipotent (that
is, may not have undergone significant cellular
differentiation) whereas the outer cells of the same
embryo may have begun differentiation to an extent where
they are not totipotent and not suitable for use here.
Methods of in vitro maturation, fertilization
and development also can be used to generate donor
embryos. Donor embryos at the 16-64 cell stage are
sometimes referred to as morula rather than blastula.
Nevertheless, for convenience the term blastula will be
used herein to refer to the embryo, regardless of age,
and the term blastomere will be used to refer to a single
cell from any such embryo.
The nucleus of the donor embryo should be
membrane-bounded to be used optimally in the procedure.
Such a membrane-bounded nucleus may either consist of an
entire blastomere or may consist of a karyoplast, which
is an aspirated cellular subset including a nucleus and a
small amount of cytoplasm bounded by a plasma membrane.
Micromanipulation of the embryos is performed
in a manner similar to the methods of McGrath and Solter
tsupra.), which is incorporated herein for details of the
micromanipulation technique. Micromanipulation is
performed using a cell holding pipette, having an outer
diameter of about 120 to 180 micrometers and an inner
diameter of approximately 25 to 35 micrometers, and a
beveled, sharpened enucleation and transfer pipette
having an outer diameter of approximately 10 to 45
micrometers, depending upon the size of the blastomere.
The donor embryos may optionally be treated
with cytochalasin B. The nuclei of the donor embryos are
prepared for transfer either by aspirating a part of the
blastomere which contains the nucleus, thus creating a
karyoplast, or by aspirating the entire blastomere.
Aspirating the entire blastomere is preferred.
9 ~ 3 ~
Culture and Maintenance Media
There are a vaxiety of embryo culture and
maintenance media routinely used for the collection of
embryos, and specifically bovine embryos. Examples of
known media, which may be used for bovine embryo culture
and maintenance, include Ham's F-10 + 10% fetal calf
serum, Iissue Culture Medium-199 (TC~ 199) + 10% fetal
calf serum, Tyrodes's-Albumin-Lactate-Pyruvate,
Dulbecco's Phosphate Buffered Saline, Eagle's and
Whitten's media. One of the most common media for the
collection and freezing of embryos is Dulbecco's
Phosphate Buffered Saline (PBS) incorporating 1 to 20%
fetal calf serum, new born serum or steer serum. If the
embryos are to be kept in an embryo culture medium for a
substantial period of time, the PBS is normally
supplemented with 10 to 20% serum.
Eyestone, et al., "Culture of One- and Two-Cell
Bovine Embryos to the Blastocyst Stage in the Ovine
Oviduct", Therioqenoloqv, Vol. 28, pp. 1-7 (1987),
reported that ligated ovine oviducts would support
development of bovine embryos from the l-cell to
blastocyst stage. Pregnancies and live calves were
produced after transfer of cultured embryos to recipient
heifers. Cultures of 1- and 2-cell embryos in the
oviducts of intact cycling, ovariectomized or anestrous
ewes produced morphologically normal morulae and
blastocysts followed by pregnancies in recipient heifers,
suggesting that ovarian activity was not required for
normal embryo development in the oviduct.
Another embryo culture and maintenance medium
is described in parent patent application serial number
07/558,969 to Rosenkrans, Jr. et al., entitled "Bovine
Embryo Medium," which is incorporated herein by
reference. This embryo medium, named CR1, comprises a
culture solution containing the nutritional substances
necessary to support an embryo and is the preferred
maintenance medium for use with this invention. The
- l o - ~ 8
medium contains hemicalcium L-~actate in amounts ranging
from 1.0 m~, to 10 mM, preferably 1.0 mM to 5.0 mM.
~emicalcium L-lactate is L-lactate with a hemicalcium
salt incorporated thereon. Hemicalcium L-lactate is
significant in that a single component satisfies two
major requirements in the culture medium: 1) the calcium
requirement necessary for compaction and cytoskeleton
arrangement; and 2) the lactate requirement necessary for
metabolism and electron transport. Hemicalcium L-lactate
also serves as valuable mineral and energy source for the
medium necessary for viability of the embryos.
Advantayeously, CRl medium does not contain
serum, such as fetal calf serum, and does not require the
use of a co-culture of animal cells or other biological
media, i.e, media comprising animal cells, e.g.,
oviductal cells. Biological media can sometimes be
disadvantageous in that they may contain trace factors
which may be harmful to the embryos and which are
difficult to detect, characterize and eliminate.
Examples of the main components in CR1 medium
include hemicalcium L-lactate, sodium chloride, potassium
chloride, sodium bicarbonate and a minor amount of fatty-
acid free bovine serum albumin. Additionally, a defined
quantity of essential and non-essential amino acids may
be added to the medium.
Salts are added to the medium to maintain a
proper osmotic pressure or osmolarity of the medium. The
preferred osmotic pressure is 265 milli-osmoles (mOSM).
The concentration of salt generally ranges from 0 Mm to
155 Mm, preferably 110 mM to 115 mM. Examples of salts
include sodium chloride and potassium chloride,
preferably sodium chloride.
The fatty-acid free BSA is added as a
surfactant, i.e., to prevent the embryos from adhering 'o
each other. Unlike culture media known to the art, the
CRl medium requires only a minimal amount of the fatty-
acid free BSA. The fatty-acid free BSA is added in
( S
amounts ranging from 1 mg/ml to 6 mg/ml, preferably 1
mg/ml to 3 mg/ml, and most preferably 3.0 mg/ml.
Antibiotically effective amounts of an agent, such as
gentamicin sulfate, penicillin, streptomycin, fungizone
or other antibiotics known to the art. Additionally, a
cryopreservation agent, known to the art, may be added to
protect the cellular integrity of the bovine embryo
during freezing operations. Further, chemical buffers,
such as HEPES, may by added to maintain the proper pH in
10 the absence of CO2.
CRl medium preferably contains the following
components in the following quantities:
sodium chloride - 114.7 mM
potassium chloride - 3.1 mM
sodium bicarbonate - 26.2 mM
hemicalcium L-lactate - 5 m~
fatty-acid free BSA - 3 mg/ml
Oocyte
The "oocyte," as used here for the recipient
oocyte, means an oocyte which develops from an oogonium
and, following meiosis, becomes a mature ovum. It has
been found that not all oocytes are equally optimal cells
for efficient nuclear transplantation in mammals. For
25 purposes of the present invention, metaphase II stage
oocytes, matured either in vivo or in vitro, have been
found to be optimal. Mature metaphase II oocytes may be
collected surgically from either nonsuperovulated or
superovulated cows or heifers 35 to 48 hours past the
30 onset of estrus or past an injection of human Chorionic
Gonadotrophin (hCG) or similar hormone. Alternaiively,
immature oocytes may be recovered by aspiration from
ovarian follicles obtained from slaughtered cows or
heifers and then may be matured in vitro in a maturation
35 medium by appropriate hormonal treatment and culturing.
As stated above, the oocyte is allowed to mature in a
known maturation medium until the oocyte enters the
-12~ u i~
metaphase II stage, generally 18-24 hours post
aspiration. For purposes o~ the present invention, this
period Or time is known as the "maturation period." As
used herein for calc~1lation of time periods, "aspiration"
refers to aspiration of the immature oocytes from ovarian
follicles.
Mature oocytes can be first treated with
cytochalasin B at about 7.5 micrograms per milliliter, or
an effectively similar microtubal inhibitor at a
concentration sufficient to allow the enucleation and
transfer pipette to be inserted through the zona
pellucida to allow for removal of a portion of the
cytoplasm without, at any point, actually rupturing the
plasma membrane. The mature oocyte is first held in
place by mild suction by the cell holding pipette. The
enucleation and transfer pipette is then inserted through
the zona pellucida of the oocyte at the point of either
the metaphase II bulge or adjacent to the first polar
body, i.e., in a location intended to be adjacent to the
metaphase chromosomes. The pipette does not penetrate
the plasma membrane. Aspiration applied through the
pipette draws a portion of the cytoplasm into the pipette
which includes, in the case of the metaphase II bulge,
the entire bulge surrounding cytoplasm, or, in the case
of the firs1- polar body, the cytoplasm adjacent to the
polar body. This process is intended to draw all the
metaphase chromosomes into the pipette. As the pipette
is withdrawn, with suction maintained, the plasma
membrane is stretched and then seals itself leaving a
competent plasma membrane on the enucleated oocyte.
Maintenance Period
Prior to fusing the membranes of the donor
membrane-bounded nucleus and the enucleated ~ecipient
oocyte together, it has been found that maintaining the
nucleus and the oocyte together in the embryo maintenance
medium significantly improves the development of the
-13-
embryo post fusion. The nucleus and the oocyte are
allowed to remain in the maintenance medium for a period
of about 6-2~ hours, preferably about 10-18 hours, after
the oocyte maturation period to align the oocyte adjacent
the membrane of the nucleus. Calculated from oocyte
aspiration, the maintenance period will be about 24-52
hours, preferably 78-42 hours, post aspiration.
Without wishing to be held to one particular
explanation for the advantages of maintaining the donor
embryo nucleus and the recipient oocyte in a maturation
medium for an extended time prior to fusion, it is
believed that the maintenance period increases the
intercellular surface area contact by the creation of
tiny microvilli on the surface of the donor membrane-
bounded nucleus and the enucleated recipient oocyte.
With the increased amounts of microvilli, there are
increased intercellular connections, i.e., projections
which will attract and adhere the donor embryo nucleus to
the recipient oocyte.
The transfer pipette, carr~ing the aspirated
membrane-bounded nucleus, is then inserted through ~he
zona pellucida of the recipient enucleated oocyte; and
the membrane-bounded nucleus is deposited under the zona
pellucida with lts membrane abutting the plasma membrane
of the recipient oocyte.
Fusion of the Nucleus and the Oocvte
At approximately 24-52 hours, preferably 28-42
hours post aspiration or, stated differently, 6-28 hours,
preferably 10-18 hours, after the nucleus is positioned
such that its membrane is adjacent the enucleated oocyte,
the cell surface contact appears to be at its peak. It
is on this basis that membrane fusion may be enhanced
with the use of the maintenance period of this invention.
The onset of electricity, via the electrofusion
technique, serves to induce the fusion process. Although
electrofusion is preferred, it is within the scope of the
~ 3 ~ ~3 i3 ~ 8
-14-
present invention to utilize other fusion techniques.
For example, fusion can be accomplished using Sendai
virus as a fusigenic agent.
In the preferred embodiment, fusion of the
membrane-bounded nucleus with the enucleated recipient
oocyte and simultaneous activation of the recipient
oocyte are carried out by a single dielectrophoresis step
using commercially available electrofusion equipment
which is described below. Prior to electrofusing the
donor embryo nucleus and enucleated recipient oocyte
together, it is necessary to orient the cell membranes in
the electric field. The term "orientation" as used
herein is defined as the placement of the two cells such
that the plane of contact of the two membranes, i.e., the
plasma membrane of the body carrying the donor nucleus
and the plasma membrane of the recipient oocyte, which
will become fused together, is perpendicular to the
electrical field. It has been found that random
orientation results in a marked reduction in the
s~ccessful fusion rate. If cells are oriented such that
the fusion membranes are parallel, or at approximately a
45~ angle, to the electrical field, the rate of
successful fusion will decrease. The alignment may be
done electrically or mechanically. If the size of the
two cells is not greatly disproportionate, a small
alignment alternating-current voltage (-5 volts per
millimeter at 1000 KHz) for a short time (10 seconds)
will cause the cells to reorient with their membranes
apposed. Repeated pulses may be needed. If the cells
vary greatly in size, mechanical manipulation ~ay be
required to properly orient the membranes.
The actual incorporation of a donor nucleus
into an enucleated oocyte is conducted by a
dielectrophoretic method of cell fusion, using a DC
current and using a non-conductive, i.e., non-ionic,
media such as a mannitol solution, sorbitol or sucrose
(Zimmerman) based cell fusion media. The fusion
~30~
-15-
phenomenon is the result of cell membrane breakdown and
pore formation between properly oriented opposing cells.
The pores, or small channels, created between the two
cells are thermodynamically unstable because of the high
surface curvature of the channels and the associated high
tension in the membrane. This instability causes the
channels to merge and enlarge until the membranes form a
single cell which contains the nucleus from the donor
embryo cell.
Post-Fusion Culture
~he embryonic single-cell clones produced as
described herein preferably are cultured, either in vitro
or in vivo, to the morula or blastula stage. For
example, the clones may be cultured in the oviduct of
sheep, or other suitable animal, or in a suitable culture
medium. The embryos then may be transplanted into the
uteri of cattle at a suitable stage of the estrous cycle.
The procedures for transplantation are commonly known and
practiced in the embryo transfer field. A percentage of
these transplants will initiate pregnancies in the
maternal surrogates. Animals born of these pregnancies
will be genetically identical where the donor cells were
from a single embryo or a clone thereof.
The following example is offered by way of
illustration and not by way of limitation~
EXPERIMENTAL PROCEDURES
Source of Recipient Oocvtes and Donor Em~rvos. The
oocyte-cumulus complexes (OCC) were aspirated from bovine
ovaries recovered from a slaughterhouse in Milwaukee, WI.
The ovaries were transported from the slaughterhouse to
the laboratory in insulated containers with saline (.9%;
30 + 2C). After arrival, the ovaries were washed with
30C water and placed into a flask containing fresh
saline (.9%) in a water bath maintained at 30C.
Follicular contents were harvested by aspiration using an
18-gauge needle and accumulated in a 50 ml sterile
conical tube (Falcon #2070). Following aspiration, OCC
were located using 100 mm plates in a warm room ~30
2C) using a stereomicroscope. The OCC were washed 4
times through low bicarbonate-TALP (Parrish, J. J. et
al., (1988, "Capacitation of Bovine Sperm by Heparin,"
Biol. Re~rod. 38, 1171-1180, incorporated herein by
reference~ and placed into maturation plates (10 OCC per
50 ul drop). Maturation plates consist of ten 50 ul
drops of maturation medium with estradiol-17~ (1 ug/ml),
FSH (.5 ug/ml~ and LH (.5 ug/ml) (Sirard et al., Biol.
Reprod., 39, pgs. 546-552, 1988) in a 60 mm petri dish
with 10 ml of paraffin oil.
Those oocytes not used as recipient oocytes
were fertilized to provide donor embryos in subsequent
experiments. Fertilization of oocytes was performed
according to the procedure described in Sirard et al.
(su~ra., 1988). Briefly, OCC were in vitro matured 22 +
2 hours, then washed 2 times through Sp-TALP (Parrish, J.
J. et al, supra. 1988) and moved into fertilization
plates. Fertilization was performed in 60 mm plates with
10 drops (40 ~l each) of fertilization medium (5 ml TL
Stock-no glucose, 30 mg fatty-free BSA-6 mglml final, 50
~1 pyruvate stock-0.2 mM final, 2.5 ~l gentamicin-25
~g/ml final-optional) covered with oil and 10 OCC for
each drop. The OCC were added, followed by 5 X 104
motile sperm, .2 ug of heparin, and 2 ul of PHE stock (20
~m penicillamine, 10 ~m hypotaurine, 1 ~m epinephrine, 21
~m sodium metabisulfate, and 118 ~m DL-lactate syrup in
.9% sodium chloride according to Leibfried, M. L. and B.
D. Bavister, 1982, "Effect of Epinephrine and Hypotaurine
on in vitro Fertilization in the Golden Hamster," J.
Reprod. Fertil., 66, 87-93).
Motile sperm cells were prepared by the Percoll
separation of frozen sperm procedure. A 45~ percoll
solution (3.090 ml/100 ml KCl, 2.920 ml/100 ml NaH2PO4,
` -17- 2~3~8
4.675 g/100 ml NaCl, 2.380 g/100 ml HEPES, adjust the pH
to 7.3) was prepared by a 1:1 dilution with 90% percoll
using Sperm TL Stock Solution (SPTL) (2.10 mM CaCl22H2O,
3.1 mM KCl, 0.4 mM MgC126H20, 100 mM NaCl, 0.29 mM
NaH2P04H20, 21.6 mM Lactic Acid, 10 mM Hepes, 25 mM
NaHCO3, adjust pH to 7.4 before bringing to final volume;
check osmolarity: 290-300 mOSM) according to Parrish,
J.J. et al., supra. 1988. The SPTL did not contain BSA.
Two - three ml of 90% percoll was placed on the bottom of
a conical tube (Falcon 2095). Two ml of 45% percoll was
layered carefully on the top of the 90% percoll. The
procedure was performed at room temperature. One unit of
semen was thawed (35OC, l min.) and layered on top of the
percoll gradients. The tube was centrifuged at 700 x g
for 30 min. The top layers were removed, and the
concentrated motile sperm was available for use.
Following 44 + 2 hours after insemination, the
oCc were stripped of cumulus cells, and the eggs were
categorized as cleaved (> 2 cells) or uncleaved. The
cleaved eggs were cultured in CRl-L-Glutamine medium for
3 to 5 days until they reach the morula or blastocyst
stage.
',
~ Embryo ~a~d nipulation.
Recipient oocytes were enucleated by aspirating
approximately one-eighth to one-fourth the cytoplasm
juxtaposed to the polar body or the metaphase bulge using
a 10-45 micron transfer pipette, leaving an enucleated
membrane-bounded oocyte. Embryos were manipulated in
calcium and magnesium-free TL Hepes buffered modified
Tyrodes medium prepared according to Bavister et 21.,
"Development of Preimplantation Embryos of the Golden
Hamster in a Defined Culture Medium," Biol. Reprod., 28:
235, 1983). Nuclei from later stage donor embryos were
removed by aspirating the nucleus and some surrounding
membrane-bounded cytoplasm from a blastomere or by
aspirating an entire blastomere. Micromanipulation was
~ ~r
conducted using a holding pipette having an outer
diameter of approximately 120-180 microns anà an inner
diameter of approximately 30 microns and a beveled,
sharpened enucleation and transfer pipette having an
outer diameter of approximately 10 to 45 microns. Whole
blastomeres, containing nuclei, were removed from donor
embryos and positioned in the perivitelline space of the
recipient oocytes by the method of McGrath and Solter
(supra.).
Maintenance Procedures
Embryos were maintained in vitro in CR1-L-
Glutamine medium for 6-18 hours prior to fusion.
Zimmerman Cell Fusion Medium (GCA Corporation, Chicago,
IL), was used for fusing donor and recipient cells.
Cells from donor embryos were washed in the medium then
placed in the fusion chamber with the Zimmerman medium.
Following the fusion treatment, oocytes were placed in
CR1-L-Glutamine medium, in 50 microliter drops, under
paraffin oil in a humidified 5% C02 in air incubator and
monitored for fusion and allowed to develop for five
days.
Activation and fusion of the intact, membrane-
bounded nuclei to the enucleated oocytes were carried out
in Zimmerman Cell Fusion Medium by dielectrophoresis
using a Zimmerman ~lectrofusion Instrument, GCA
Corporation, Chicago, IL. The fusion chamber consisted
of two parallel electrodes 1 mm apart on a glass slide.
The instrument was adjusted in the following manner:
Fusion voltage: 80-120 volts (DC)
Electrode distance: 1 mm
Alignment voltage: 1-5 volts (AC)
Alignment frequency: 1000 KHz
Pulse duration: 10-~0 microseconds
Postfusion alignment time: 5 seconds
Number of Pulses: 1-6
--19-
Experiment ~ ~' $
The experiment was designed to determine
whether the development of nuclear transfer embryos (NTs)
could be improved by maintaining the donor embryo cells
and recipients in maintenance medium for an extended
period o~ time. NTs were produced according to Prather
et al., 1987, Biol. Reprod., 37,859-866, which is
incorporated herein by reference. Specifically, in vitro
matured oocytes were used as recipients, in vitro
developed embryos were used as donors, and the
subsequently formed NTs were developed in vitro in CR1
with 1 mM L-glutamine medium (CR1-L-GLN).
The treatment structure was a blocked one-way
design with 3 treatments. Each donor embryo served as
the block, i.e., every treatment was imposed on donor
embryo cells from each donor embryo. Enucleation and
transfer of donor blastomeres was performed at 24 hours
post-oocyte aspiration. The three treatments were as
follows: 1) fusio~ at 30 hours; Z) those that did not
fuse at 30 hours and were then refused at 42 hours; and
3) fusion at 42 hours. During the manipulation process,
the donor and recipient embryo cells were handled in
calcium and magnesium free TALP-hepes. The cells were
maintained in CR1-L-GLN during the waiting period before
fusion as well as is th~ post-fusion development period.
Embryos were developed in 50 microliter drops of this
maintenance medium under paraffin oil in a humidified
incubator with an atmosphere of 5% C02 in air. This
experiment was replicated on three different days using
three morula stage donor embryos (30-4~ cells each) on
each day of nuclear transfer. The results are
illustrated in the following table:
-20- ~ t ~
Nuclear Transfer - Fusion at 30 Hours
Versus 42 hours
-
~ 1
IFUS~IOIY ACTIV~.TION ¦CLEAVAGE DEVELOPMENT
MORULA +
30 hr _ BLASTOCYST
N 59/104 68/97 19/59 2/59
% 56.9 71 32.0 4 7+3.7b
N 31/42 l9J31 7J31
~ 80.1 6309 22.2+6.7C
N 87/108 80/99 62/87 28/87
% 80.4 81 71.0 32.6+5.3C
a cultured 5 days in CR1 + L-Glutamine (1 mM)
b,c uncommon superscripts differ (P<0.05)
The results indicate that when the fusion
regime is changed to a later time period, i.e., following
the maintenance period of this invention, development of
NTs is simi]ar to that of normal in vitro matured,
fertilized and developed oocytes. Thus, the nuclear
transfer embryo appears to have increased fusion and
developmental rates when fusion is delayed by
approximately 4-18 hours after the time fusion would
normally take place, i.e., 34-48 hours after aspiration
of the recipient oocytes.
It is understood that the invention is not
confined to the pzrticular construction and arrangement
herein described, but embraces such modlfied forms
thereof as come within the scope of the following claims.
