Note: Descriptions are shown in the official language in which they were submitted.
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PRODUCTION OF TRANSGENIC ANIMALS USING NUCLEAR
TRANSFER AND OOCYTES RECOVERED BY LOPU
Background of the Invention
The field of the invention is the development and propagation of
transgenic animals.
Techniques to generate transgenic animals by the introduction of a
recombinant DNA into zygotes, fetal cells, or oocytes are well known
(reviewed by Wall, Theriogenology 45:57-68, 1996). Methods to develop
transgenic animals carrying a gene fused to a tissue-specific promoter, such
as
milk, are also known (WO 93/25567). The use of transgenic animals carrying
such transgenes makes it possible to produce desired peptides in the animals.
These peptides can be produced in larger quantities and with less expense than
those produced using more traditional methods of protein production in
microorganisms or animal cells. Once transgenic animals are generated by
nuclear transfer, they or their offspring may be used in efficient, large
quantity
production of desired polypeptides. In this context, tissue-specific
expression
and production of proteins has been demonstrated.
Summary of the Invention
The invention features methods for the generation of a transgenic
animal using oocytes recovered through laparoscopic aspiration of follicles
and
nuclear transfer (NT) techniques. This oocyte recovery procedure is also
known as laparoscopic ovum pick up (LOPU), laparoscopic follicle aspiration,
or laparoscopic oocyte aspiration. A transgene may be introduced into the
oocytes by enucleating the oocytes and fusing them with a donor cell
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containing the gene.
Accordingly, in a first aspect, the invention features a method for
generating a non-human transgenic animal containing a desired gene, involving
the steps of:
(a) recovering an oocyte from a donor animal by laparoscopic ovum
pick up;
(b) enucleating the oocyte;
(c) providing a cell containing the desired gene;
(d) fusing the cell with the oocyte to form a fused couplet;
(e) activating the couplet to form a zygote;
(f) transferring the zygote, or a cleaved embryo, morulae, or
blastocyst formed from culturing said zygote, into a recipient animal; and
(g) allowing the zygote, cleaved embryo, morulae, or blastocyst to
develop to term.
In a second aspect, the invention features a method for generating a
non-human transgenic animal containing a desired gene, said method
involving:
(a) recovering an oocyte from a donor animal by laparoscopic ovum
pick up;
(b) activating the oocyte;
(c) enucleating the activated oocyte;
(d) providing a cell containing a desired gene;
(e) fusing the cell with the enucleated activated oocyte to form a
fused couplet;
(f) allowing the couplet to form a zygote;
(g) transferring the zygote, or a cleaved embryo, morulae, or
blastocyst formed from culturing the zygote, into a recipient animal; and
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(h) allowing the zygote, cleaved embryo, morulae, or blastocyst to
develop to term.
In one embodiment of the above aspects of the invention, the cell and
the oocyte are derived from the same animal or different animals. The animals
can be from the same or different breeds. In another embodiment of the above
aspects of the invention, the animal is a ruminant, such as a goat or a sheep.
By "transgenic animal" is meant a non-human animal containing a
transgene.
By "transgene" is meant a DNA sequence introduced into the
germline of a non-human animal by way of human intervention using any of the
methods described herein.
By "tissue-specific expression" is meant the expression within a
specifically desired tissue or a product which is released from a specific
tissue.
As used herein, "tissue-specific expression" refers to the production of a
protein in the milk, urine, or blood specifically.
As referred to herein, by "prepubertal" is meant less than 5 months of
age.
By "laparoscopic ovum pick up" or "LOPU" is meant a procedure
used to recover oocytes.
The invention provides a number of advantages. The LOPU
procedure may be repeated on the same animal for several months without a
reduction in the number of oocytes recovered. Also, no apparent adhesions
develop as a result of LOPU procedures. In addition, the ability to generate a
transgenic animal using oocytes generated from a prepubertal donor is
advantageous because it reduces the time required for propagating generations
of transgenic animals in the case that the same animal is the donor of both
the
oocyte (recovered by LOPU) and the nucleated cell to be used for the nuclear
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transfer process.
Other features and advantages of the invention will be apparent from
the following detailed description, and from the claims.
Brief Description of the Drawing
Figure 1 is a schematic representation of steps involved in the
generation of transgenic animals using oocytes recovered by the LOPU
technique.
Detailed Description
Example 1
Synchronization and Gonadotrophin Stimulation of Goats to be Used as
Donors of Oocytes Recovered by LOPU
Adult Goats: Adult goats may be subjected to LOPU without any hormonal
stimulation. However, higher numbers of oocytes are obtained if donor goats
are synchronized and stimulated with gonadotrophins. Synchronization of
donor goats may be achieved using established protocols known to those skilled
in the art. The following is an example of a synchronization protocol which
may be used.
Intravaginal sponges containing 60 mg of medroxyprogesterone
acetate were inserted into the vagina of donor goats and left in place for 7
to 10
days, with an injection of 125 ~ g cloprostenol given 48 hours before sponge
removal. Typically, for recovery of immature oocytes, the sponge was left in
place until the oocyte collection, while for the recovery of oocytes more
advanced in maturation, the sponge was removed up to 48 hours before the
oocyte collection.
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The priming of the ovaries was achieved using gonadotrophin
preparations including follicle stimulating hormone (FSH), equine chorionic
gonadotrophin (eCG), and human menopausal gonadotrophin (hMG). Any
established regime for superovulation known by those skilled in the art may be
used. The following hormonal regimes are examples of methods which may be
used. A total dose equivalent to 120 mg of NIH-FSH-Pl was given twice daily
in decreasing doses (35 mg/dose on the first day, 25 mg/dose on the second
day) starting 48 hours before sponge removal. Alternatively, 70 mg of NIH-
FSH-P1 may be given together with 400 IU of eCG 36 to 48 hours before
LOPU. The recovered oocytes were then matured in vitro as described in
Example 3.
An alternative strategy for the recovery of oocytes is to aspirate
oocytes which have been matured in vivo. For this purpose it is essential to
control the number of hours between the luteinizing hormone (LH) peak and
the time at which the oocytes are collected. This may be achieved by drug-
induced depletion of the endogenous LH peak. For example, the FSH/LH
contents of the hypophysis may be depleted using gonadotrophin releasing
hormone (GnRH) agonists such as buserelin or deslorelin. Alternatively, the
hypophysis may be made refractory to hypothalamic GnRH using a GnRH
antagonist such as cetrorelix. The desired GnRH agonist/antagonist may be
administered by means of repeated injections, or more appropriately, by means
of drug release devices such as subcutaneous implants or pumps. The GnRH
agonist/antagonist is administered to the donor goats for at least 7 days
prior to
the start of gonadotrophin stimulation, and the treatment is continued until
the
LOPU procedure occurs. Follicular development is then stimulated by means
of administration of gonadotrophins using a similar protocol as described
above.
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Prepubertal Goats: To recover oocytes from prepubertal goats,
synchronization is not required. However, for recovering high numbers of
oocytes, donor goats may need to be stimulated with gonadotrophin. This may
be achieved by applying the same regimes used for superovulation of adult
goats, as described above.
Example 2
Laparoscopic Ovum Pick-Up
Oocytes from donor goats were recovered by aspiration of follicle
contents (puncture or folliculocentesis) under laparoscopic observation. The
laparoscopy equipment used (commercially available from Richard Wolf,
Germany) was composed of a 7 mm telescope, light cable, light source, 7 mm
trocar for the laparoscope, atraumatic grasping forceps, and two 5 mm "second
puncture" trocars. The follicle puncture set was composed of a puncture
pipette, tubing, a collection tube, and a vacuum pump. The puncture pipette
was made using a PVC pipette (5 mm external diameter, 2 mm internal
diameter) and a 20G short bevel hypodermic needle, which was cut to a length
of 5 mm and fixed into the tip of the pipette with instant glue. The
connection
tubing was made of silicon with an internal diameter of 5 mm, and connected
the puncture pipette to the collection tube. The collection tube was a 50 ml
centrifuge tube with an inlet and an outlet available in the cap. The inlet
was
connected to the pipette, and the outlet was connected to a vacuum line.
Vacuum was provided by a vacuum pump connected to the collection tube by
means of PVC 8 mm tubing. The vacuum pressure was regulated with a flow
valve and measured as drops of collection media per minute entering the
collection tube, and was usually adjusted to 50-70 drops/minute.
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The complete puncture set was washed and rinsed ten times with
tissue culture quality distilled water before gas sterilization, and one time
with
collection medium before use. The collection medium was TCM 199
supplemented with 0.05 mg/ml of heparin and 1 % (v/v) fetal calf serum (FCS).
The collection tube contained approximately 0.5 ml of this medium to receive
the oocytes.
The goats were fasted 24 hours prior to laparoscopy. Anaesthesia
was induced by intravenous administration of diazepam (0.35 mg/kg body
weight) and ketamine (5 mg/kg body weight), and maintained with isofluorane
via endotrachial intubation. The animals were restrained in a cradle position
for laparoscopic artificial insemination as described by Evans and Maxwell
(Salomon's Artificial Insemination of Sheep and Goats, Sydney: Butterworths,
1987). The 3 trocars described above were inserted and the abdominal cavity
was filled with filtered air. The ovary surface was visualized and the
follicles
were punctured by pulling the fimbria in different directions with the
grasping
forceps. The needle was inserted into the follicle and rotated gently to
ensure
that as much of the follicle contents as possible were aspirated. After
aspiration of 3 to 5 follicles, the pipette and tubing were rinsed using
sterile
collection media.
Results from LOPU performed on two types of goats, standard dairy
breeds (STD) and dwarf breeds (BELE), receiving hormonal treatments (as
described in Example 1 ) in terms of number of follicles (FL) aspirated, and
cumulus-oocyte complexes (COCs) recovered per donor (average ~ standard
deviation) are presented in Table 1.
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Table 1. COCs Recovered from Goats Given Different Hormonal Treatments
Prior to the LOPU Procedure
Breed Sponge Treatment n avg. FL avg. %
COCs recovery
BELE 10 days FSHx4 12 14.05.3 12.75.6 90.71
BELE 7-10 daysOS* 36h 15 11.25.0 10.05.7 89.29
BELE 7-10 daysOS 48h 14 11.75.4 10.15.6 86.32
STD 7-10 daysOS 36h 17 27.510.8 24.010.7 87.27
STD 7-10 daysOS 48h 14 24.48.9 20.16.9 82.38
STD None None 12 15.95.1 6.32.0 39.62
BELE"= Breed Early Lactate Early
STD= standard breed
OS* = one shot; all the gonadotrophic stimulation was given in a single
injection, either 36 or
48 hours before LOPU
Example 3
Culture and Enucleation of Oocytes Recovered from Goats by LOPU
Oocyte preparation: Cumulus-oocyte complexes (COCs) were recovered
from primed follicles by LOPU. The COCs were washed once in 2 ml of M 199
containing 0.5% BSA, placed into 50 ~,1 drops of maturation medium, covered
with an overlay of mineral oil (Sigma), and incubated at 38.5°C-
39°C in 5%
CO2. The maturation medium consisted of M 199 supplemented with bLH
(0.02 U; Sioux Biochemicals), bFSH (0.02 U; Sioux Biochemicals), estradiol ~3-
17 (1 ~,g/ml; Sigma), sodium pyruvate (0.2 mM; Sigma), kanamycin (50
~,g/ml), and 10% heat-inactivated fetal calf serum (ImmunoCorp), goat serum,
or estrous goat serum. After 23-24 hours of maturation, the cumulus cells were
removed from the matured oocytes by placing the COCs in a 1.5 ml
microcentrifuge tube containing 250 ~,l of EmCare supplemented with
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hyaluronidase (1 mg/ml), and vortexing for 1-2 minutes. The cumulus cells
may be used in subsequent manipulations, for example, gene transfer, as donor
cells for oocytes derived from the same animal or a different animal.
The denuded oocytes were washed in EmCare containing 1 % FCS
and returned to maturation medium. Fifteen to twenty denuded oocytes were
placed into a microdrop (50 ~.1) containing 5 ~ g of the fluorescent DNA dye
Hoeschst 33342 (stock solution 1 mg/ml saline) in 1 ml of EmCare containing 1
% FCS. The oocytes were incubated in the Hoeschst-EmCare solution for 20-
30 minutes at 30°C-36°C.
Manipulation of Oocytes: One manipulation drop (150 ~.l) of EmCare
supplemented with 1 % FCS was placed into a 100 mm Optics dish (Falcon),
centered, and covered completely with mineral oil. Oocytes stained with the
Hoeschst dye were placed into the center of the manipulation drop. Each
oocyte was picked up using the holding pipette and rotated until the polar
body
(PB) was visualized between 3- and 6 o'clock. The edge of the oocyte-
containing polar body was moved into a fluorescent UV light path and the
location of the chromosomes were noted. The oocyte was pulled slightly out of
the UV light path, and the cytoplasm in the area containing the chromosomes
and polar body was removed using the manipulation pipette. The removed
cytoplasm was checked for the presence of chromosomes and the polar body by
moving the pipette into the UV light path. The process was repeated until all
oocytes were enucleated. The enucleated oocytes were then placed into a
droplet of EmCare containing 1 % FCS, and overlaid with 2 ml of mineral oil in
a Falcon 1008 dish. These dishes were kept on a warm surface (30°C-
36°C).
Alternatively, the enucleated oocytes were returned to the maturation drop if
the nuclear transfer procedure was not immediate.
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Results: In 11 sessions, 734 LOPU-derived and in vitro-matured oocytes were
prepared for enucleation. The denuded oocytes were stained with Hoeschst
33342 and their stage of nuclear maturation was observed. Sixty-nine percent
(235/338) of the oocytes matured in maturation medium supplemented with
FCS were in metaphase II (MII), while 86°Io (341/396) of the oocytes
matured
in maturation medium supplemented with goat serum were in MII. A total of
462 karyoplast-cytoplast couplets were produced using the LOPU-derived
enucleated oocytes (462 cytoplasts/576 enucleated MII oocytes; 80%
production).
Isolation of Activated Oocytes: Alternatively, if desired, an activated oocyte
may be used to carry out the present invention. To activate an oocyte, one
would carry out the oocyte preparation and manipulation procedures as
described above. Upon observation of the denuded oocytes stained with
Hoeschst 33342, oocytes which are in the telophase stage of nuclear maturation
are considered to be activated. These oocytes may be selected and fused with a
cell to form a fused couplet which does not require further activation.
Example 4
Transgenes Used for the Generation of Transgenic Goats and the
Production of Heterologous or Homologous Protein in Milk, Urine, or
Blood of the Transgenic Animal
A genetic construct suitable for use in the present invention generally
includes the following elements:
(a) a promoter or transcription initiation regulatory unit;
(b) a transcription termination codon;
(c) DNA encoding a useful protein, or a nucleotide consisting of a
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ribozyme or antisense oligonucleotide;
(d) optionally, a naturally-occurring or synthetic sequence encoding a
signal polypeptide directing the secretion of the recombinant protein from the
cell if secretion is desired; and
(e) optionally, an insulator element (e.g., chicken /3-globin or chicken
lysozyme MARS elements) which may result in a gene dosage effect (i.e., more
copies of the transgene yield increased protein expression) or may allow for
position-independent expression which is a result of the insulating effect
frqm
surrounding chromatin.
Conventional molecular biology methods are used to generate and
assemble the above elements.
Milk-specific expression of a heterologous or homologous protein: Useful
promoters include asl-casein (as described, for example, in U.S. Patent No.
5,304,489), as2-casein, ~3-casein, K-casein, (3-lactoglobulin (as described,
for
example, in U.S. Patent No. 5,322,773), a-lactalbumin, and whey acidic protein
(WAP). If desired, the promoter may be linked to enhancer elements (such as
CMV or SV40) or insulator elements (such as chicken (I-globin).
An example of a DNA expression cassette using the WAP promoter,
for example, as described in WO 92/22644, and insulator elements operably
linked to a heterologous gene (in this case, a gene from a spider encoding
components of spider silk) can be used as illustrated in WO 99/47661A2. This
genetic construct also includes a transcription termination region.
Preferably,
the termination region includes a poly-adenylation site at the 3' end of the
gene
from which the promoter region of the genetic construct was derived. The
heterologous or homologous gene may be either a cDNA or genomic clone
containing introns (all or a subset). If the gene is a cDNA clone, the genetic
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construct preferably also includes an intron which may increase the level of
expression of the particular gene. Useful introns, for example, are those
found
in genes encoding caseins.
Urine-specific expression of a heterologous or homologous protein: Useful
promoters for the urine-specific expression of a heterologous or homologous
protein are those disclosed in PCT/US96/08233, and U.S. Patent No.
5,824,543, such as uroplakins I, II, and III, hereby incorporated by
reference.
The uroplakin II promoter, for example, has been shown to direct the
expression of hGH in the urine of transgenic mice in detectable levels. Other
useful promoters include kidney-specific promoters such as rennin and
uromodulin.
Blood-specific expression of human immunoglobulin: A genetic construct
that directs the blood-specific expression of human immunoglobulin includes
human Ig loci containing plural variable Vh and V~ regions, either as a mini-
locus region or as a large portion of the Ig locus, as described in
PCT/US97/23091, and references cited therein. Such a construct can be
created using, for example, yeast artificial chromosomes (VACS) or
mammalian artificial chromosomes.
Preferably a construct containing the Ig locus or loci is introduced
into a cell in which the endogenous Ig loci have been interrupted and rendered
non-functional. This cell may then be used for embryo reconstruction using
nuclear transfer techniques. This will allow the development of functional,
mature B-cells expressing high affinity antibodies in the animal. Upon
challenge with a specific antigen (e.g., anthrax) human antibodies will be
produced, which may be purified from the plasma of these animals and
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subsequently used for the treatment of animals according to techniques
described in WO 98/24,893, hereby incorporated by reference.
Example 5
Introduction of a Trans ene into a Cell Line from which a Donor Nucleus
is Used in Nuclear Transfer Experiments
In all of the above examples, the genetic construct may be introduced
into a cell type of interest, for example, a fetal fibroblast (using, for
example,
the methods of Cibelli et al., Science 280:1256-1528, 1998) or cumulus cells
(using, for example, the methods of Kato et al., Science 282:2095-2098, 1998)
by a variety of techniques, including electroporation, lipofection, calcium
phosphate transfection, viral infection, and microinjection. Preferably the
transgene is transfected with a selectable marker so selection of cells
containing
the transgene may be achieved. Such selection markers include, but are not
limited to 6418, hygromycin, and puromycin. It may also be desirable for the
transgene to specifically target an area of the genome of the cell by using,
for
example, the Cre-lox system (Melton, Bioessays 16:633-638, 1994; Guo et al.,
Nature 389:40-46, 1997). In all of the examples described above the selected
cell line is used in the subsequent step of fusion with an enucleated LOPU-
derived oocyte.
_Example 6
Nuclear Transfer (Fusion and Activ nd Culture of the Nuclear
Transfer-derived Embryo Culture
Preparation of donor cells by serum starvation to generate GO cells: Fetal
fibroblasts were isolated from day 27 to day 30 fetuses from the dwarf breed
of
goat BELE° (Breed Early Lactate Early). The cells were transfected with
a
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construct containing the enhanced green fluorescent protein (GFP) gene. Four
GFP positive clones (one male and three female, including the donor cell line
BELE-FF3-GFP) and one non-transfected male line (BELE-FF4) were used as
donor cells in nuclear transfer.
Eight days prior to the nuclear transfer, 2.5 x 104 donor cells were
plated in one well of a 24-well plate in 1.5 ml of complete media (DMEM
supplemented with 10% FBS, 0.1 mM ~3-mercaptoethanol, and 0.1 %
gentamycin) and incubated in a humidified atmosphere at 37°C and 5%
CO2.
The next day, fresh complete media was added to the well. Two days later the
media was again replaced with fresh media. Four to eight days prior to nuclear
transfer, the cells were washed twice, placed into low serum media (DMEM
supplemented with 0.5% FBS, 0.1 mM (3-mercaptoethanol, and 0.1%
gentamycin), and returned to the incubator (37°C and 5% CO~) until the
day of
nuclear transfer. Low serum media was replaced with fresh low serum media
every 24-48 hours.
On the day of nuclear transfer the donor cells were prepared as
follows. Thirty minutes before they were needed, the cells were rinsed quickly
with pre-warmed 0.05% trypsin/EDTA, and incubated with 200 ~.1 of the same
solution for 3 minutes in the incubator. The cells were recovered from the
well
and placed into a cryovial with EmCare supplemented with 1% FCS. The cells
were pelleted by centrifugation (875xg for 3 min) and resuspended twice in
EmCare supplemented with 1 % FCS. The final donor cell suspension (500 ~.l
per ml of EmCare containing 1 % FCS) was placed in a 35 mm suspension dish
and the cells were used immediately for nuclear transfer.
Oocyte preparation: Cumulus-ooctyes complexes (COCs) were recovered
from primed follicles by LOPU as described in Example 2.
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Manipulation of Oocytes: The enucleation of LOPU-derived oocytes was
achieved as described in Example 3.
Fusion: A donor cell was picked up with the manipulation tool and slipped
into the perivitelline space. Cell-cytoplast couplets were fused using
electrofusion as soon after enucleation of the oocytes as possible. The
couplets
were moved through dishes containing (i) EmCare supplemented with 1 mg of
BSA/ml; (ii) a 1:l dilution of sorbitol fusion medium (0.25 M sorbitol, 0.1 mM
calcium acetate, 0.5 mM magnesium acetate, 0.1 % bovine serum albumin) and
EmCare; and (iii) sorbitol fusion medium. Groups of four to six couplets were
aligned between the electrodes of a BTX fusion chamber (catalog No. 450) in a
100 mm plate containing sorbitol fusion medium. A brief fusion pulse was
administered by a BTX and optimizer. A typical pulse of 17 .sec at 2.39
kV/cm (90 V peak) was applied.
The couplets were moved through the sorbitol fusion
medium/EmCare solution and the EmCare/BSA solution, and then placed in
microdrops of EmCare supplemented with 1 % FCS. After all couplets had
been exposed to the fusion pulse they were placed into culture drops of the
appropriate medium (SOFM according to Tervit et al. (J. Reprod. Fertility
30:493-497, 1972); G 1 according to Gardner and Lane (Human Reprod.,
Update 3:367-382, 1997); or TCM containing 10% fetal calf serum, and
incubated at 38.5°C-39°C in 5% CO2, 7% OZ, and 88% N2.
After 2-3 hours, the fused couplets were activated using the calcium
ionophore and DMAP method of Susko-Parrish et al. (Biol. Reprod. 51:1099-
1108, 1994) or by application of additional electrical pulses ( 1.26 kV/cm, 80
,sec), followed by incubation in nocodozole or cytochalasin B (Campbell et
al.,
Nature 380:64-66, 1996). After being cultured for 2.5 to 4 hours in DMAP,
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nocodazole, or cytochalasin B, activated nuclear transfer-derived zygotes were
returned to culture drops containing SOFM or G 1. Cleavage development (2-
to 4-cell stages) was observed at 22 hours (the night before embryo transfer)
and 36 hours (the morning of embryo transfer). Nuclear transfer-derived
embryos were transferred into synchronized recipients between days 1 and 12
post fusion (day 0 = day of fusion).
In Vitro Culture: Reconstructed embryos were placed into microdrops of 25
~,1 of G1 or low phosphate (0.35 mM) SOFM embryo culture medium (Gardner
et al., Biol. Reprod. 50:390-400, 1994) under an oil overlay. After 48-72
hours,
cleaved embryos were moved to fresh microdrops of embryo culture medium.
On day 4 or 5 (day 0 = day of fusion) embryos were moved to microdrops of
G2 medium or high phosphate (1.2 mM) SOFM.
Embryo transfer: Nuclear transfer-derived zygotes, or cleaved embryos at the
2- to 8-cell stage were transferred into the oviduct of a synchronized
recipient.
Morulae and blastocysts were transferred into the uterus of a synchronized
recipient. Pregnancies were determined at 30 and 60 days of gestation.
Results: A total of 462 karyoplast-cytoplast couplets received one or more
fusion pulses, with 54% appearing fused and 43% undergoing initial cleavage
(2- to 8-cell stages). A total of 153 nuclear transfer-derived embryos were
transferred into 21 recipients; 21 morula-staged embryos into 4 recipients and
132 cleavage-staged embryos into 17 recipients. Five of the recipients
receiving cleavage-staged embryos were confirmed to be pregnant by
ultrasound. Table 2 summarizes the effects of donor cell source on pregnancy
rates. These results indicate that this system is in fact efficient.
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Table 2. Effect of Donor Cell Source on Pregnancy Rates
Donor cell # of embryosRecipients Offspring% of
line transferred*pregnant/total embryos
(% pregnancy transferred
rate)
BELE-FF4 70 4/8 (50%) 5 7.1
BELE-FF4-GFP 5 0/1 (0%) 0 0
BELE-FF3-GFP 13 I/z (50%) 1 7.6
BELE-FF1-GFP 9 0/2 (0%) 0 0
Kinder-GFP 35 0/4 (0%) 0 0
* cleavage-stage transfers
Example 7
Synchronization of Animals to be Used as Recipients of Nuclear Transfer-
reconstructed Embryos Derived Using Oocytes From LOPU Procedures
Recipients are synchronized by any established regime known by
those skilled in the art. They should be observed on standing heat during the
day that the oocytes are enucleated. The following hormonal protocol is one
example of a method which may be used. Intravaginal sponges containing 60
mg of medoxyprogesterone acetate were inserted into the vagina of recipient
goats and left in place for 7 to 10 days with an injection of 125 ~.g
closprostenol given 48 hours before sponge removal. Sponges were removed
and an injection of 400 IU of eCG was administered on the same day as the
LOPU takes place.
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Example 8
Transfer of Embryos Reconstructed by Nuclear Transfer Using LOPU-
derived Oocytes to Recipient Goats
Reconstructed nuclear transfer embryos were either incubated for a
short period (42-48 hours) or 5 days and then transferred to synchronized
recipient goats. The recipient goats were fasted 24 hours prior to surgery.
Anesthesia was induced by intravenous administration of diazepam (0.35
mg/kg body weight) and ketamine (5 mg/kg body weight), and maintained with
isofluorane via endotrachial intubation.
A laparoscopic exploration was then performed to confirm if the
recipient had one or more recent ovulations/corpora lutea (CL) present in the
ovaries and a normal oviduct and uterus. The laparoscopic exploration was
carried out to avoid performing a laparotomy on an animal which had not
responded properly to the hormonal synchronization protocol and to which an
embryo should not be transferred. If the short culture period is preferred
(overnight following nuclear transfer/fusion), the embryos may be transferred
to the oviduct of recipient goats. For this purpose, a mid-ventral laparotomy
of
approximately 10 cm in length is established, the reproductive tract is
exteriorized, and the embryos are implanted into the oviduct ipsilateral to
ovulation/s by means of a Tomcat catheter threaded into the oviduct from the
fimbria.
If embryos are cultured for 5 days, the resulting morula/blastocyst-
staged embryos may be transferred to the uterus. For this purpose, a mid-
ventral laparotomy of approximately 5 cm in length is established and the
uterine horn ipsilateral to the CLs is exteriorized using a surgical clamp
under
laparoscopic observation. A small perforation is made with an 18G needle in
the oviductal third of the horn, and the embryos are then implanted by means
of
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WO 01/32854 cA o23aa~22 2002-04-22 pCT/US00/29555
a Tomcat catheter threaded into the uterine lumen.
Example 9
Pregnancies and Births
A total of 21 recipients were transferred, of which 5 (24% of
embryos transferred) became pregnant and gave birth to 6 kids. Four of these
pregnancies (5.7% of embryos transferred) were derived from the non-
transfected male line (BELE-FF4), while one (7.6% of embryos transferred)
was derived from a GFP transfected female line (BELE-FF3-GFP). The
BELE-FF4 donor cells used to generate the embryos which were transferred to
recipients, which then became pregnant, were from passage 2 cells maintained
in low serum for 4 days. The BELE-FF3-GFP donor cells which were used to
produce cleavage-stage nuclear transfer-reconstructed embryos which were
then transferred to a donor recipient, which then became pregnant, were
derived from passage 5 donor cells maintained in low serum for 6 days. Four
recipients receiving morula-staged embryos did not become pregnant. The
source of the oocyte (Dwarf/Pygmy or Saanen/Alpine) did not affect the initial
pregnancy rate (P>0.05).
In an additional set of experiments, 155 nuclear transfer-
reconstructed embryos using LOPU-derived oocytes and cumulus, granulosa, or
fetal fibroblast lines as the donor cells were transferred to 15 recipients.
Of
these recipients and other subsequent sets of recipients, 6 kids have been
born.
Of these 6 kids, 2 express biosteel (a spider silk protein) in their milk.
What is claimed is:
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