U.S. patent application number 12/615750 was filed with the patent office on 2010-05-13 for method of constructing nucleus-implanted egg, parthenogenetic embryo and parthenogenetic mammal.
Invention is credited to Manabu Kawahara, Tomohiro KONO, Yayoi Obata.
Application Number | 20100122364 12/615750 |
Document ID | / |
Family ID | 40313051 |
Filed Date | 2010-05-13 |
United States Patent
Application |
20100122364 |
Kind Code |
A1 |
KONO; Tomohiro ; et
al. |
May 13, 2010 |
METHOD OF CONSTRUCTING NUCLEUS-IMPLANTED EGG, PARTHENOGENETIC
EMBRYO AND PARTHENOGENETIC MAMMAL
Abstract
Disclosed is a method for constructing a nucleus-implanted egg,
a parthenogenetic embryo and for producing a parthenogenetic mammal
each having 2 haploid genome sets originating in mammalian ova, and
provides methods of constructing a nucleus-implanted egg having a
haploid genome set derived from primitive ovarian follicle egg (ng
ovum) and a haploid genome set from MII phase (second meiosis
metaphase) egg (fg ovum), a parthenogenetic embryo and a
parthenogenetic mammal, including steps (1) introducing ng ovum
into a nucleus-deleted deleted germinal vesicle stage (GV) egg,
developing the obtained egg to MII phase by in vitro maturing and
culturing to prepare a first nucleus-implanted egg, and (2)
extracting MII phase chromosome from the first nucleus-implanted
egg and introducing it into other fg ovum to prepare a second
nucleus-implanted egg, wherein a ng or fg ovum from which an
imprinted gene undergoing epigenetic modification during sperm
generation is used.
Inventors: |
KONO; Tomohiro; (Tokyo,
JP) ; Obata; Yayoi; (Tokyo, JP) ; Kawahara;
Manabu; (Tokyo, JP) |
Correspondence
Address: |
BIRCH STEWART KOLASCH & BIRCH
PO BOX 747
FALLS CHURCH
VA
22040-0747
US
|
Family ID: |
40313051 |
Appl. No.: |
12/615750 |
Filed: |
November 10, 2009 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12213385 |
Jun 18, 2008 |
7659443 |
|
|
12615750 |
|
|
|
|
10566724 |
Feb 2, 2006 |
|
|
|
PCT/JP2004/011491 |
Aug 4, 2004 |
|
|
|
12213385 |
|
|
|
|
Current U.S.
Class: |
800/24 |
Current CPC
Class: |
C12N 15/8775 20130101;
A01K 67/02 20130101 |
Class at
Publication: |
800/24 |
International
Class: |
C12N 15/873 20100101
C12N015/873 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 5, 2003 |
JP |
2003-286543 |
Jun 29, 2007 |
JP |
2007-171707 |
Claims
1. A method of constructing a nucleus-implanted egg of a non-human
mammal, the nucleus-implanted egg having a haploid genome set
derived from ng ovum and a haploid genome set from fg ovum, which
comprises the steps of (1) introducing a primitive ovarian follicle
egg (ng ovum) into a nucleus-deleted egg in a germinal vesicle
stage (GV stage egg) and then developing them to MII phase (second
meiosis metaphase) by in vitro maturing and culturing to prepare a
first nucleus-implanted egg, and (2) extracting MII phase
chromosome from said first nucleus-implanted egg and introducing it
into other MII phase egg (fg ovum) to prepare a second
nucleus-implanted egg, wherein ovum from which an imprinted gene
that undergoes gene modification posteriori during the generation
of sperm is deleted is used as the ng ovum or fg ovum.
2. The method of claim 1, wherein the primitive ovarian follicle
egg (ng ovum) is an oocyte of a neonate.
3. The method of claim 1, wherein the other MII phase egg is an
ovulation ovum.
4. The method of claim 1, wherein the imprinted gene is at least
one gene selected from the group consisting of H19, Gtl2 and
Ras-grf1 genes.
5. The method of claim 1 or 4, wherein the ovum from which the
imprinted gene is deleted is derived from a gene-deleted non-human
mammal.
6. A method of constructing a parthenogenetic embryo, which
comprises activating the second nucleus-implanted egg obtained by
the method recited in claim 1 and then culturing the second
nucleus-implanted egg in vitro to develop the same.
7. A method of constructing a parthenogenetic non-human mammal,
which comprises implanting the parthenogenetic embryo obtained by
the method recited in claim 6 into the uterus of a non-human mammal
and growing the same.
Description
[0001] This application is a Continuation of application Ser. No.
12/213,385, filed Jun. 18, 2008, now allowed, which is a
continuation-in-part of application Ser. No. 10/566,724, filed Feb.
2, 2006, now abandoned, which is a PCT national stage application
of PCT/JP2004/011491, now abandoned, which claims the benefit of
Japanese application No. 2003-286543 filed Aug. 5, 2003 and
Japanese application No. 2007-171707 filed Jun. 29, 2007.
TECHNICAL FIELD
[0002] The present invention relates to a method for constructing a
nucleus-implanted egg. More specifically, it relates to a method of
constructing a nucleus-implanted egg produced from maternal genomes
alone. The present invention also relates to a method for
constructing a parthenogenetic embryo from a nucleus-implanted egg.
Further, the present invention relates to a method for producing a
parthenogenetic mammal from the above parthenogenetic embryo.
TECHNICAL BACKGROUND
[0003] Mammals perform ontogeny by fertilization of ova and sperm,
and the ontogeny is never completed by ova alone, which means that
the genomes of sperm and eggs are vitally different in function. It
is said that the above functional difference is due to the
existence of groups of genes (imprinted genes) which are identical
but exhibit entirely different expressions depending as a result of
chemical DNA modification imprinted posteriori during the
generation of germ cells. In fact, oocytes of neonates have not
undergone the above gene modification, and a number of genes
exhibit gene expression patterns like those derived from sperm.
[0004] For analyzing the expression patterns of imprinted genes, a
method of constructing a nucleus-implanted ovum from a genome
derived from an oocyte of a neonate of a mouse and a genome of an
ovum derived from a maturated female mouse has been proposed. (see
"Genomic Imprinting during Oogenesis and Embryonic Development" by
Tomihiro Kono, Proteins, Nucleic Acid and Enzymes Vol. 43, No. 4
(1998), pp. 267-274.) The above method comprises the steps of (1)
introducing neonate oocyte (ng ovum) into a nucleus-deleted egg at
a germinal vesicle stage (GV stage) and then developing the oocyte
to an MII phase (second meiosis metaphase) by in vitro culture for
maturation to prepare a first nucleus-implanted egg, and (2)
extracting MII phase chromosomes from the above first
nucleus-implanted egg and introducing it into other MII phase egg
(fg ovum) to prepare a second nucleus-implanted egg. The second
nucleus-implanted egg obtained by this method has a haploid genome
set derived from the ng ovum and a haploid genome set derived from
the fg ovum.
[0005] Neonate oocytes (ng ova) do not resume any meiosis by nature
until they reach the last stage (mouse ova having a diameter of 60
.mu.m) of ovum growth process, and their cell cycles are at a stop
at a diplotene stage in the beginning phase of the first meiosis.
We have found that when the above neonate oocyte (ng ovum) whose
cell cycle is at a stop is introduced into the cytoplasm of a fully
grown oocyte, it resumes meiosis, and the above method has been
accordingly proposed.
[0006] The introduced gene derived from the ng ovum has not
undergone chemical DNA modification imprinted posteriori during the
ovum growth period, and it is expected that the second
nucleus-implanted egg will be a useful material for analyzing the
expression control of an imprinted gene. It has been confirmed that
parthenogenetic embryo from the above second nucleus-implanted egg
develops to a fetus at day 13.5 of gestation, which fetus
morphologically normal comparison with a fetus derived from a
fertilized egg. However, its growth thereafter could not been
confirmed.
DISCLOSURE OF THE INVENTION
[0007] It is an object of the present invention to provide a method
for constructing a nucleus-implanted egg that is a
nucleus-implanted egg having 2 (two) haploid genome sets derived
from mammal ova and is able to grow up to adulthood, a method for
constructing a parthenogenetic embryo from the above
nucleus-implanted egg and a method for producing a parthenogenetic
mammal from the above parthenogenetic embryo.
[0008] The gene of the ng ovum for use in the method for
constructing a second nucleus-implanted egg, described in the
above-noted document, has not undergone chemical DNA modification
imprinted posteriori during an ovum growth period and is close to a
gene derived from sperm, but it differs from the gene derived from
sperm.
[0009] We have made diligent studies for bringing a genomic gene of
the second nucleus-implanted egg having 2 haploid genome sets
derived from ova close into a genomic gene in the fertilization of
sperm and ovum and, as a result, have arrived at the present
invention by finding the use, as one of the haploid genome sets
derived from ova, of a gene from which an imprinted gene that is to
undergo gene modification posteriori during spermatogenesis is
deleted.
[0010] That is, in mammals, identical genes or alleles are arranged
in the same sequence on homologous chromosomes derived from
paternal and maternal genes, genic expressions are equally
exhibited from biparental alleles to take part in gene expressions
of individuals.
[0011] However, some genes exhibit paternal expressions and some
genes exhibit maternal expressions. For example, H19 gene is a gene
that regulates the expression of IGF2 (insulin like growth factor
II) that is an embryo growth factor, and it is expressed from a
maternal gene but is not expressed from a paternal gene. That is
because the H19 gene undergoes posterior gene modification during
the generation of sperm and is inhibited from paternal expression.
Such a gene is called an imprinted gene.
[0012] On the other hand, IGF2 gene is expressed from a paternal
gene but is not expressed from a maternal gene. That is because the
IGF2 gene and the H19 gene have in common an enhancer (gene
expression enhancing sequence) located in a downstream to the H19
gene. This enhancer generally works dominantly over the H19 gene,
and when it undergoes gene modification posteriori during the
generation of sperm, it can no longer work on the H19 gene, and it
comes to work on the IGF2 gene. As a result, there is established a
relationship in which the expression of the H19 gene from a
paternal gene is inhibited and the IGF2 gene is expressed from a
paternal gene.
[0013] In the general fertilization of sperm and ovum, the H19 gene
is expressed from a maternal gene and the IGF2 gene is expressed
from a paternal gene, whereby normal embryogenesis is performed. In
case that parthenogenesis between ovum and ovum is conducted like
the present invention and when the genes of both of the ova are
derived from maternal genes, it is predicted that H19 gene alone,
which is expressed from a maternal gene, is expressed, and that
IGF2 gene, which is expressed from a paternal gene, is not
expressed.
[0014] We have found, however, that when one of H19 genes from ova
is deleted for the parthenogenesis of ovum and ovum, the IGF gene
which is expressed from a paternal gene by nature is expressed from
a gene derived from the ovum (maternal gene) to perform normal
embryogenesis and generation of a mammal, and the present invention
has been accordingly completed.
[0015] According to the present invention, (call as `First
invention` hereinafter) there is provided a method for constructing
a nucleus-implanted egg of a mammal, the nucleus-implanted egg
having a haploid genome set derived from ng ovum and a haploid
genome set derived from fg ovum, which comprises the steps of
[0016] (1) introducing a primitive ovarian follicle egg (ng ovum)
into a nucleus-deleted egg in a germinal vesicle stage (GV stage
egg) and then developing them to MII phase (second meiosis
metaphase) by in vitro culture for development to prepare a first
nucleus-implanted egg, and
[0017] (2) extracting all of MII phase chromosome from said first
nucleus-implanted egg and introducing it into other MII phase egg
(fg ovum) to prepare a second nucleus-implanted egg,
[0018] wherein ovum from which an imprinted gene that undergoes
gene modification posteriori during the generation of sperm is
deleted is used as the ng ovum or fg ovum.
[0019] First invention includes a method for constructing a
parthenogenetic embryo, which comprises activating said second
nucleus-implanted egg and developing the same in vitro culture for
development.
[0020] First invention also includes a method for producing a
parthenogenetic mammal, which comprises implanting said
parthenogenetic embryo in the uterus of a female mammal and growing
the same.
[0021] According to First invention, there is provided a method for
constructing a nucleus-implanted egg that is a nucleus-implanted
having 2 haploid genome sets derived from ova of mammals and that
is able to grow up to adulthood. According to the present
invention, further, there are provided a method for constructing a
parthenogenetic embryo from said nucleus-implanted egg and a method
for producing a parthenogenetic mammal from said parthenogenetic
embryo.
[0022] According to First invention, particularly, there can be
provided the above nucleus-implanted egg having an ability to grow
up to adulthood and a parthenogenetic embryo, and First invention
is technically more significant than a conventional method in which
a nucleus-implanted egg can be developed only up to a fetus
approximately at day 13.5 of gestation.
[0023] The contents of First invention have been disclosed in a
document (Tomohiro Kono et al, Nature Vol. 428. No. 6985, pp.
860-864, 22 Apr. 2004), after the filing of Japanese patent
application No. 2003-286543 on which the priority of the present
application is based.
[0024] Although First invention is an excellent method in that a
nucleus-implanted egg having an ability to grow up to adulthood is
obtained, the proportion of produced adults to parthenogenetic
embryos is only about 1.5%, and an improvement in production
efficiency of adults have been desired.
[0025] An object of Second invention is to provide a
nucleus-implanted egg that is a nucleus-implanted egg having 2
(two) haploid genome sets derived from mammal ova and shows
excellent production efficiency of adults. Another object of Second
invention is to provide a method for constructing a parthenogenetic
embryo from the nucleus-implanted egg and a method for producing a
parthenogenetic mammal from the parthenogenetic embryo.
[0026] The fg ovum or ng ovum used in First invention is close to a
gene derived from a sperm in that an imprinted gene, particularly
an H19 gene, is missing. However, it differs from the gene derived
from a sperm, and production efficiency of adults from an obtained
nucleus-implanted egg is low.
[0027] Thus, we have focused our attention on paternal expressions
and maternal expressions of genes and made studies for bringing a
genomic gene of the second nucleus-implanted egg having 2 haploid
genome sets derived from ova close to a genomic gene in the
fertilization of sperm and ovum.
[0028] As a result, we have found that, in First invention, the
production efficiency of adults can be improved significantly by
use of an ovum in which both (A) a DNA methylation imprint region
(region A) that controls expressions of H19 gene and Igf2 gene and
(B) a DNA methylation imprint region (region B) that controls
expressions of Gtl2 gene and Dlk1 gene are missing, as the ng ovum
or fg ovum, and has completed an improved invention (call as
`Second invention`) based on this finding.
[0029] According to Second invention, there is provided a method
for constructing a nucleus-implanted egg of a mammal, the
nucleus-implanted egg having a haploid genome set derived from an
ng ovum and a haploid genome set derived from an fg ovum, which
comprises the steps of
(1) introducing a non-grown stage egg (ng ovum) into a
nucleus-deleted egg in a germinal vesicle stage (GV stage egg) and
then developing the obtained egg to an MII phase (second meiosis
metaphase) by in vitro culture for development to prepare a first
nucleus-implanted egg, and (2) extracting all of MII phase
chromosomes from said first nucleus-implanted egg and introducing
it into other MII phase egg (fg ovum) to prepare a second
nucleus-implanted egg, wherein an ovum in which both (A) a DNA
methylation imprint region (region A) that controls expressions of
H19 gene and Igf2 gene and (B) a DNA methylation imprint region
(region B) that controls expressions of Gtl2 gene and Dlk1 gene are
missing is used as the ng ovum or fg ovum.
[0030] Further, Second invention includes a method for constructing
a parthenogenetic embryo, which comprises activating said second
nucleus-implanted egg and then developing the same in vitro culture
for development.
[0031] Further, Second invention includes a method for producing a
parthenogenetic mammal, which comprises implanting and growing said
parthenogenetic embryo in the uterus of a female mammal.
[0032] The reason why the production efficiency of adults can be
improved in Second invention can be estimated as follows. That is,
in mammals, identical genes or alleles are arranged in the same
sequence on homologous chromosomes derived from paternal and
maternal genes, genic expressions are equally exhibited from
biparental alleles to take part in gene expressions of
individuals.
[0033] However, some genes exhibit paternal expressions and some
genes exhibit maternal expressions. For example, an embryo growth
factor Igf2 (insulin like growth factor II) gene is expressed from
a paternal locus (derived from a sperm) but is not expressed from a
maternal locus (derived from an ovum). Meanwhile, an H19 gene
located in a downstream to the Igf2 gene is expressed only from a
maternal locus. Such a gene is called an imprinted gene. This is
because the Igf2 gene and the H19 gene have in common an enhancer
(gene expression enhancing sequence) located in a downstream to the
H19 gene. This enhancer generally works dominantly over the H19
gene, and when an expression regulating region (region A) in an
upstream of the H19 gene undergoes gene modification posteriori
(DNA methylation) during the generation of sperm, it can no longer
work on the H19 gene, and it comes to work on the Igf2 gene. As a
result, there is established a relationship in which the expression
of the H19 gene from a paternal gene is inhibited and the Igf2 gene
is expressed from a paternal gene.
[0034] Similarly, a Dlk1 gene and a Gtl2 gene are imprinted genes
expressed from a paternal locus and a maternal locus, respectively.
An expression regulating region (region B) located in an upstream
of the Gtl2 gene undergoes methylation during the generation of
sperm, whereby expression regulation is established.
[0035] That is, in the general fertilization of sperm and ovum, the
H19 and Gtl2 genes are expressed from a maternal gene and the Igf2
and Dlk1 genes are expressed from a paternal gene, whereby normal
embryogenesis is performed, as shown in Table 1.
TABLE-US-00001 TABLE 1 Gene Igf2 H19 Dlk1 Gtl2 Sperm .largecircle.
X .largecircle. X Ovum X .largecircle. X .largecircle.
.largecircle.: Expression is promoted. X: Expression is
inhibited.
[0036] In parthenogenesis between ova, it is expected that since
the genes of both of the ova are derived from maternal genes, the
H19 and Gtl2 genes expressed from a maternal gene are expressed
excessively and the Igf2 and Dlk1 genes expressed from a paternal
gene are not expressed, as shown in Table 2.
TABLE-US-00002 TABLE 2 Gene Igf2 H19 Dlk1 Gtl2 Ovum X .largecircle.
X .largecircle. Ovum X .largecircle. X .largecircle. .largecircle.:
Expression is promoted. X: Expression is inhibited.
[0037] Thus, it is expected that when an ovum in which both the
region A and the region B are missing is used as the ng ovum or fg
ovum, expression regulation close to general fertilization of sperm
and ovum is performed and an adult production rate can be improved
significantly, as shown in Table 3.
TABLE-US-00003 TABLE 3 Gene Igf2 H19 Dlk1 Gtl2 Ovum Having No
Regions .largecircle. X .largecircle. X A and B Ovum X
.largecircle. X .largecircle. .largecircle.: Expression is
promoted. X: Expression is inhibited.
BRIEF DESCRIPTION OF DRAWINGS
[0038] FIG. 1 is a drawing for schematically showing the method for
constructing a nucleus-implanted egg, provided by First
invention.
[0039] FIG. 2 is a photograph of a parthenogenetic mouse obtained
according to First invention and an offspring thereof.
[0040] FIG. 3 is a drawing for schematically showing the method for
constructing a nucleus-implanted egg, provided by Second
invention.
[0041] FIG. 4 is a photograph of a parthenogenetic mouse obtained
according to Second invention and an offspring thereof.
[0042] In FIG. 1 to FIG. 4, symbols a to i represent as follows.
[0043] a: ng ovum derived from a neonate [0044] b: fg ovum derived
from a matured female [0045] c: Nuclear implanting [0046] d:
Maturing in vitro by culturing [0047] e: Matured
nucleus-substituted ovum [0048] f: Ovulation ovum [0049] g:
Implanting of MII phase mitotic apparatus [0050] h: Artificial
activation of ovum [0051] i: Reconstructed ng/fg parthenogenetic
embryo [0052] a to e correspond to the first step of nuclear
implantation in the present invention, and f to i correspond to the
second step of nuclear implantation in the present invention.
BEST MODE OF EMBODIMENT OF THE INVENTION
First Step of Nuclear Implantation
[0053] This is a step in which a primitive ovarian follicle ovum
(ng ovum) is introduced into a nucleus-deleted germinal vesicle
stage egg (GV stage egg) and matured in vitro by culturing to
develop it up to MII phase (second meiosis metaphase).
[0054] As a GV stage egg, there can be used an in vivo grown ovum
obtained by administering a matured mammal with a pregnant mare
ciliary gonadotropic hormone and carrying out super-ovulation
treatment or an in vivo grown ovum obtained from the ovary of a
matured mammal without any treatment. The above in vivo grown ovum
can be collected by incision of an ovarian follicle from a female
mammal ovary obtained by super-ovulation treatment or without any
treatment with an injection needle using a PBS solution or by
suction from an ovarian follicle with an injection needle, or the
like. In a GV stage egg having cumulus cells adhering thereto,
preferably, the cumulus cells are removed by pipetting with a glass
pipette or by oxygen treatment with trypsin-EDTA or the like.
[0055] The GV stage egg is subjected to the cutting of zona
pellucida and deletion of nucleus to prepare a recipient egg. The
zona pellucida can be cut off with a glass knife while observing it
through a microscope. The deletion of nucleus can be performed by
inserting a nucleus-deleting pipette through a zone pellucida
ablation portion and removing it together with a small amount of
cytoplasm.
[0056] While the ng ovum is preferably an oocyte in a fetal life or
an oocyte of a neonate, it can be also collected from the ovary of
a matured mammal.
[0057] The introduction is preferably carried out by cell fusion.
Preferably, the ng ovum is injected into the subzone of the
recipient egg together with Sendai virus of Japan (HVJ) and
fused.
[0058] Then, the ng ovum is matured in vitro by culturing to be
developed to MII phase (second meiosis metaphase). The maturation
in vitro by culturing can be carried out in an .alpha.MEM culture
medium containing 5% of fetal bovine serum (FBS) in a carbon
dioxide culturing apparatus. There can be also sued an M16 culture
medium containing 5% of fetal bovine serum (FBS). In the in vitro
maturation by culturing, the egg performs the disintegration of
karyotheca, the formation of a mitotic division apparatus, meiosis
and the releasing of the first polocyte, whereby there can be
obtained the first nucleus-implanted egg that has reached MII
phase.
[0059] The mammal preferably includes non-human animals such as a
mouse, a swine, a cow, a sheep, a goat, a rat, a rabbit, and the
like.
Second Step of Nuclear Implantation
[0060] This step is a step in which MII phase chromosome is
extracted from the first nucleus-implanted egg and introduced into
other MII phase egg (fg ovum) to prepare a second nucleus-implanted
egg.
[0061] The fg ovum is preferably an ovulation ovum from a female
matured mammal. The above ovulation ovum can be obtained by
administering a matured mammal with a pregnant mare ciliary
gonadotropic hormone or human ciliary gonadotropic hormone and
carrying out ovulation treatment. On the other hand, as the fg
ovum, there can be also used an egg that is developed to MII phase
by obtaining an in vivo grown ovum from the ovary of a matured
mammal without any treatment and maturing the ovum in a state where
it is covered with cumulus cells by in vitro culturing. In the fg
ovum, preferably, part of its zona pellucida is cut beforehand.
[0062] The introduction can be carried out by the following method.
An MII phase chromosome (mitotic apparatus) is sucked from the zona
pellucida ablation portion of the first nucleus-implanted egg with
a nucleus-deleting pipette. Then, hemagglutinating virus of Japan
is sucked in a tip of a pipette and injected into the MII phase
chromosome by inserting the tip through the zone pellucida ablation
portion of the fg ovum. These procedures are preferably carried out
in a nuclear implanting medium such as an M2 culture medium. Then,
culturing is carried out in the M2 culture medium for a
predetermined period of time for fusion, so that the second
nucleus-implanted egg can be obtained. The second nucleus-implanted
egg has a haploid genome set derived from the ng ovum and a haploid
genome set derived from the fg ovum.
(Imprinted Gene)
[0063] In First invention, the ng ovum or the fg ovum is an ovum
having, deleted, an imprinted gene that is to undergo gene
modification posteriori during the spermatogenesis. The ng ovum or
the fg ovum is preferably an ovum from which the imprinted gene and
its expression, regulating region are deleted.
[0064] The imprinted gene includes H19 gene (Leighton P. A. et al,
Nature 375, 34-39, 1995), Igf2 gene (Leighton et al, Nature 375,
34-39, 1995), Dlk1 gene (Schmidt, J. V. et al, Genes Dev. 14,
1997-2002, 2000), Gtl2 gene (Schmidt, J. V. et al, Genes Dev. 14,
1997-2002, 2000) and Ras-grf1 gene.
[0065] It is therefore preferred to use an ovum from which at least
one gene selected from the group consisting of H19, Igf2, Dlk1,
Gtl2 and Ras-grf1 genes is deleted. It is more preferred to use an
ovum from which at least one gene selected from the group
consisting of H19, Gtl2 and Ras-grf1 genes is deleted. It is
particularly preferred to use an ovum from which one or both of H19
and Gtl2 genes are deleted.
[0066] The ovum having the imprinted gene deleted can be obtained
from a gene-deleted mammal. The gene-deleted mammal can be produced
by the use of a known target gene recombination method
(gene-targeting: e.g., Methods in Enzymology 225: 803-890, 1993),
and for example, such a mouse can be obtained as follows.
[0067] First, the target sequence of an imprinted gene such as an
isolated H19 gene, Gtl2 gene, or the like, is replaced with
neomycin resistance gene (Neo.sup.r gene), and a thymidine kinase
gene (HSV-tk gene) that is a herpes virus is added to the terminal
portion of the imprinted gene to prepare a targeting vector. The
targeting vector is introduced into mouse embryo-stem cells (ES
cells), and there are selected cells in which the imprinted gene of
cellular genome DNA is homologously recombined with the mutant
sequence of the targeting vector.
[0068] The selection of the above gene-recombined cells can be made
by adding G418 to a cell culture medium, removing non-recombined
cells having no Neo.sup.r gene, further adding ganciclovir and
removing random-recombined cells in which the HSV-tk gene remains.
The imprinted gene of the thus-selected gene-recombined cells is a
mutant sequence obtained by inserting the Neo.sup.r gene into the
sequence thereof, and it cannot express the imprinted gene at
all.
[0069] Then, the above gene-recombined ES cells are injected into
the initial embryo (blastocyst) of a mouse, and the initial embryo
is developed in vivo to an individual to produce a chimera mouse.
And, the chimera mouse and a wild type mouse are allowed to mate to
produce offspring mice, and individual mice having a mutant
sequences in one or both of alleles are selected from the offspring
mice, whereby gene-deleted mice can be obtained.
(Region A and Region B)
[0070] In Second invention, an ng ovum or fg ovum is an ovum in
which both a region A and a region B are missing.
[0071] The region A is a DNA methylation imprint region that
controls expressions of H19 gene and Igf2 gene. The region A is a
region having a length of 10 kb and situated between the Igf2 gene
and the H19 gene, in the case of a mouse. It is a region from an
EcoRI restriction enzyme cleavage site nearest to the 5' side of
the H19 gene to an EcoRI restriction enzyme cleavage site in an
upstream of about 10 kb.
[0072] The H19 gene is described in Leighton P. A. et al, Nature
375, 34-39, 1995. The length of the H19 gene is 3 kb. The Igf2 gene
is described in Leighton et al, Nature 375, 34-39, 1995.
[0073] The region B is a DNA methylation imprint region that
controls expressions of Gtl2 gene and Dlk1 gene. The region B is a
region having a length of 4.2 kb and situated between the Dlk1 gene
and the Gtl2 gene, in the case of a mouse. It is a region from an
Sca1 restriction enzyme cleavage site nearest to the 5' side of the
Gtl2 gene to an Sac1 restriction enzyme cleavage site in an
upstream of about 4.2 kb.
[0074] The Gtl2 gene is described in maternally expressed gene
3/gene-trap locus 2, Schmidt, J. V. et al, Genes Dev. 14,
1997-2002, 2000. The Dlk1 gene is described in Schmidt, J. V. et
al, Genes Dev. 14, 1997-2002, 2000.
[0075] The ovum in which both the region A and the region B are
missing can be obtained from neonates born by mating the respective
gene-deleted mammals with each other. The gene-deleted mammal can
be constructed by the use of a known target gene recombination
method (gene-targeting: e.g., Methods in Enzymology 225: 803-890,
1993), and for example, such a mouse can be obtained as
follows.
[0076] First, the target sequence of isolated regions A and B is
replaced with a neomycin resistance gene (Neor gene), and a
thymidine kinase gene (HSV-tk gene) that is a herpes virus is added
to the terminal portion of the regions A and B to prepare a
targeting vector. The targeting vector is introduced into mouse
embryo-stem cells (ES cells), and there are selected cells in which
the regions A and B of cellular genome. DNA are homologously
recombined with the mutant sequence of the targeting vector.
[0077] The selection of the above gene-recombined cells can be made
by adding G418 to a cell culture medium, removing non-recombined
cells having no Neor gene, further adding ganciclovir and removing
random-recombined cells in which the HSV-tk gene remains. The
regions A and B of the thus-selected gene-recombined cells are a
mutant sequence obtained by inserting the Neor gene into the coding
sequence thereof and cannot be expressed.
[0078] Then, the above gene-recombined ES cells are injected into
the initial embryo (blastocyst) of a mouse, and the initial embryo
is developed in vivo to an individual to produce a chimera mouse.
Then, the chimera mouse and a wild-type mouse are allowed to mate
to produce offspring mice, and individual mice having a mutant
sequence in the regions A and B are selected from these offspring
mice, whereby gene-deleted mice can be obtained.
[0079] Although Second invention uses an ovum in which the regions
A and B that are expression regulating regions are missing, an ovum
in which an H19 gene itself in addition to the regions A and B is
missing may be used.
(Construction of Parthenogenetic Embryo)
[0080] The present invention includes a method for constructing a
parthenogenetic embryo, which comprises activating the above second
nucleus-implanted egg and then developing it in vitro by culturing.
Preferably, the ovum is activated with strontium. Specifically, the
ovum can be activated by culturing it in an M16 culture medium
containing 10 mM of SrCl.sub.2. Alternatively, the ovum can be
activated by electric pulse, ethanol or the like. The development
in vitro by culturing can be carried out under conditions of a 5%
CO.sub.2, 5% O.sub.2 and 90% N.sub.2 gaseous phase and 37 to
39.degree. C.
(Production of Parthenogenetic Mammal)
[0081] The present invention includes a method for producing a
parthenogenetic mammal, which comprises implanting the above
parthenogenetic embryo in the uterus of a mammal and allowing it to
grow. The mammal is naturally a same kind of mammal from which
parthenogenetic embryo was constructed. While the mammal for the
implantation is not specially limited, it is preferred to use a
mammal that is artificially inseminated and then induced to abort
with prostaglandin F2.alpha., or the like during the early stage of
gestation for synchronization. The mammal preferably includes
non-human mammals such as a mouse, a swine, a cow, a sheep, a goat,
a rat, a rabbit, and the like.
EXAMPLES
[0082] The present invention will be explained with reference to
Examples hereinafter. In Examples, mice were used as a mammal.
Example 1
Collection of Ng Ova
[0083] Ovaries were collected from one-day-old neonates of mice
(Leighton et al, Nature 375: 34-39, 1995) from which 13 Kb of H19
genes and upstream regions thereof had been deleted according to a
target gene recombination method (Methods in Enzymology 225:
803-890, 1993). The collected ovaries were transferred into a 0.02%
EDTA solution and cultured at 37.degree. C. for 10 minutes. Then,
the ovaries were cut apart with an injection needle and dissociated
non-grown stage ova were collected and used as ng ova as a
donor.
Collection of Gv Stage Egg
[0084] Matured mice (8 to 12 weeks old, B6D2F1, Charles
River/Claire) were administered with pregnant mare ciliary
gonadotropic hormone at a dose of 5 to 7.5 IU, and then grown
ovarian follicles in the ovaries were cut apart with a 27-gage
injection needle to collect GV stage eggs covered each with cumulus
cells. The cumulus cells were removed by pipetting, and then the GV
stage eggs were cultured in an M2 culture medium (containing 240
.mu.M of dbcAMP and 5% FBS) at 37.degree. C. for 2 hours.
Nuclear Removal
[0085] A micromanipulator (supplied by Narishige Co., Ltd.) was
fixed to an inverted microscope for an operation. First, the zona
pellucida each of the GV stage eggs was 15 to 20% cut off with a
glass knife. Then, the GV stage eggs were transferred to a
nucleus-implanting M2 medium (containing 10 .mu.g/ml of
cytochalasin B, 100 ng/ml of colcemid, 240 .mu.M of dbcAMP and 5%
FBS) and cultured at 37.degree. C. for 15 minutes. By
micromanipulation, a nucleus-removing pipette (having a diameter of
25 .mu.m) was inserted through the ablation portion each of the
zona pellucida, and the nucleus each of the GV stage eggs was
removed together with a small amount of cytoplasm, to prepare
recipient eggs.
First Step of Nuclear Implantation
[0086] Then, the ng ova were sucked into an implanting pipette
(having a diameter of 15 .mu.m), and then hemagglutinating virus of
Japan (HVJ: Cosmobio) was sucked into the tip portion of the
pipette. The pipette was inserted through the ablation portion of
the zona pellucida each of the recipient eggs and pressed to each
recipient egg for injection. The thus-obtained nucleus-implanted
eggs were transferred to an .alpha.MEM culture medium containing 5%
FCS and cultured in a carbon dioxide gas incubator at 37.degree. C.
for 14 hours. The nucleus-implanted eggs developed to the second
meiosis metaphase (MII phase) through the steps of disintegration
of the nuclear membrane, generation of the mitotic apparatus,
meiosis and releasing of the first polocyte, to give the first
nucleus-implanted eggs.
Second Step of Nuclear Implantation
[0087] Matured female mice (8 to 12 weeks old, B6D2F1, Charles
River/Claire) were administered with pregnant mare ciliary
gonadotropic hormone and human ciliary gonadotropic hormone at a
dose of 5 to 7.5 IU each at an interval of 48 hours, and 14 hours
after the administration of the human ciliary gonadotropic hormone,
oviducts were collected. A mass of ovulation ova covered with
cumulus cells were collected from the oviducts, then, the cumulus
cells were removed in an M2 culture medium containing 300 .mu.g/ml
of hyaluronidase by pipetting, and then, ovulation ova (fg ova)
were collected. Part of zona pellucida was cut apart by
micromanipulation. The ovulation ova and the first
nucleus-implanted eggs were transferred into an implanting M2
culture medium (containing 5 .mu.g/ml of cytochalasin B). A
nucleus-deleting pipette (having a diameter of 25 .mu.m) was
inserted through the ablation portion of the zona pellucida each of
the above first nucleus-implanted eggs, and MII phase chromosome
(mitotic apparatus) was sucked therein. Then, hemagglutinating
virus of Japan was sucked into the tip portion of the pipette, and
the pipette was inserted into the ablation portion of zone
pellucida each of the fg ova to inject MII phase chromosome. They
were transferred to an M2 culture medium and cultured at 37.degree.
C. for 30 minutes to fuse them, whereby second nucleus-implanted
eggs were obtained.
[0088] The following Table 4 shows production efficiency of the
nucleus-implanted eggs.
TABLE-US-00004 TABLE 4 First nucleus- Second nucleus- implanted
eggs implanted eggs Number of fused 287/350 212/249 eggs/number of
(82%) (85%) manipulated eggs (percent) Number of maturation 249 --
(percent) (87%)
Example 2
Construction of Parthenogenetic Embryo
[0089] The thus-obtained 212 second nucleus-implanted eggs were
cultured in an M16 culture medium containing M of strontium
chloride at 37.degree. C. for 3 hours to artificially induce
activation of the ova. As a result, 189 ova were activated. 158 Ova
in which two each of the second polocytes and pronuclei were
generated were in vitro cultured in an M16 culture medium at
37.degree. C. for 3 days. As a result, 131 blastocysts were
produced.
Example 3
Production of Parthenogenetic Mouse
[0090] The thus-obtained 131 blastocysts were implanted in an
uterus of a female mouse at day 2.5 of pseudopregnancy (female
mouse that was mated with a vasoligated male according to a
conventional method and a day when its copulatory plug was
confirmed was a day 0.5), to give birth to 2 normal female
neonates. Analysis of genes of these showed that they had H19 gene
(derived from the fg ovum) and Neo.sup.r gene (derived from ng
ovum), so that it was confirmed that the above neonates were
neonates from the second nucleus-implanted eggs.
[0091] One of these was sacrificed by euthanasia for gene analysis
after its anabiosis was confirmed. The other one was named
"Kaguya", and it normally grown to adulthood. "Kaguya" delivered
neonates by mating with a male, so that it was confirmed that
"Kaguya" had normal reproduction ability. FIG. 2 shows a photograph
taken when "Kaguya" safely delivered the neonates.
Example 4
Collection of Ng Ova
[0092] Ovaries were collected from one-day-old female neonates
obtained by mating mice (Leighton et al., Nature 375: 34-39, 1995)
from which 13 Kb (H19 genes and region A) of H19 genes (3 kb) and
upstream regions thereof (10 kb) had been deleted according to a
target gene recombination method (Methods in Enzymology 225:
803-890, 1993) with mice (Schmidt, J. V. et al, Genes Dev. 14,
1997-2002, 2000) from which a Dlk1-Gtl2 expression regulating
region (region B, length: 4.15 Kb, Gtl2 gene upstream regions) had
been deleted. The collected ovaries were transferred into a 0.02%
EDTA solution and cultured at 37.degree. C. for 10 minutes. Then,
the ovaries were cut apart with an injection needle and dissociated
non-grown stage ova where collected. Ova from which both the region
A and the region B had been deleted were selected from the obtained
ova and used as ng ova as a donor.
Collection of GV Stage Egg
[0093] Matured mice (8 to 12 weeks old, B6D2F1, Charles
River/Claire) were administered with pregnant mare ciliary
gonadotropic hormone at a dose of 5 to 7.5 IU, and then grown
ovarian follicles in the ovaries were cut apart with a 27-gage
injection needle to collect GV stage eggs covered each with cumulus
cells. The cumulus cells were removed by pipetting, and then the GV
stage eggs were cultured in an M2 culture medium (containing 240
.mu.M of dbcAMP and 5% FBS) at 37.degree. C. for 2 hours.
Nuclear Removal
[0094] A micromanipulator (supplied by Narishige Co., Ltd.) was
fixed to an inverted microscope for an operation. First, the zona
pellucida each of the GV stage eggs was 15 to 20% cut off with a
glass knife. Then, the GV stage eggs were transferred to a
nucleus-implanting M2 medium (containing 10 .mu.g/ml of
cytochalasin B, 100 ng/ml of colcemid, 240 .mu.M of dbcAMP and 5%
FBS) and cultured at 37.degree. C. for 15 minutes. By
micromanipulation, a nucleus-removing pipette (having a diameter of
25 .mu.m) was inserted through the ablation portion each of the
zona pellucida, and the nucleus each of the GV stage eggs was
removed together with a small amount of cytoplasm, to prepare
recipient eggs.
First Step of Nuclear Implantation
[0095] Then, the ng ova were sucked into an implanting pipette
(having a diameter of 15 .mu.m), and then hemagglutinating virus of
Japan (HVJ: Cosmobio) was sucked into the tip portion of the
pipette. The pipette was inserted through the ablation portion of
the zona pellucida each of the recipient eggs and pressed to each
recipient egg for injection. The thus-obtained nucleus-implanted
eggs were transferred to an .alpha.MEM culture medium containing 5%
FCS and cultured in a carbon dioxide gas incubator at 37.degree. C.
for 14 hours. The nucleus-implanted eggs developed to the second
meiosis metaphase (MII phase) through the steps of disintegration
of the nuclear membrane, generation of the mitotic apparatus,
meiosis and releasing of the first polocyte, to give the first
nucleus-implanted eggs.
Second Step of Nuclear Implantation
[0096] Matured female mice (8 to 12 weeks old, B6D2F1, Charles
River/Claire) were administered with pregnant mare ciliary
gonadotropic hormone and human ciliary gonadotropic hormone at a
dose of 5 to 7.5 IU each at an interval of 48 hours, and 14 hours
after the administration of the human ciliary gonadotropic hormone,
oviducts were collected. A mass of ovulation ova covered with
cumulus cells were collected from the oviducts, then, the cumulus
cells were removed in an M2 culture medium containing 300 .mu.g/ml
of hyaluronidase by pipetting, and then, ovulation ova (fg ova)
were collected. Part of zona pellucida was cut apart by
micromanipulation. The ovulation ova and the first
nucleus-implanted eggs were transferred into an implanting M2
culture medium (containing 5 mg/ml of cytochalasin B). A
nucleus-deleting pipette (having a diameter of 25 .mu.m) was
inserted through the ablation portion of the zona pellucida each of
the above first nucleus-implanted eggs, and an MII phase chromosome
(mitotic apparatus) was sucked therein. Then, hemagglutinating
virus of Japan was sucked into the tip portion of the pipette, and
the pipette was inserted into the ablation portion of zone
pellucida each of the fg ova to inject the MII phase chromosome.
They were transferred to an M2 culture medium and cultured at
37.degree. C. for 30 minutes to fuse them, whereby second
nucleus-implanted eggs were obtained.
[0097] The following Table 5 shows production efficiency of the
nucleus-implanted eggs.
TABLE-US-00005 TABLE 5 First nucleus- Second nucleus- implanted
eggs implanted eggs Number of Fused 322/383 238/274 Eggs/Number of
(84.1%) (86.9%) Manipulated Eggs (percent) Number of Mature Eggs
274 (85.1%) -- (percent)
Example 5
Construction of Parthenogenetic Embryo
[0098] The thus-obtained 238 second nucleus-implanted eggs were
cultured in an M16 culture medium containing 10 M of strontium
chloride at 37.degree. C. for 3 hours to artificially induce
activation of the ova. As a result, 221 ova were activated. The ova
were in vitro cultured in an M16 culture medium at 37.degree. C.
for 3 days. As a result, 206 blastocysts were produced.
Example 6
Production of Parthenogenetic Mouse
[0099] The thus-obtained 206 blastocysts were implanted in an
uterus of a female mouse at day 2.5 of pseudopregnancy (female
mouse that was mated with a vasoligated male according to a
conventional method and a day when its copulatory plug was
confirmed was a day 0.5), to give birth to 14 normal female
neonates, all of which grew to adulthood. Thus, production
efficiency was as high as 7%. Analysis of their genes showed that
they had deficiencies of expression regulating methylation regions
(regions A and B) of H19-IGF2 gene and GTL-DLK1 gene in hetero, so
that it was confirmed that the above neonates were neonates from
the second nucleus-implanted eggs.
[0100] As a result of examining the reproduction abilities of five
mice out of these 14 parthenogenetic mice, it was found that all of
the tested parthenogenetic mice normally mated, became pregnant and
gave birth and it was confirmed that they all had normal
reproduction ability.
ADVANTAGEOUS EFFECTS OF INVENTION
[0101] According to First invention, there can be constructed an
adult parthenogenetic mammal having 2 haploid genome sets derived
from ova.
[0102] According to Second invention, there is provided a method
for constructing a nucleus-implanted egg that is a
nucleus-implanted egg having 2 haploid genome sets derived from
mammal ova and shows excellent production efficiency of adults.
Further, according to Second invention, there are also provided a
method for constructing a parthenogenetic embryo from the
nucleus-implanted egg and a method for producing a parthenogenetic
mammal from the parthenogenetic embryo. Mammals obtained by the
present invention have a normal reproduction ability.
[0103] In particular, according to Second invention, production
efficiency of adults with respect to a parthenogenetic embryo is
about 7%, and the present invention is technically more significant
than First invention showing a production efficiency of about
1.5%.
INDUSTRIAL UTILITY
[0104] According to the present invention, there can be produced an
adult parthenogenetic mammal having 2 haploid genome sets derived
from ova. Such a mammal is useful as a laboratory animal for
analyzing functions of genes. According to the present invention,
obtained mammals are all female, so that there can be efficiently
produced, for example, milking cows that have excellent genes and
that are genetically uniform. Further, there can be efficiently
produced cows that provide excellent beef cattle. The present
invention so promises its use in the livestock industry. In case
parthenogenetic mammals are produced according to the present
invention, non-human mammals are main objects thereof, while it is
expected that the present invention will be applied to production
of internal organs for implantation in the medical field.
* * * * *