U.S. patent application number 12/647316 was filed with the patent office on 2010-06-10 for unactivated oocytes as cytoplast recipients for nuclear transfer.
Invention is credited to Keith Henry Stockman Campbell, Ian Wilmut.
Application Number | 20100146654 12/647316 |
Document ID | / |
Family ID | 10779997 |
Filed Date | 2010-06-10 |
United States Patent
Application |
20100146654 |
Kind Code |
A1 |
Campbell; Keith Henry Stockman ;
et al. |
June 10, 2010 |
UNACTIVATED OOCYTES AS CYTOPLAST RECIPIENTS FOR NUCLEAR
TRANSFER
Abstract
A method of reconstituting an animal embryo involves
transferring a diploid nucleus into an oocyte which is arrested in
the metaphase of the second meiotic division. The oocyte is not
activated at the time of transfer, so that the donor nucleus is
kept exposed to the recipient cytoplasm for a period of time. The
diploid nucleus can be donated by a cell in either the GO or G1
phase of the cell cycle at the time of transfer. Subsequently, the
reconstituted embryo is activated. Correct ploidy is maintained
during activation, for example, by incubating the reconstituted
embryo in the presence of a microtubule inhibitor such as
nocodazole. The reconstituted embryo may then give rise to one or
more live animal births. The invention is useful in the production
of transgenic animals as well as non-transgenics of high genetic
merit.
Inventors: |
Campbell; Keith Henry Stockman;
(Midlothian, GB) ; Wilmut; Ian; (Midlothian,
GB) |
Correspondence
Address: |
LAW OFFICE OF SALVATORE ARRIGO
1050 CONNECTICUT AVE. NW, 10TH FLOOR
WASHINGTON
DC
20036
US
|
Family ID: |
10779997 |
Appl. No.: |
12/647316 |
Filed: |
December 24, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09658862 |
Sep 8, 2000 |
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12647316 |
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08803165 |
Feb 19, 1997 |
6252133 |
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09658862 |
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PCT/GB96/02098 |
Aug 30, 1996 |
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08803165 |
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Current U.S.
Class: |
800/18 ;
800/14 |
Current CPC
Class: |
C12N 15/873 20130101;
C12N 15/8771 20130101; A61K 35/54 20130101; C12N 15/8773
20130101 |
Class at
Publication: |
800/18 ;
800/14 |
International
Class: |
A01K 67/027 20060101
A01K067/027 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 31, 1995 |
GB |
GB 9517779.6 |
Claims
1. A method of reconstituting an animal embryo, the process
comprising transferring a diploid nucleus into an oocyte which is
arrested in the metaphase of the second meiotic division without
concomitantly activating the oocyte, keeping the nucleus exposed to
the cytoplasm of the recipient for a period of time sufficient for
the embryo to become capable of giving rise to a live birth and
subsequently activating the reconstituted embryo while maintaining
correct ploidy.
2. A method as claimed in claim 1, in which the animal is an
ungulate species.
3. A method as claimed in claim 2, in which the animal is a cow or
bull, pig, goat, sheep, camel or water buffalo.
4. A method as claimed in any one of claims 1 to 3, in which the
donor nucleus is genetically modified.
5. A method as claimed in any one of claims 1 to 4, wherein the
diploid nucleus is donated by a quiescent cell.
6. A method as claimed in any one of claims 1 to 5, wherein the
recipient oocyte is enucleate.
7. A method as claimed in any one of claims 1 to 6, wherein nuclear
transfer is achieved by cell fusion.
8. A method as claimed in any one of claims 1 to 7, wherein the
animal is a cow or bull and wherein the donor nucleus is kept
exposed to the recipient cytoplasm for a period of from 6 to 20
hours prior to activation.
9. A method as claimed in any one of claims 1 to 8, wherein correct
ploidy is maintained during activation by microtubule
inhibition.
10. A method as claimed in claim 9, wherein microtubule inhibition
is achieved by the application of nocodazole.
11. A method as claimed in any one of claims 1 to 8, wherein
correct ploidy is maintained during activation by microtubule
stabilisation.
12. A method as claimed in claim 11, wherein microtubule
stabilisation is achieved by the application of taxol.
13. A method of preparing an animal, the method comprising: (a)
reconstituting an animal embryo as claimed in any preceding claim;
(b) causing an animal to develop to term from the embryo; and (c)
optionally, breeding from the animal so formed.
14. A method as claimed in claim 13, wherein the animal embryo is
further manipulated prior to full development. of the embryo.
15. A method as claimed in claim 14, wherein more than one animal
is derived from the embryo.
16. A reconstituted animal embryo which is capable of giving rise
to a live birth and is prepared by a method as claimed in any one
of claims 1 to 12.
17. An animal prepared by a method as claimed in any one of claims
13 to 15.
18. An animal developed from an embryo as claimed in claim 16.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. application Ser. No.
09/658,862, filed Sep. 8, 2000, which is a division of U.S.
application Ser. No. 08/803,165, filed Feb. 19, 1997 (now U.S. Pat.
No. 6,252,133), which is a continuation of International
Application No. PCT/GB96/02098, filed Aug. 30, 1996, which claims
the benefit of GB 9517779.6, filed Aug. 31, 1995, in Great Britain,
all of which are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] This invention relates to the generation of animals
including but not being limited to genetically selected and/or
modified animals, and to cells useful in their generation.
[0003] The reconstruction of mammalian embryos by the transfer of a
donor nucleus to an enucleated oocyte or one cell zygote allows the
production of genetically identical individuals. This has clear
advantages for both research (i.e. as biological controls) and also
in commercial applications (i.e. multiplication of genetically
valuable livestock, uniformity of meat products, animal
management).
[0004] Embryo reconstruction by nuclear transfer was first proposed
(Spemann, Embryonic Development and Induction 210-211 Hofner
Publishing Co., New York (1938)) in order to answer the question of
nuclear equivalence or `do nuclei change during development?`. By
transferring nuclei from increasingly advanced embryonic stages
these experiments were designed to determine at which point nuclei
became restricted in their developmental potential. Due to
technical limitations and the unfortunate death of Spemann these
studies were not completed until 1952, when it was demonstrated in
the frog that certain nuclei could direct development to a sexually
mature adult (Briggs and King, Proc. Natl. Acad. Sci. USA 38
455-461 (1952)). Their findings led to the current concept that
equivalent totipotent nuclei from a single individual could, when
transferred to an enucleated egg, give rise to "genetically
identical" individuals. In the true sense of the meaning these
individuals would not be clones as unknown cytoplasmic
contributions in each may vary and also the absence of any
chromosomal rearrangements would have to be demonstrated.
[0005] Since the demonstration of embryo cloning in amphibians,
similar techniques have been applied to mammalian species. These
techniques fall into two categories: 1) transfer of a donor nucleus
to a matured metaphase II oocyte which has had its chromosomal DNA
removed and 2) transfer of a donor nucleus to a fertilised one cell
zygote which has had both pronuclei removed. In ungulates the
former procedure has become the method of choice as no development
has been reported using the latter other than when pronuclei are
exchanged.
[0006] Transfer of the donor nucleus into the oocyte cytoplasm is
generally achieved by inducing cell fusion. In ungulates fusion is
induced by application of a DC electrical pulse across the
contact/fusion plane of the couplet. The same pulse which induces
cell fusion also activates the recipient oocyte. Following embryo
reconstruction further development is dependent on a large number
of factors including the ability of the nucleus to direct
development i.e. totipotency, developmental competence of the
recipient cytoplast (i.e. oocyte maturation), oocyte activation,
embryo culture (reviewed Campbell and Wilmut in Vth World Congress
on Genetics as Applied to Livestock 20 180-187 (1994)).
[0007] In addition to the above we have shown that maintenance of
correct ploidy during the first cell cycle of the reconstructed
embryo is of major importance (Campbell et al., Biol. Reprod. 49
933-942 (1993); Campbell et al., Biol. Reprod. 50 1385-1393
(1994)). During a single cell cycle all genomic DNA must be
replicated once and only once prior to mitosis. If any of the DNA
either fails to replicate or is replicated more than once then the
ploidy of that nucleus at the time of mitosis will be incorrect.
The mechanisms by which replication is restricted to a single round
during each cell cycle are unclear, however, several lines of
evidence have implicated that maintenance of an intact nuclear
membrane is crucial to this control. The morphological events which
occur in the donor nucleus after transfer into an enucleated
metaphase II oocyte have been studied in a number of species
including mouse (Czolowiska et al., J. Cell Sci. 69 19-34 (1984)),
rabbit (Collas and Robl, Biol. Reprod. 45 455-465 (1991)), pig
(Prather et al., J. Exp. Zool. 225 355-358 (1990)), cow (Kanka et
al., Mol. Reprod. Dev. 29 110-116 (1991)). Immediately upon fusion
the donor nuclear envelope breaks down (NEBD), and the chromosomes
prematurely condense (PCC). These effects are catalysed by a
cytoplasmic activity termed maturation/mitosis/meiosis promoting
factor (MPF). This activity is found in all mitotic and meiotic
cells reaching a maximal activity at metaphase. Matured mammalian
oocytes are arrested at metaphase of the 2nd meiotic division
(metaphase II) and have high MPF activity. Upon fertilisation or
activation MPF activity declines, the second meiotic division is
completed and the second polar body extruded, the chromatin then
decondenses and pronuclear formation occurs. In nuclear transfer
embryos reconstructed when MPF levels are high NEBD and PCC occur;
these events are followed, when MPF activity declines, by chromatin
decondensation and nuclear reformation and subsequent DNA
replication. In reconstructed embryos correct ploidy can be
maintained in one of two ways; firstly by transferring nuclei at a
defined cell cycle stage, e.g. diploid nuclei of cells in G1, into
metaphase II oocytes at the time of activation; or secondly by
activating the recipient oocyte and transferring the donor nucleus
after the disappearance of MPF activity. In sheep this latter
approach has yielded an increase in the frequency of development to
the blastocyst stage from 21% to 55% of reconstructed embryos when
using blastomeres from 16 cell embryos as nuclear donors (Campbell
et al., Biol. Reprod. 50 1385-1393 (1994)).
[0008] These improvements in the frequency of development of
reconstructed embryos have as yet not addressed the question of
nuclear reprogramming. During development certain genes become
"imprinted" i.e. are altered such that they are no longer
transcribed. Studies on imprinting have shown that this
"imprinting" is removed during germ cell formation (i.e.
reprogramming). One possibility is that this reprogramming is
affected by exposure of the chromatin to cytoplasmic factors which
are present in cells undergoing meiosis. This raises the question
of how we may mimic this situation during the reconstruction of
embryos by nuclear transfer in order to reprogram the developmental
clock of the donor nucleus.
BRIEF DESCRIPTION OF THE DRAWING
[0009] FIG. 1 depicts the maturation of oocytes.
DETAILED DESCRIPTION OF THE INVENTION
[0010] It has now been found that nuclear transfer into an oocyte
arrested in metaphase II can give rise to a viable embryo if normal
ploidy (i.e. diploidy) is maintained and if the embryo is not
activated at the time of nuclear transfer. The delay in activation
allows the nucleus to remain exposed to the recipient
cytoplasm.
[0011] According to a first aspect of the present invention there
is provided a method of reconstituting an animal embryo, the method
comprising transferring a diploid nucleus into an oocyte which is
arrested in the metaphase of the second meiotic division without
concomitantly activating the oocyte, keeping the nucleus exposed to
the cytoplasm of the recipient for a period of time sufficient for
the reconstituted embryo to become capable of giving rise to a live
birth and subsequently activating the reconstituted embryo while
maintaining correct ploidy. At this stage, the reconstituted embryo
is a single cell.
[0012] In principle, the invention is applicable to all animals,
including birds such as domestic fowl, amphibian species and fish
species. In practice, however, it will be to non-human animals,
especially non-human mammals, particularly placental mammals, that
the greatest commercially useful applicability is presently
envisaged.
[0013] It is with ungulates, particularly economically important
ungulates such as cattle, sheep, goats, water buffalo, camels and
pigs that the invention is likely to be most useful, both as a
means for cloning animals and as a means for generating transgenic
animals. It should also be noted that the invention is also likely
to be applicable to other economically important animal species
such as, for example, horses, llamas or rodents, e.g. rats or mice,
or rabbits.
[0014] The invention is equally applicable in the production of
transgenic, as well as non-transgenic animals. Transgenic animals
may be produced from genetically altered donor cells. The overall
procedure has a number of advantages over conventional procedures
for the production of transgenic (i.e. genetically modified)
animals which may be summarised as follows:
[0015] (1) fewer recipients will be required;
[0016] (2) multiple syngeneic founders may be generated using
clonal donor cells;
[0017] (3) subtle genetic alteration by gene targeting is
permitted;
[0018] (4) all animals produced from embryos prepared by the
invention should transmit the relevant genetic modification through
the germ line as each animal is derived from a single nucleus; in
contrast, production of transgenic animals by pronuclear injection
or chimerism after inclusion of modified stem cell populations by
blastocyst injection produces a proportion of mosaic animals in
which all cells do not contain the modification and may not
transmit the modification through the germ line; and
[0019] 5) cells can be selected for the site of genetic
modification (e.g. integration) prior to the generation of the
whole animal.
[0020] It should be noted that the term "transgenic", in relation
to animals, should not be taken to be limited to referring to
animals containing in their germ line one or more genes from
another species, although many transgenic animals will contain such
a gene or genes. Rather, the term refers more broadly to any animal
whose germ line has been the subject of technical intervention by
recombinant DNA technology. So, for example, an animal in whose
germ line an endogenous gene has been deleted, duplicated,
activated or modified is a transgenic animal for the purposes of
this invention as much as an animal to whose germ line an exogenous
DNA sequence has been added.
[0021] In embodiments of the invention in which the animal is
transgenic, the donor nucleus is genetically modified. The donor
nucleus may contain one or more transgenes and the genetic
modification may take place prior to nuclear transfer and embryo
reconstitution. Although microinjection, analogous to injection
into the male or female pronucleus of a zygote, may be used as a
method of genetic modification, the invention is not limited to
that methodology: mass transformation or transfection techniques
can also be used e.g. electroporation, viral transfection or
lipofection.
[0022] In the method of the invention described above, a diploid
nucleus is transferred from a donor into the enucleated recipient
oocyte. Donors which are diploid at the time of transfer are
necessary in order to maintain the correct ploidy of the
reconstituted embryo; therefore donors may be either in the G1
phase or preferably, as is the subject of our co-pending PCT patent
application No. PCT/GB96/02099 filed today (claiming priority from
GB 9517780.4), in the GO phase of the cell cycle.
[0023] The mitotic cell cycle has four distinct phases, G, S, G2
and M. The beginning event in the cell cycle, called start, takes
place in the G1 phase and has a unique function. The decision or
commitment to undergo another cell cycle is made at start. Once a
cell has passed through start, it passes through the remainder of
the G1 phase, which is the pre-DNA synthesis phase. The second
stage, the S phase, is when DNA synthesis takes place. This is
followed by the G2 phase, which is the period between DNA synthesis
and mitosis. Mitosis itself occurs at the M phase. Quiescent cells
(which include cells in which Quiescence has been induced as well
as those cells which are naturally quiescent; such as certain fully
differentiated cells) are generally regarded as not being in any of
these four phases of the cycle; they are usually described as being
in a GO state, so as to indicate that they would not normally
progress through the cycle. The nuclei of quiescent GO cells, like
the nuclei of G1 cells, have a diploid DNA content; both of such
diploid nuclei can be used in the present invention.
[0024] Subject to the above, it is believed that there is no
significant limitation on the cells that can be used in nuclear
donors: fully or partially differentiated cells or undifferentiated
cells can be used as can cells which are cultured in vitro or
abstracted ex vivo. The only limitation is that the donor cells
have normal DNA content and be karyotypically normal. A preferred
source of cells is disclosed in our co-pending PCT patent
application No. PCT/GB95/02095, published as WO 96/07732. It is
believed that all such normal cells contain all of the genetic
information required for the production of an adult animal. The
present invention allows this information to be provided to the
developing embryo by altering chromatin structure such that the
genetic material can re-direct development.
[0025] Recipient cells useful in the invention are enucleated
oocytes which are arrested in the metaphase of the second meiotic
division. In most vertebrates, oocyte maturation proceeds in vivo
to this fairly late stage of the egg maturation process and then
arrests. At ovulation, the arrested oocyte is released from the
ovary (and, if fertilisation occurs, the oocyte is naturally
stimulated to complete meiosis). In the practice of the invention,
oocytes can be matured either in vitro or in vivo and are collected
on appearance of the 1st polar body or as soon as possible after
ovulation, respectively.
[0026] It is preferred that the recipient be enucleate. While it
has been generally assumed that enucleation of recipient oocytes in
nuclear transfer procedures is essential, there is no published
experimental confirmation of this judgement. The original procedure
described for ungulates involved splitting the cell into two
halves, one of which was likely to be enucleated (Willadsen Nature
320 (6) 63-65 (1986)). This procedure has the disadvantage that the
other unknown half will still have the metaphase apparatus and that
the reduction in volume of the cytoplasm is believed to accelerate
the pattern of differentiation of the new embryo (Eviskov et al.,
Development 109 322-328 (1990)).
[0027] More recently, different procedures have been used in
attempts to remove the chromosomes with a minimum of cytoplasm.
Aspiration of the first polar body and neighbouring cytoplasm was
found to remove the metaphase II apparatus in 67% of sheep oocytes
(Smith & Wilmut Biol. Reprod. 40 1027-1035 (1989)). Only with
the use of DNA-specific fluorochrome (Hoechst 33342) was a method
provided by which enucleation would be guaranteed with the minimum
reduction in cytoplasmic volume (Tsunoda et al., J. Reprod. Fertil.
82 173 (1988)). In livestock species, this is probably the method
of routine use at present (Prather & First J. Reprod. Fertil.
Suppl. 41 125 (1990), Westhusin et al., Biol. Reprod. (Suppl.) 42
176 (1990))
[0028] There have been very few reports of non-invasive approaches
to enucleation in mammals, whereas in amphibians, irradiation with
ultraviolet light is used as a routine procedure (Gurdon Q. J.
Microsc. Soc. 101 299-311 (1960)). There are no detailed reports of
the use of this approach in mammals, although during the use of
DNA-specific fluorochrome it was noted that exposure of mouse
oocytes to ultraviolet light for more than 30 seconds. reduced the
developmental potential of the cell (Tsunoda et al., J. Reprod.
Fertil. 82 173 (1988)).
[0029] As described above enucleation may be achieved physically,
by actual removal of the nucleus, pro-nuclei or metaphase plate
(depending on the recipient cell), or functionally, such as by the
application of ultraviolet radiation or another enucleating
influence.
[0030] After enucleation, the donor nucleus is introduced either by
fusion to donor cells under conditions which do not induce oocyte
activation or by injection under non-activating conditions. In
order to maintain the correct ploidy of the reconstructed embryo
the donor nucleus must be diploid (i.e. in the GO or G1 phase of
the cell cycle) at the time of fusion.
[0031] Once suitable donor and recipient cells have been prepared,
it is necessary for the nucleus of the former to be transferred to
the latter. Most conveniently, nuclear transfer is effected by
fusion. Activation should not take place at the time of fusion.
[0032] Three established methods which have been used to induce
fusion are:
[0033] (1) exposure of cells to fusion-promoting chemicals, such as
polyethylene glycol;
[0034] (2) the use of inactivated virus, such as Sendai virus;
and
[0035] (3) the use of electrical stimulation.
[0036] Exposure of cells to fusion-promoting chemicals such as
polyethylene glycol or other glycols is a routine procedure for the
fusion of somatic cells, but it has not been widely used with
embryos. As polyethylene glycol is toxic it is necessary to expose
the cells for a minimum period and the need to be able to remove
the chemical quickly may necessitate the removal of the zona
pellucida (Kanka et al., Mol. Reprod. Dev. 29 110-116 (1991)). In
experiments with mouse embryos, inactivated Sendai virus provides
an efficient means for the fusion of cells from cleavage-stage
embryos (Graham Wistar Inst. Symp. Monogr. 9 19 (1969)), with the
additional experimental advantage that activation is not induced.
In ungulates, fusion is commonly achieved by the same electrical
stimulation that is used to induce parthogenetic activation
(Willadsen Nature 320 (6) 63-(1986), Prather et al., Biol. Reprod.
37 859-866 (1987)). In these species, Sendai virus induces fusion
in a proportion of cases, but is not sufficiently reliable for
routine application (Willadsen Nature 320 (6) 63-65 (1986)).
[0037] While cell-cell fusion is a preferred method of effecting
nuclear transfer, it is not the only method that can be used. Other
suitable techniques include microinjection (Ritchie and Campbell,
J. Reproduction and Fertility Abstract Series No. 15, p60).
[0038] In a preferred embodiment of the invention, fusion of the
oocyte karyoplast couplet is accomplished in the absence of
activation by electropulsing in 0.3M mannitol solution or 0.27M
sucrose solution; alternatively the nucleus may be introduced by
injection in a calcium free medium. The age of the oocytes at the
time of fusion/injection and the absence of calcium ions from the
fusion/injection medium prevent activation of the recipient
oocyte.
[0039] In practice, it is best to enucleate and conduct the
transfer as soon as possible after the oocyte reaches metaphase II.
The time that this will be post onset of maturation (in vitro) or
hormone treatment (in vivo) will depend on the species. For cattle
or sheep, nuclear transfer should preferably take place within 24
hours; for pigs, within 48 hours; mice, within 12 hours; and
rabbits within 20-24 hours, although transfer can take place later,
it becomes progressively more difficult to achieve as the oocyte
ages. High MPF activity is desirable.
[0040] Subsequently, the fused reconstructed embryo, which is
generally returned to the maturation medium, is maintained without
being activated so that the donor nucleus is exposed to the
recipient cytoplasm for a period of time sufficient to allow the
reconstructed embryo to become capable, eventually, of giving rise
to a live birth (preferably of a fertile offspring).
[0041] The optimum period of time before activation varies from
species to species and can readily be determined by
experimentation. For cattle, a period of from 6 to 20 hours is
appropriate. The time period should probably not be less than that
which will allow chromosome formation, and it should not be so long
either that the couplet activates spontaneously or, in extreme
cases that it dies.
[0042] When it is time for activation, any conventional or other
suitable activation protocol can be used. Recent experiments have
shown that the requirements for parthogenetic activation are more
complicated than had been imagined. It had been assumed that
activation is an all-or-none phenomenon and that the large number
of treatments able to induce formation of a pronucleus were all
causing "activation". However, exposure of rabbit oocytes to
repeated electrical pulses revealed that only selection of an
appropriate series of pulses and control of the Ca.sup.2+ was able
to promote development of diploidized oocytes to mid-gestation
(Ozil Development 109 117-127 (1990)). During fertilization there
are repeated, transient increases in intracellular calcium
concentration (Cutbertson & Cobbold Nature 316 541-542 (1985))
and electrical pulses are believed to cause analogous increases in
calcium concentration. There is evidence that the pattern of
calcium transients varies with species and it can be anticipated
that the optimal pattern of electrical pulses will vary in a
similar manner. The interval between pulses in the rabbit is
approximately 4 minutes (Ozil Development 109 117-127 (1990)), and
in the mouse 10 to 20 minutes (Cutbertson & Cobbold Nature 316
541-542 (1985)), while there are preliminary observations in the
cow that the interval is approximately 20 to 30 minutes (Robl et
al., in Symposium on Cloning Mammals by Nuclear Transplantation
(Seidel ed.), Colorado State University, 24-27 (1992)). In most
published experiments activation was induced with a single
electrical pulse, but new observations suggest that the proportion
of reconstituted embryos that develop is increased by exposure to
several pulses (Collas & Robl Biol. Reprod. 43 877-884 (1990)).
In any individual case, routine adjustments may be made to optimise
the number of pulses, the field strength and duration of the pulses
and the calcium concentration of the medium.
[0043] In the practice of the invention, correct ploidy must be
maintained during activation. It is desirable to inhibit or
stabilise microtubule polymerisation in order to prevent the
production of multiple pronuclei, thereby to maintain correct
ploidy. This can be achieved by the application of a microtubule
inhibitor such as nocodazole at an effective concentration (such as
about 5 mg/m1). Colchecine and colcemid are other microtubule
inhibitors. Alternatively, a microtubule stabiliser, such as, for
example, taxol could be used.
[0044] The molecular component of microtubules (tubulin) is in a
state of dynamic equilibrium between the polymerised and
non-polymerised states. Microtubule inhibitors such as nocodazole
prevent the addition of tubulin molecules to microtubules, thereby
disturbing the equilibrium and leading to microtubule
depolymerisation and destruction of the spindle. It is preferred to
add the microtubule inhibitor a sufficient time before activation
to ensure complete, or almost complete, depolymerisation of the
microtubules. Twenty to thirty minutes is likely to be sufficient
in most cases. A microtubule stabiliser such as taxol prevents the
breakdown of the spindle and may also therefore prevent the
production of multiple pronuclei. Use of a microtubule stabiliser
is preferably under similar conditions to those used for
microtubule inhibitors.
[0045] The microtubule inhibitor or stabiliser should remain
present after activation until pronuclei formation. It should be
removed thereafter, and in any event before the first division
takes place.
[0046] In a preferred embodiment of the invention at 30-42 hours
post onset of maturation (bovine and ovine, i.e. 6-18 hours post
nuclear transfer) the reconstructed oocytes are placed into medium
containing nocodazole (5 .mu.g/ml) and activated using conventional
protocols. Incubation in nocodazole may be continued for 4-6 hours
following the activation stimulus (dependent upon species and
oocyte age).
[0047] According to a second aspect of the invention, there is
provided a viable reconstituted animal embryo prepared by a method
as described previously.
[0048] According to a third aspect of the invention, there is
provided a method of preparing an animal, the method
comprising:
[0049] (a) reconstituting an animal embryo as described above;
and
[0050] (b) causing an animal to develop to term from the embryo;
and
[0051] (c) optionally, breeding from the animal so formed.
[0052] Step (a) has been described in depth above.
[0053] The second step, step (b) in the method of this aspect of
the invention is to cause an animal to develop to term from the
embryo. This may be done directly or indirectly. In direct
development, the reconstituted embryo from step (a) is simply
allowed to develop without further intervention beyond any that may
be necessary to allow the development to take place. In indirect
development, however, the embryo may be further manipulated before
full development takes place. For example, the embryo may be split
and the cells clonally expanded, for the purpose of improving
yield.
[0054] Alternatively or additionally, it may be possible for
increased yields of viable embryos to be achieved by means of the
present invention by clonal expansion of donors and/or if use is
made of the process of serial (nuclear) transfer. A limitation in
the presently achieved rate of blastocyst formation may be due to
the fact that a majority of the embryos do not "reprogram"
(although an acceptable number do). If this is the case, then the
rate may be enhanced as follows. Each embryo that does develop
itself can be used as a nuclear donor at the 32-64 cell stage;
alternatively, inner cell mass cells can be used at the blastocyst
stage. If these embryos do indeed reflect those which have
reprogrammed gene expression and those nuclei are in fact
reprogrammed (as seems likely), then each developing embryo may be
multiplied in this way by the efficiency of the nuclear transfer
process. The degree of enhancement likely to be achieved depends
upon the cell type. In sheep, it is readily possible to obtain 55%
blastocyst stage embryos by transfer of a single blastomere from a
16 cell embryo to a preactivated "Universal Recipient" oocyte. So
it is reasonable to hypothesise that each embryo developed from a
single cell could give rise to eight at the 16 cell stage. Although
these figures are just a rough guide, it is clear that at later
developmental stages the extent of benefit would depend on the
efficiency of the process at that stage.
[0055] Aside from the issue of yield-improving expediencies, the
reconstituted embryo may be cultured, in vivo or in vitro to
blastocyst.
[0056] Experience suggests that embryos derived by nuclear transfer
are different from normal embryos and sometimes benefit from or
even require culture conditions in vivo other than those in which
embryos are usually cultured (at least in vivo). The reason for
this is not known. In routine multiplication of bovine embryos,
reconstituted embryos (many of them at once) have been cultured in
sheep oviducts for 5 to 6 days (as described by Willadsen, In
Mammalian Egg Transfer (Adams, E. E., ed.) 185 CRC Press, Boca
Raton, Fla. (1982)). In the practice of the present invention,
though, in order to protect the embryo it should preferably be
embedded in a protective medium such as agar before transfer and
then dissected from the agar after recovery from the temporary
recipient. The function of the protective agar or other medium is
twofold: first, it acts as a structural aid for the embryo by
holding the zona pellucida together; and secondly it acts as
barrier to cells of the recipient animal's immune system. Although
this approach increases the proportion of embryos that form
blastocysts, there is the disadvantage that a number of embryos may
be lost.
[0057] If in vitro conditions are used, those routinely employed in
the art are quite acceptable.
[0058] At the blastocyst stage, the embryo may be screened for
suitability for development to term. Typically, this will be done
where the embryo is transgenic and screening and selection for
stable integrants has been carried out. Screening for
non-transgenic genetic markers may also be carried out at this
stage. However, because the method of the invention allows for
screening of donors at an earlier stage, that will generally be
preferred.
[0059] After screening, if screening has taken place, the
blastocyst embryo is allowed to develop to term. This will
generally be in vivo. If development up to blastocyst has taken
place in vitro, then transfer into the final recipient animal takes
place at this stage. If blastocyst development has taken place in
vivo, although in principle the blastocyst can be allowed to
develop to term in the pre-blastocyst host, in practice the
blastocyst will usually be removed from the (temporary)
pre-blastocyst recipient and, after dissection from the protective
medium, will be transferred to the (permanent) post-blastocyst
recipient.
[0060] In optional step (c) of this aspect of the invention,
animals may be bred from the animal prepared by the preceding
steps. In this way, an animal may be used to establish a herd or
flock of animals having the desired genetic characteristic(s).
[0061] Animals produced by transfer of nuclei from a source of
genetically identical cells share the same nucleus, but are not
strictly identical as they are derived from different oocytes. The
significance of this different origin is not clear, but may affect
commercial traits. Recent analyses of the mitochondrial DNA of
dairy cattle in the Iowa State University Breeding Herd revealed
associated with milk and reproductive performance (Freeman &
Beitz, In Symposium on Cloning Mammals by Nuclear Transplantation
(Seidel, G. E. Jr., ed.) 17-20, Colorado State University, Colorado
(1992)). It remains to be confirmed that similar effects are
present throughout the cattle population and to consider whether it
is possible or necessary in specific situations to consider the
selection of oocytes. In the area of cattle breeding the ability to
produce large numbers of embryos from donors of hick genetic merit
may have considerable potential value in disseminating genetic
improvement through the national herd. The scale of application
will depend upon the cost of each embryo and the proportion of
transferred embryos able to develop to term.
[0062] By way of illustration and summary, the following scheme
sets out a typical process by which transgenic and non-transgenic
animals may be prepared. The process can be regarded as involving
five steps:
[0063] (1) isolation of diploid donor cells;
[0064] (2) optionally, transgenesis, for example by transfection
with suitable constructs, with or without selectable markers;
[0065] (2a) optionally screen and select for stable
integrants--skip for micro-injection;
[0066] (3) embryo reconstitution by nuclear transfer;
[0067] (4) culture, in vivo or in vitro, to blastocyst; [0068] (4a)
optionally screen and select for stable integrants--omit if done at
2a--or other desired characteristics;
[0069] (5) transfer if necessary to final recipient.
[0070] This protocol has a number of advantages over previously
published methods of nuclear transfer:
1) The chromatin of the donor nucleus can be exposed to the meiotic
cytoplasm of the recipient oocyte in the absence of activation for
appropriate periods of time. This may increase the "reprogramming"
of the donor nucleus by altering the chromatin structure. 2)
Correct ploidy of the reconstructed embryo is maintained when GO/G1
nuclei are transferred. 3) Previous studies have shown that
activation responsiveness of bovine/ovine oocytes increases with
age. One problem which has previously been observed is that in
unenucleated aged oocytes duplication of the meiotic spindle pole
bodies occurs and multipolar spindles are observed. However, we
report that in embryos reconstructed and maintained with high MPF
levels although nuclear envelope breakdown and chromatin
condensation occur no organised spindle is observed. The
prematurely. condensed chromosomes remain in a tight bunch,
therefore we can take advantage of the ageing process and increase
the activation response of the reconstructed oocyte without
adversely affecting the ploidy of the reconstructed embryo.
[0071] According to a fourth aspect of the invention, there is
provided an animal prepared as described above.
[0072] Preferred features of each aspect of the invention are as
for each other aspect, mutatis mutandis.
[0073] The invention will now be described by reference to the
accompanying Examples which are provided for the purposes of
illustration and are not to be construed as being limiting on the
present invention. In the following description, reference is made
to the accompanying drawing, in which: FIG. 1 shows the rate of
maturation of bovine oocytes in vitro.
Example 1
"MAGIC" Procedure using Bovine Oocytes
[0074] Recipient oocytes the subject of this experimental procedure
are designated MAGIC (Metaphase Arrested GI/GO Accepting Cytoplast)
Recipients.
[0075] The nuclear and cytoplasmic events during in vitro oocyte
maturation were studied. In addition the roles of fusion and
activation in embryos reconstructed at different ages were also
investigated. The studies have shown that oocyte maturation is
asynchronous; however, a population of matured oocytes can be
morphologically selected at 18 hours (FIG. 1)
Morphological Selection of Oocytes
[0076] In FIG. 1 ovaries were obtained from a local abattoir and
maintained at 28-32.degree. C. during transport to the laboratory.
Cumulus oocyte complexes (COC's) were aspirated from follicles 3-10
mm in diameter using a hypodermic needle (1.2 mm internal diameter)
and placed into sterile plastic universal containers. The universal
containers were placed into a warmed chamber (35.degree. C.) and
the follicular material allowed to settle for 10-15 minutes before
pouring off three quarters of the supernatant. The remaining
follicular material was diluted with an equal volume of dissection
medium (TCM 199 with Earles salts (Gibco), 75.0 mg/1 kanamycin,
30.0 mM Hepes, pH 7.4, osmolarity 280 mOsmols/Kg H.sub.20)
supplemented with 10% bovine serum, transferred into an 85 mm petri
dish and searched for COC's under a dissecting microscope.
[0077] Complexes with at least 2-3 compact layers of cumulus cells
were selected washed three times in dissection medium and
transferred into maturation medium (TC medium 199 with Earles salts
(Gibco), 75 mg/1 kanamycin, 30.0 mM Hepes, 7.69 mM NaHCO.sub.3, pH
7.8, osmolarity 280 mOsmols/Kg H.sub.2O) supplemented with 10%
bovine serum and 1.times.10.sup.6 granulosa cells/ml and cultured
on a rocking table at 39.degree. C. in an atmosphere of 5% CO.sub.2
in air. Oocytes were removed from the maturation dish and wet
mounted on ethanol cleaned glass slides under coverslips which were
attached using a mixture of 5% petroleum jelly 95% wax. Mounted
embryos were then fixed for 24 hours in freshly prepared methanol:
glacial acetic acid (3:1), stained with 45% aceto-orcein (Sigma)
and examined by phase contrast and DIC microscopy using a Nikon
Microphot-SA, the graph in FIG. 1 shows the percentage of oocytes
at MII and those with a visible polar body.
Activation of Bovine Follicular Oocytes
[0078] If maturation is then continued until 24 hours these oocytes
activate at a very low rate (24%.sup.-) in mannitol containing
calcium (Table 1a). However, removal of calcium and magnesium from
the electropulsing medium prevents any activation.
[0079] Table 1a shows activation of bovine follicular oocytes
matured in vitro for different periods. Oocytes were removed from
the maturation medium, washed once in activation medium, placed
into the activation chamber and given a single electrical pulse of
1.25 kV/cm for 80 .mu.s.
TABLE-US-00001 TABLE 1a Hours post onset of Pronuclear No. of
oocytes maturation (hpm) age formation (N) (hrs)) (% activation) 73
24 24.6 99 30 84.8 55 45 92.7* *many 2 or more pronuclei
Activation Response of Sham Enucleated Bovine Oocytes
[0080] Table 1b shows activation response of in vitro matured
bovine oocytes sham enucleated at approximately 22 hours post onset
of maturation (hpm). Oocytes were treated exactly as for
enucleation, a small volume of cytoplasm aspirated not containing
the metaphase plate. After manipulation the oocytes were given a
single DC pulse of 1.25 KV/cm and returned to the maturation
medium, at 30 hpm and 42 hpm groups of oocytes were mounted, fixed
and stained with aceto-orcein. The results show the number of
oocytes at each time point from five individual experiments as the
number of cells having pronuclei with respect to the total number
of cells.
TABLE-US-00002 TABLE 1b No. cells No. cells having having
pronuclei/Total pronuclei/ no. of cells Total no. of EXPERIMENT 30
hpm cells 1 1/8 -- 2 0/24 0/30 3 0/21 0/22 4 0/27 0/25 5 0/19 0/1
hpm = hours post onset of maturation
Pronuclear Formation in Enucleated Oocytes
[0081] Table 2 shows pronuclear formation in enucleated oocytes
fused to primary bovine fibroblasts (24 hpm) and subsequently
activated (42 hpm). The results represent five separate
experiments. Oocytes were divided into two groups, group A were
incubated in nocodazole for 1 hour prior to activation and for 6
hours following activation. Group B were not treated with
nocodazole. Activated oocytes were fixed and stained with
aceto-orcein 12 hours post activation. The number of pronuclei (PN)
in each parthenote was then scored under phase contrast. The
results are expressed as the percentage of activated oocytes
containing 1 or more pronuclei.
TABLE-US-00003 TABLE 2 TOTAL 1 PN 2 PN 3 PN 4 PN >4 PN GROUP A
52 100 0 0 0 0 GROUP B 33 45.2 25.8 16.1 3.2 9.7
[0082] The absence of an organised spindle and the absence of a
polar body suggests that in order to maintain ploidy in the
reconstructed embryo then only a diploid i.e. GO/G1 nucleus should
be transferred into this cytoplasmic situation. Incubation of
activated oocytes in the presence of the microtubule inhibitor
nocodazole for 5 hours, 1 hour prior to and following the
activation stimulus prevents the formation of micronuclei (Table 2)
and thus when the donor nucleus is in the GO/G1 phase of the cell
cycle the correct ploidy of the reconstructed embryo is
maintained.
Results
[0083] These results show that:
[0084] i) these oocytes can be enucleated at 18 hours post onset of
maturation (FIG. 1);
[0085] ii) enucleated oocytes can be fused to donor
blastomeres/cells in either 0.31 mannitol or 0.27M sucrose
alternatively the donor the cells or nuclei can be injected in
calcium free medium in the absence of any activation response;
[0086] iii) reconstructed embryos or enucleated pulsed oocytes can
be cultured in maturation medium and do not undergo spontaneous
activation;
[0087] iv) the transferred nucleus is seen to undergo nuclear
envelope breakdown (NEBD) and chromosome condensation. No organised
meiotic/mitotic spindle is observed regardless of the cell cycle
stage of the transferred nucleus;
[0088] v) such manipulated couplets will activate at 30 hours and
42 hours with a frequency equal to unmanipulated control
oocytes;
[0089] vi) no polar body is observed following subsequent
activation, regardless of the cell cycle stage of the transferred
nucleus;
[0090] viii) upon subsequent activation 1-5 micronuclei are formed
per reconstructed zygote (Table 2).
Reconstruction of Bovine Embryos Using "MAGIC" Procedure
[0091] In preliminary experiments this technique has been applied
to the reconstruction of bovine embryos using primary fibroblasts
synchronised in the GO phase of the cell cycle by serum starvation
for five days. The results are summarised in Table 3.
[0092] Table 3 shows development of bovine embryos reconstructed by
nuclear transfer of serum starved (GO) bovine primary fibroblasts
into enucleated unactivated MII oocytes. Embryos were reconstructed
at 24 hpm and the fused couplets activated at 42 hpm. Fused
couplets were incubated in nocodazole (5 .mu.g/ml) in M2 medium for
1 hour prior to activation and 5 hours post activation. Couplets
were activated with a single DC pulse of 1.25 KV/cm for 80
.mu.sec.
TABLE-US-00004 TABLE 3 NUMBER OF BLASTOCYSTS/ EXPERIMENT TOTAL
NUMBER OF FUSED NUMBER COUPLETS % BLASTOCYSTS 1 1/30 3.3 2 4/31
12.9
Example 2
"MAGIC" Procedure Using Ovine Oocytes
[0093] Similar observations to those in Example 1 have also been
made in ovine oocytes which have been matured in vivo. Freshly
ovulated oocytes can be retrieved by flushing from the oviducts of
superstimulated ewes 24 hours after prostaglandin treatment. The
use of calcium magnesium free PBS/1.0% FCS as a flushing medium
prevents oocyte activation. Oocytes can be enucleated in calcium
free medium and donor cells introduced as above in the absence of
activation. No organised spindle is observed, multiple nuclei are
formed upon subsequent activation and this may be suppressed by
nocodazole treatment
Results
[0094] In preliminary experiments in sheep, a single pregnancy has
resulted in the birth of a single live lamb. The results are
summarised in Tables 4 and 5.
[0095] Table 4 shows development of ovine embryos reconstructed by
transfer of an embryo derived established cell line to unactivated
enucleated in vivo matured ovine oocytes. Oocytes were obtained
from superstimulated Scottish blackface ewes, the cell line was
established from the embryonic disc of a day 9 embryo obtained from
a Welsh mountain ewe. Reconstructed embryos were cultured in the
ligated oviduct of a temporary recipient ewe for 6 days, recovered
and assessed for development.
TABLE-US-00005 TABLE 4 NUMBER OF MORULA, DATE OF BLASTOCYSTS/
NUCLEAR TOTAL TRANSFER PASSAGE NUMBER NUMBER 17.1.95 6 4/28 19.1.95
7 1/10 31.1.95 13 0/2 2.2.95 13 0/14 7.2.95 11 1/9 9.2.95 11 1/2
14.2.95 12 16.2.95 13 3/13 TOTAL 10/78 (12.8%)
[0096] Table 5 shows induction of pregnancy following transfer of
all morula/blastocyst stage reconstructed embryos to the uterine
horn of synchronised final recipient blackface ewes. The table
shows the total number of embryos from each group transferred the
frequency of pregnancy in terms of ewes and embryos, in the
majority of cases 2 embryo's were transferred to each ewe. A single
twin pregnancy was established which resulted in the birth of a
single live lamb.
TABLE-US-00006 TABLE 5 PASSAGE NUMBER "MAGIC" P6 4 P7 1 P11 2 P12 0
P13 3 TOTAL MOR/BL 10 TOTAL NUMBER EWES 6 PREGNANT EWES % 1 (16.7)
FOETUSES/ 2/10 (20.0) TOTAL TRANSFERRED (%)
* * * * *