Note: Descriptions are shown in the official language in which they were submitted.
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Applicant/Assignee: Agrobiogen GmbH Biotechnologie
Our ref.: 80035/L1S (AS/LS)
Efficient nuclear transfer with primordial gametes
The present invention relates to a method for breeding animals through cloning
and the animals obtainable by the method. This invention relates in particular
t~ a method
for cloning animals through efficient nuclear transfer with specific foetal
cells.
Animals, in particular economically useful animals, have long been bred and
further developed with respect to specific properties by man for a very wide
range of
purposes. Thus, for example, cows and bulls with a high breeding value for
milk output have
been mated to obtain animals with a high milk yield.
In recent years, animals, in particular members of the ungulates, such as
sheep,
cattle or cows, have become the focal point of research as producers of
substances of
importance in terms of nutrition or pharmaceuticals, since the development of
genetic
I S engineering has made it possible specifically to produce animals to which
it is possible to
impart a novel property, for example the ability to produce a specific drug.
However, the
problem with the commercial use of such animals is that the genetic construct
transferred to
them is passed on to the progeny in an integrated and stable manner.
For this purpose, attempts were made to solve the problem of gene transfer by
performing it in cells, animals being generated again from these cells by
means of cloning.
Among those skilled in the art, the term "cloning" is generally defined as
replication of genetic material, derived from a single cell, which, when
applied to
embryology, may be understood as meaning the creation of embryos or animals of
identical
genotype. In embryology, the germ during blastogenesis, i.e. up to the
development of
primordial organs, is referred to as an embryo, and in the subsequent
development stages as a
foetus. Depending on species, embryonic phases have periods of different
lengths, for
example a period of about 4 weeks in cattle, and shorter or longer periods may
be necessary
for this purpose in other species within the ungulates.
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To date, several routes have been taken for the cloning of animals, i.e. for
the
replication of a genotype peculiar to a specific animal.
On the one hand, early embryonic stages and further developments were
subjected to microsurgery and the parts isolated therefrom in each case were
grown on in
S vitro or in vivo.
Furthermore, a micromanipulatory combination of asynchronous development
stages, referred to as "chimeral clones" was performed, in which blastomer(s)
from embryos
of more advanced stages were combined with blastomers from earlier stages with
the aim of
supporting the first-mentioned in their further development capacity and thus
producing
identical multiple individuals. However, the largest number of clones obtained
thereby was
only of the order of magnitude of not more than 5-8.
A further procedure was the parthenogenic activation or the mating of
homozygous parent animals in order to obtain clones having specific
properties.
Since, however, the above-mentioned method proved relatively poor in terms
of their effectiveness and reliability, a further method was developed, which
is generally
referred to as nuclear transfer.
Here, cell nuclei which originate from multicellular embryos are transferred
to
appropriately prepared ova, it being possible to create genetically identical
embryos.
To be able to carry out cloning successfully by means of nuclear transfer.
however, some essential parameters must be taken into account.
The ovum used as the recipient cell must have completed the metaphase stage
in the 2nd meiotic division (metaphase II) and should no longer contain any of
its own
nuclear DNA, i.e. it should be present as a so-called enucleated ovum.
Furthermore, the
cytoplasm of the ovum should be influenced as little as possible, since the
substances
contained in the cytoplasm itself may be important for the further
development, for example
the division of the cell.
Moreover, the nuclear DNA of the transferred nucleus must be reprogrammed.
Since the "donor" nucleus originates from a multicellular embryo, the
respective donor cell
has already passed through some division cycles. This means that the cell is
in a stage of
development which is advanced in comparison with a totipotent fertilized ovum
and in which
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specific genes required for the early development may already have been
switched off.
For this reason, the nuclear DNA used must be reprogrammed in such a way
that the complete genetic information of the nuclear DNA is available again
and the division
process of the embryo begins again in the zygote stage. Thus, the better this
reprogramming
S or activation can be achieved, the higher is the probability of successful
cloning, with which
a fully developed, i.e. live-born cloned animal can subsequently also be
obtained.
In addition to the nuclear DNA, inter alia, the mRNA present in the cytoplasm
is also important since, at the instant of union of ovum and donor cell, said
mRNA contains
the messengers required for the present development or differentiation stage
of the donor cell
and the proteins produced therewith can influence the further development of
the cell.
The method of nuclear transfer has already been used with modest success.
Thus, Willadsen et al. (Nature 320 (1986), 63-65) report on the cloning of
lambs, the nuclei
originating from nucleus donor cells from the 8-cell stage. Robl et al. (J.
Anim. Sci. _64
( 1987), 642-647) reported on the first nuclear transfer experiment in cattle,
exclusively cattle
embryos obtained ex vivo being used as nucleus donors. In these experiments,
an in vivo
intermediate culture in sheep's oviducts was always required. In the following
years it was
also possible to show that embryonic cloning in cattle can be carried out
successfully in vitro,
i.e. using embryos produced in vitro and ova matured in vitro (Suns et al.,
Proc. Natl. Acad.
Sci. USA 91 ( 1991 ), 6143-6147).
WO 97/07668 furthermore describes a method for reproducing an animal
embryo, in which in general a nucleus having a diploid chromosome set is
transferred to an
enucleated ovum which is kept in the metaphase stage II, a certain time being
allowed to
elapse before the ovum is activated after introduction of the nucleus. As a
result of the later
activation of the ovum after introduction of the nuclear DNA, it is intended
to achieve
improved reprogramming of the nuclear DNA introduced.
WO 96/07732 likewise describes a method for reproducing an animal embryo,
in which cells from the blastoderm of an embryo are isolated in the blastocyst
stage and
allowed to mature in a suitable environment and their nuclei are then
introduced into suitable
cells.
However, a remaining problem with this technology is that of finding for the
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nuclear transfer suitable donor cells with which it is possible to produce an
animal embryo
most expediently and economically. It is known that the reprogramming of the
nuclear DNA
from the donor cell chosen in each case gives rise to the greatest difficulty
in embryo
cloning, since this influences not only the further early maturing of the
embryo but also the
later development after any implantation into a mother animal. Thus, in spite
of all successes
in this area, there are still problems with regard to effective reprogramming
of the donor
nuclear DNA in order to ensure that the manipulated ovum with the new nucleus
approaches
the state of a natural zygote. This was manifested inter alia in an extremely
low yield with
respect to the production of embryonic blastocysts and a low rate of division.
It is therefore the object of the present invention to overcome the
disadvantages of the prior art and to provide a suitable nucleus donor cell
with which an
improved method for cloning animals can be provided.
This object is achieved by a method for cloning an animal embryo, in which
primordial gametes are used as the donor cell for the nuclear transfer. The
nucleus of this
1 S cell is combined with a suitable recipient cell, and the cell thus
obtained is grown for a period
so that a blastocyst forms. The blastocysts obtainable therefrom can, if
required, then be
introduced into a mother animal for carrying to full term.
According to a preferred embodiment of the present invention, the recipient
cell is an enucleated ovum, into which the nucleus of the primordial gamete
can be
introduced for nuclear transfer or with which the primordial gamete itself is
fused. The
primordial gamete can be obtained from a foetus and used directly or after
long culture.
The animals for which the method according to the invention can be carried
out are, for example, ungulates, rabbits, rodents or birds, ungulates, in
particular pigs, sheep,
goats, cattle or cows, being preferred.
In a preferred embodiment, the primordial gametes used in the method are
transgenic, i.e. they contain one or more genes which are derived either from
an exogenous
source or which constitute an endogenous gene introduced at another nonnatural
locus in the
genome. These genes preferably code for a drug, for example an antibody, or a
nutritionally
interesting substance, for example chymosin or trypsin, it being possible for
the gene in each
case to be under the control of an or of the endogenous or of an exogenous
promoter.
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To obtain primordial gametes, foetuses are obtained from pregnant animals,
and the cells are obtained, for example, by abstraction from the foetus. The
desired
primordial gametes are then selected from the cells obtained from the foetus,
for example by
allowing existing fibroblasts to adhere to the culture vessel, allowing the
gametes to grow on
5 suitable feeder cells or mechanical selection by means of a pipette. Owing
to their
phenotype, primordial gametes can be distinguished from other cells since they
can be
identified as irregularly shaped or round cells which have a slightly
yellowish appearance
and a large nucleus and may exhibit "blebbing" phenomena. Thus, the desired
primordial
cells can then be isolated.
For the following steps, the primordial gametes obtained can be used as such,
or the nucleus can be isolated therefrom and further used.
As a rule, enucleated ova which are matured in vivo or in vitro are used as
recipient cells. Thus, for example, unfertilized ova which were matured in
vitro and from
which the surrounding cumulus cells were removed after reaching metaphase II
can be used.
The recipient cell should preferably have no nuclear DNA of its own. Several
possibilities are available in the prior art for removing the DNA of the ovum,
for example the
division of the ovum into two halves, of which one half no longer has any
nucleus and can be
further used, or exposure to ultraviolet light for destroying the endogenous
cell DNA.
Removal of the nucleus or of the pronuclei or of the metaphase plate by means
of
micromanipulation is also possible. A treatment of the ova prior to the
micromanipulation
with cytochalasin B with subsequent extraction of the cytoplasm present in the
vicinity of the
polar body with the aid of a pipette, for example guided by a Leitz
micromanipulator (Leica,
Bensheim, Germany), has proved preferable. Since the nuclear DNA of the ovum
is at this
time localized in the vicinity of the polar bodies, the enucleation rate in
this method is very
high, at the same time only a small part of the cytoplasm being abstracted.
After the cells involved in each case in the nuclear transfer have been
obtained,
in general two routes can be taken. The nucleus of the primordial gametes is
isolated using
established methods known from the prior art and is introduced into the
prepared recipient
cell, for example by means of injection, or the primordial gamete itself is
fused with the
recipient cell.
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In the case of a fusion, a primordial gamete can be pushed with the aid of a
suitable apparatus, such as a transfer pipette, under the zona pellucida of
the enucleated cell
and deposited there. For integration of the nucleus of the primordial gamete
into the cell
plasma of the ovum, the membrane of the fibroblast is fused with the membrane
of the ovum.
The techniques for the fusion of cells are well known in the prior art, for
example fusion
using the Sendai virus, treatment with PEG (polyethylene glycol), laser fusion
or
electroshock. The last-mentioned method, so-called electrofusion, in which
pores which
permit fusion of the cytoplasm are induced by brief direct current pulses of
about 1 to S
kV/cm, preferably 1 to 3 kV/cm, with a respective duration of 2 psec to 1 sec,
which may be
repeated, for example 2 to 10 times, is preferred in the present method since,
if they are of
suitable strength, the electric pulses can simultaneously result in activation
of the (fused)
ovum. The activation can also be effected a few hours (about 2-5 hours) after
the fusion, for
example by incubation of the fused cell in a 7% alcohol solution, preferably a
7% ethanol
solution, or by means of other methods known in the prior art.
Activation of the fused cell is an important step since it is the prerequisite
for
initiation of the dividing activity of the fusion product. After fusion and
activation are
complete, the gamete-ovum complexes (nuclear transfer embryos) are cultured
until they
reach a stage in which they can, if required, be transferred to a recipient.
If desired,
substances which support or inhibit the aggregation of microtubuli can be
added to the
culture medium used. Nocadazol and colcemid are examples of aggregation-
inhibiting
agents, and taxol is a microtubuli stabilizer. These substances prevent any
formation of a
plurality of pronuclei.
In the existing methods of the prior art, for further culture of the embryos
forming, it was necessary to transfer them carefully to an intermediate
recipient. This was
achieved in general by transferring the embryo, packed in a protective medium,
such as agar,
to the oviducts of an "interim mother animal" (temporary recipient), in which
a further
development until implantation in the (final) mother animal was required. In
the method
according to the invention, however, it is also possible to use existing in
vitro systems for the
cultivation, without adversely affecting the yields. Without being tied to any
theory, this fact
might be associated with a choice of the donor cell with which it is possible
to obtain
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embryos which very closely approach naturally produced embryos with respect to
their
development. The cells are cultured for a period until blastocysts form. This
comprises a
period of up to 10 days, preferably 6 to 7 days.
According to the invention, it is now possible to allow cloned embryos to grow
S into foetuses, which might then be used again as nucleus donors. In this so-
called recloning,
the number of cloned embryos can be further increased.
The primordial gametes for use in the method according to the invention can
be obtained from a multiplicity of animals, such as, for example, from
mammals, ungulates,
rabbits, rodents, such as, for example, rats or mice, or birds, such as, for
example, ducks,
geese or chickens. Inter alia from economic points of view, ungulates are
preferred, such as,
for example, cattle, sheep, goats, buffalo, camels and pigs. Sheep or cows are
most
preferred.
To facilitate the isolation of the gene product, the gene product can be
directed
to a product of the animal itself, for example to the milk in the case of cows
or sheep or to
the eggs in the case of birds. This can be achieved by the choice of suitable
promoters for
organ-specific expression, which are known from the prior art. However, the
gene product
can also be obtained from the animal itself, for example from the serum. It is
also possible
for the organ(s)/tissues of the animals to constitute the desired product, for
example for
(xeno)transplantation.
The primordial gametes used in the method according to the invention or the
foetuses or animals from which they are derived can moreover be transgenic,
the transgene
preferably coding for a nutritionally or pharmaceutically interesting product,
for example an
antibody. For example, in cows or sheep, the gene for chymosin or trypsin can
be
incorporated in a construct which permits the production of the corresponding
enzyme or of
one of its precursors in the animal's milk. The transgene of interest can,
depending on
requirements, be under the control of an exogenous, likewise transgenic
promoter, or a
known endogenous promoter can be used for this purpose.
By means of the method according to the invention, it is now possible to
achieve an improvement in the production of homologous animal proteins, a
modification of
animal products, such as milk itself, or the production of animal organs for,
for example,
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g
medical use.
A large number of proteins have been produced to date from animal organs by
purification from these organs and then used in medicine or technology.
Problems exist,
inter alia, with regard to the relative amounts in which they are present in
these tissues (for
S example FSH from pituitary glands), which leads to high production costs
since on the one
hand a large amount of starting material, i.e. many animals, are required for
production,
which, owing to the large number of animals, entails the danger of
contamination with, for
example, pathogens such as BSE or Ehec.
Examples of interesting proteins obtained from animal organs are aprotinin
from the lung, chymosin from the stomach, catalase from the liver, elastase,
pancreatin,
insulin or trypsin from the pancreas, hyaluronidase from testicles,
chrondroitin from the
trachea, collagens from the skin, fibronectin or vitronectin from plasma,
epithelial cell
growth suppl. or LH (luteinising hormone) from the pituitary gland, f broblast
growth factor
or gangliosides from the brain, and haemoglobin, thrombin, transferrin, etc.
This list should
not be regarded as imposing any restrictions.
For all these products, an ectotopic expression, i.e. an expression in another
tissue, for example in the mammary gland, can be achieved if an additive gene
transfer was
carried out beforehand, for example via injection, transformation,
transfection or by another
method known from the prior art, in the cells used for cloning, a gene
construct recombined
in vitro additionally being integrated into the genome. In addition, by
homologous
recombination in the cells, it is possible to ensure the gene present
endogenously is coupled
with a promoter which gives another expression pattern for this structural
gene, for example
production of chymosin in the udder with associated secretion in the milk
instead of
endogenous synthesis in the stomach. Furthermore, a promoter present
endogenously. for
example the casein or lactoglobulin promoter can be coupled with a new
structural gene so
that optimum conditions are present for the expression. In both above-
mentioned cases.
promoter and structural gene, which are recombined into the genome are
isolated beforehand
from a gene bank which has been obtained, for example, from primordial
gametes, so that the
DNA used is not only endogenous to the species (self cloning) but also
isogenous DNA.
In this way, the composition of foods obtained from animal products, such as,
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for example, milk, can be altered in the desired manner so that they have
positive alimentary,
dietary, health-promoting properties or a lower allergenic potential or better
shelf life or
processing properties. Thus, for example, milk containing Ehec antibodies or
milk having
properties specially tailored to diseases, such as, for example, lactose
intolerance, can be
produced.
In addition, by using MACs (mammalian artificial chromosomes),
integrational mutations can be avoided and large DNA fragments transferred.
These MACs
are replicated as additional mini- or microchromosomes in the nucleus in
exactly the same
way as the endogenous chromosomes. Thus, it is possible, for example, to
transfer gene
clusters beyond the species, for example complete human immunoglobulin gene
clusters to
economically useful animals, and these economically useful animals would then
be capable
of producing human antibodies, which could be recovered and used. The transfer
of specific
MACs from the same species furthermore means that, with cumulative genetic
effects, there
would be an increase in the synthesis of the gene product.
Also important is an expression of homologous proteins or of possibly
transgenic tissues or organs in economically useful animals, in the case of
proteins in the
same organs in which these proteins are also expressed in humans. The proteins
are then
obtained by methods known in the prior art, and the tissues or organs are
removed from the
animal before eventual transplantation. The advantage of this procedure is a
high identity of
the expressed proteins since they are processed or post-translationally
modified in the correct
organ, for example the expression of erythropoietin in the kidney. The result
of this is that
the proteins obtained from the different tissues have the same glycosylation
as the substances
in humans themselves, their activity very closely approaching that of the
natural protein. It is
thus possible to obtain transgenic animals, for example pigs, cattle, etc.,
which produce
human insulin, erythropoietin, etc., which can then be better used in
medicine.
On the basis of the method according to the invention, an economically useful
animal produced transgenically and having, for example, the above-mentioned
properties can
then be propagated in a stable manner.
Thus, efficient production of relatively large clone groups of transgenic
animals can be achieved by the following procedure, as shown schematically
below:
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1. Micromanipulation of zygotes
A prepared gene construct (for example as described in German
Offenlegungsschrift 40 12 526) is introduced into a fertilized ovum and
integrates into the
genome. This is a normal gene transfer with cumulative, random integration of
the gene
5 construct into the genome of the zygote. It is also possible to carry out a
homologous
recombination in which simultaneously an endogenous gene can be eliminated or
an
endogenous promoter/an endogenous gene can be used.
2. in vitro culture
The zygote is cultivated in a suitable medium briefly, i.e. for 1-5 hours or
for
10 several days, for example 1 to 8 days. It can also be transferred
immediately into the oviduct
of a suitable recipient animal.
3. Transfer to recipient
The zygote is transferred into the oviduct or the uterus of a recipient animal
for
further development into an embryo, it being possible to transfer up to 4
embryos.
4. Isolation of the embryo or foetus
The foetus is isolated, for example by sacrificing the recipient animal and
removing the foetus. Other methods of isolating the embryo without sacrificing
the recipient
animal are also possible.
S. Selection for transgenic foetuses
Cells of the embryo are isolated and are investigated with respect to
integration
of the gene construct.
6. Isolation of primordial gametes from the foetuses
The primordial gametes from foetuses which have integrated the gene
construct are isolated and further used.
7. Cloning
The nuclei of transgenic, primordial gametes are combined with suitable
recipient cells (nucleus transfer, fusion) and treated as explained in more
detail here.
8. in vitro culture
The cells are cultured in vitro for a period of about 4 to 10 days.
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9. Transfer to recipient
The cell clusters (blastocysts) forming are again transferred to recipient
animals. Before the transfer, the zona pellucida is slit, which has proved
particularly
suitable.
10. Repetition of the method from 6. to 9.
The present invention also relates to the cloned animals which are obtainable
by the method according to the invention and which, as explained above, may or
may not be
transgenic.
Another advantage of the method according to the invention, in addition to
increased efficiency, i.e. a higher success rate compared with the methods of
the prior art, in
the reproduction of genetically identical embryos, is that larger clone groups
are obtainable.
Thus, an already cloned foetus can be used for producing further clones, since
the foetal cells
can be recovered and efficiently reused in vitro (point 10 of the above-
mentioned method).
Thus, using the method according to the invention, it was possible to obtain
results which were even better than those obtained from punctured oocytes with
subsequent
maturation and fertilization - but without cloning. The higher in vivo
development capacity
observed considerably increases the efficiency of the cloning programmes.
The so-called gravidity rate (gestation rate) can serve as a measure of the
efficiency of such methods and can be determined as the proportion of animals
which have
become pregnant after transfer of embryos cultivated in vitro for 6-7 days to
synchronized
recipient animals. The respective gravidity rates can be determined by
measurement of the
progesterone level, ultrasonic investigations or rectal palpation.
For cattle as an example, the following results were obtained by different
methods:
Gravidity rates:
Ooctye puncture with subsequent IVM and IVF 34%
Embryo cloning (average) 25%
Embryo cloning with primordial gametes > 50%
IVM = in vitro maturation
IVF = in vitro fertilization
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The invention is now explained in detail with reference to the example which
is given merely for illustration and is not intended to restrict the scope of
the invention.
Example
Isolation of primordial gametes in the bovine foetus
Foetuses from uteri of sacrificed female calves or cows were isolated and
placed in PBS (phosphate buffered saline, without Ca2+/Mg2+, with
penicillin/streptomycin,
plus 10% of foetal calfs serum (FCS)) on ice water in the laboratory. The
foetuses were then
washed several times with fresh PBS. Each foetus was divided transversely,
caudally with
respect to the anterior limbs, followed by medial opening of the abdominal
wall and cranial
displacement of liver and intestinal convolution. Thereafter, the now exposed
mesonephros
and the median gonads were isolated and were washed in fresh PBS. The gonads
were
removed by means of forceps and again washed in PBS. They were then incubated
in 0.02%
EDTA for 10 to 20 minutes. Incubation in 0.4% protease (Sigma, P 6911 ) for 3-
8 minutes at
37-39°C achieved the same effect.
The gonad cells were transferred by means of a pipette to culture medium
containing added growth factors (Dulbecco's modified Eagle Medium (Gibco),
supplemented
with 15% of FCS, 2 mM L-glutamine, 10-' mmol B-mercaptoethanol, 2 mmol
nonessential
amino acids, LIF (Leukemia Inhibitory Factor, 1000 units/ml), bFGF (basic
Fibroblast
Growth Factor, 10 ng/ml) and penicillin/streptomycin), a cell culture being
obtained.
Alternatively, the gonads were punctured several times with a 30 G needle
(gauge) and the
cells taken up and released by means of a pipette until a cell suspension was
present. The
cells were then carefully separated off by centrifuging in a tabletop
centrifuge at 1100 rpm
( 160 g) for 4 minutes and then resuspended in culture medium.
The resuspended cells were then cultured in 35 mm Petri dishes at 37 to
39°C
with 5% of C02 in water vapour-saturated air until required for use.
For isolation of the cells for cloning, the cell suspension was transferred to
a 4
cm cell culture dish and kept for 20-22 hours in Dulbecco's modified Eagle
Medium (Gibco)
(supra) at 37-39°C with 5% of C02. 15 minutes prior to the beginning of
cloning, the cells
were treated briefly (1-2 minutes) with a 0.1% trypsin solution (Sigma) to
bring them
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13
completely into suspension.
For further selection with respect to primordial gametes and their
proliferation,
the culture (containing gametes, fibroblasts, erythrocytes, etc.) was then
transferred to feeder
cells. Bovine fibroblasts, untransfected STO cells (ATCC CRL-1503) or
deactivated
S primordial gonads were successfully used as feeder cells. The culture medium
was DMEM
(Gibco, No. 074-2100A) (high glucose), which had been supplemented with 1 S%
FCS, 2
mmol nonessential amino acids, 2 mmol L-glutamine, 10-4 13-mercaptoethanol,
penicillin/
streptomycin, 1000 IU/ml LIF and 10 ng/ml bFGF.
Alternatively, the primordial gametes were transferred selectively from the
cell
culture using a pipette to a new dish, with the result that the number of
"foreign" cells could
be minimized.
The primordial gametes which are to be used as nucleus donors can be
identified in the cell suspension as large ( 15-25 ~,m), irregularly shaped or
round cells which
have a yellowish appearance and a large nucleus and sometimes exhibit
"blebbing"
phenomena. These cells could be selectively isolated.
Additive gene transfer
Gene constructs recombined in vitro, as described in German
Offenlegungsschrift 40 12 526, which is hereby incorporated by reference, are
integrated in a
stable manner by conventional DNA microinjection (Brem G., Transgenic Animals,
Genetic
Engineering of Animals, VCH Weinheim (1993), 83-170) into the nuclei of
isolated
primordial gametes or by known transformation methods (Maniatis et al.,
Molecular
Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 1989). The
detection of the
integration into the cells is performed by means of PCR and/or Southern
Blotting (Maniatis.
above). An expression in well differentiated cells shows that the gene
transfer has been
successful.
Cloning
18-20 hours after the beginning of maturation, bovine oocytes isolated from
the ovary were freed from the cumulus cells surrounding them and were
enucleated within
two hours (Molecular Reproduction and Development 42 ( 1995), 53-57). About 20-
22 hours
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after the beginning of maturation, primordial gametes isolated as above were
transferred by
means of a transfer pipette into the perivitelline space of enucleated oocytes
and the resulting
karioplast-cytoplast complexes (KCC) were each exposed to a double electrical
pulse of 2.1
kV/cm for 10 psec in order to induce fusion. The KCCs were cultivated in Ham's
F-12
Medium (Sigma) with 10% FCS in an incubator. The fusion was assessed 30 to 60
minutes
after the fusion pulse, by microscopic investigation.
24 hours after the beginning of maturation, the KCCs were activated by
incubation for 5 minutes in 7% ethanol and then cultivated for 5 hours in 10
pg/ml
cyclohexamide (Sigma C-7698) and 5 pg/ml cytochalasin B (Sigma, C-6762). The
KCCs
were then transferred in a 100 ~l drop of CR-1 Medium (Rosenkrans and First,
1991) with
10% of oestrus cow serum. The drops were covered with a layer of liquid
paraffin and
cultivated for 7 to 8 days at 39°C in water vapour-saturated atmosphere
comprising 5% of
CO2, 5% of O~ and 90% of N,.
Table: Primordial gametes (PG) as nucleus donors
KCC Fused KCC Division Blastocysts
(%) (%) (%)
PG foetus 139 109 (74%) 77 (71%) 38 (3~%)
50-57 days
PG foetus 143 128 (90%) 81 (63%) 32 (25%)
65-76 days
PG foetus 171 171 (87%) 90 (60%) 30 (20%)
95-105 days
Embryonic 111 108 (97%) 66 (61%) 3 (3%)
blastomers
As is evident from the above Table, it was possible as early as the first
experiment to obtain a rate of division of up to 71% and a blastocyst rate of
up to 35%.
Embryo transfer
CA 02318117 2000-07-14
Management of recipients
The recipients used were female calves which fulfilled the following criteria:
1. Bred in farms where no IBR (bovine Herpes virus Type 1 ) was suspected;
2. serological investigation for BHV-1 antibodies (infectious bovine
5 rhinotracheitis/infectious pustular vulvovaginitis) negative;
3. serological investigation for BVD (bovine virus diarrhoea)/MD antigen
(mucosal
disease) negative;
4. body mass development corresponding to the age ( 13-16 months);
5. sexual maturity reached; in animals which are to carry the embryo to term,
breeding
10 maturity reached;
6. gynaecological examination without pathological results.
Immediately after stabling, all recipients received mineral boli (All Trace,
Ranching Consult GmbH) in order to supplement the selenium, copper and cobalt
supplied,
which experience has shown to be insufficient (Wittkowsi et al., Zur
Selensupplementierung
15 bei Farsen [Supplementation in heifers]; Jahrestagung der
Arbeitsgemeinschaft
Embryotransfer Deutschland (AET-d), 13.06.-14.06.1996, Marktredwitz).
BVD antibody-negative animals were immunized against BVD/MD (Rumilis~,
Intervet) in order to minimize the risk of infection on transfer of embryos
(Modl et al.,
Control of bovine viral diarrhea virus in abbatoir ovaries for in vitro
fertilization (IVF) or
cloning programs. 11 a Reunion A.E.T.E. Hanover, 8-9 September 1995) or after
placing in
stables with unknown BVD status. The animals were fed ad libitum with grass
silage, hay
and straw. Deworming was performed in the spring and autumn with ivermectin
(Ivornec~,
MSD Agvet). Some of the recipients were housed in covered cattle yards (open-
sided cattle
sheds, group size 6 animals) and some in rearing housing.
Preparation of recipients
The embryos prepared in vitro were transferred to recipients with synchronized
menstruation, i.e. the stage of the sexual cycle corresponds to the age of the
embryo to be
transferred. The day of oestrus is regarded as day 0 of the cycle. The oestrus
synchronization was carried out in the dioestrus by a single intramuscular
administration of a
CA 02318117 2000-07-14
16
prostaglandin F2-a-analogue (2.0 ml of Estrumate° Mallinckrodt
Veterinary). Female calves
in which no functional corpus luteum was diagnosable by means of rectal
palpation were not
used for the oestrus synchronization. Experience shows that oestrus occurs 2-3
days after
administration and is assessed on the basis of the oestrus behaviour and of
the finding in the
vaginal examination.
Embryo transfer
The embryos produced in vitro were transferred after culture for 7 days to
suitable recipients. For this purpose, the embryos were identified and
qualitatively assessed,
the zona was slit and said embryos were transferred to a suitable transfer
medium and then
drawn into minipaillettes (Minitiib). The transfer media used were PBS + 10%
of foetal
calfs serum (FCS, Biochrom), Ovum Culture Medium (ICP, New Zealand) + 10% of
FCS or
TL-Hepes + 10% of FCS.
The closed paillettes were stored at 37.8°C in a miniincubator until
the
transfer, which was to take place within about 90 minutes.
1 S The suitability of the recipients was assessed on the basis of the
following
criteria:
The animals were observed in oestrus about 7 days before transfer, and the
asynchronism
should not exceed 24 hours (Hasler et al., Theriogenology 43 (1995), 141-152).
The
presence and the size of a functional corpus luteum were evaluated accordingly
(Assey et al.,
Theriogenology 39 (1993), 1321-1330).
The animals used showed no signs of disease of the genital tract.
After the selection, an epidural anaesthetic (2.0 ml of Lidocain°,
Albrecht) was
given and the external genital organ was carefully cleaned with dry paper.
Thereafter, the
transfer catheter (Minitiib) at body heat was loaded with a paillette and
provided with a
plastic protective sheath (Sanisheath, Minitiib). The transfer was performed
bloodlessly with
rectal monitoring of the cervical passage and of the catheter position into
the apex of the
ipsilateral uterine cornu (Reichenbach et al., J. Reprod. Fertil. 95 (1992),
363-370). The
plastic protective sheath was perforated with the transfer catheter only at
the external os
uteri, in order to avoid entrainment of germs from the vagina into the uterus.
If it was
intended to transfer a plurality of embryos to one recipient, these were
transferred bilaterally.
CA 02318117 2000-07-14
17
For this purpose, the transfer catheter was drawn back into the body of the
uterus, the guide
with the emptied paillette was removed, a new paillette containing embryos)
was pushed
into the catheter and the latter was positioned in the contralateral uterine
cornu. Immediately
after the transfer, all relevant data (vital number of the recipient, origin,
number and quality
of the embryos, etc.) were documented.
Gestation examination
21 days after oestrus, i.e. 14 days after the embryo transfer, an oestrus
check
was performed and the progesterone content of the blood serum was determined.
Values of
less than 0.1 ng/ml are regarded with certainty as indicating the absence of
gestation. At
progesterone values of more than 2.0 ng/ml, gestation can be expected. The
first direct
gestation examination was carried out on about the 35th day by means of
ultrasonics and the
second was carried out manually on about the 42nd day of gestation.
