Note: Descriptions are shown in the official language in which they were submitted.
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Method for Generating Cloned Animals Using Chromosome Shuffling
Cross Reference to Related Application
This application claims priority from U.S. Provisional Application Serial No.
60/238,014, filed October 6, 2000, which is incorporated herein in its
entirety.
Field of Invention
This invention concerns methods of cloning animals that incorporate methods
for manipulating or shuffling chromosomes. The methods find important use in
the
fields of agriculture, xenotransplantation, laboratory science and species
conservation,
where shuffling of chromosomes can be used to correct chromosomal
abnormalities,
and to create autosomally isogenic, sexually non-isogenic cloned animals.
Background of the Invention
Microcell-mediated chromosome transfer has been used for many years in
order to introduce a single chromosome into a target cell. For instance, in
1986,
Saxon et al demonstrated the complete suppression of tumorigenicity in HeLa
cells by
introducing a single human chromosome via microcell fusion. See Saxon et al.,
1986,
"Introduction of human chromosome 11 via microcell transfer controls
tumorigenic
expression of HeLa cells," EMBO J. 5: 3461-66. This technology was used by
others
to create libraries of human chromosomes, by fusing single human chromosomes
carrying the HGPRT gene with HGPRT-deficient mouse cells. See Koi et al.,
1989,
"Construction of mouse A9 clones containing a single human chromosome
(X/autosome translocation) via Micro-cell fusion," Jpn. J. Cancer Res. 80: 122-
25.
Other investigators have also used microcell fusion to demonstrate that
tumorigenicity
of various tumor cell lines is suppressed by introducing a single human
chromosome.
See Yoshida et al., 1994, "Alteration of tumorigenicity in undifferentiated
thyroid
carcinoma cells by introduction of normal chromosome 11," J. Surg. Oncol.
55:170-
74; see also Dong et al., 1996, "Prostate cancer - biology of metastasis and
its clinical
implications," World J. Urol. 14: 182-89. However, there have been no reports
of the
use of microcell mediated fusion for the replacement of chromosomes in cells
and
animals, or the simultaneous removal of chromosomes in addition to microcell-
mediated chromosome transfer.
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Other techniques have been used to replace entire chromosomes or very large
fragments of chromosomes. For instance, U.S. Patent No. 5,721,367 by Kay et al
describes methods for replacing greater than SO kb of a mammalian genome, and
transgenic mammals comprising >50 kb transgenes integrated into their genome.
S However, 50 kb is only a small fraction of the length of a typical mammalian
chromosome, and such replacements will not result in substitutions of entire
chromosomes, or correction of chromosomal defects that are at opposite ends of
a
chromosome. U.S. Patent No. 6,077,697 by Hadlaczky and Szalay describes
mammalian artificial chromosomes (MACs) which are stable and self replicating,
and
I 0 may be used to permit targeted integration of megabase pair size DNA
fragments.
However, MACS do not themselves replace native chromosomes or correct existing
chromosomal defects. Thus, it would be advantageous to have a system for
replacing
entire chromosomes in mammalian cells, particularly in the context of cloned
and
transgenic mammals.
15 The recent showing that somatic cells may be used as donors for nuclear
transfer enables the development of complex genetic manipulations in the
context of
cloning that were not considered possible before. For instance, experiments
performed in the early 1990's suggested that when an embryo progresses to the
blastocyst stage (the embryonic stage where the first two cell lineages
separate) the
20 efficiency of nuclear transfer decreases dramatically. See Collas and Robl,
1991,
"Relationship between nuclear remodeling and development in nuclear transplant
rabbit embryos," Biol. Reprod. 45: 455-465. For example, inner cell mass cells
(cells
from the blastocyst which form both somatic and germ cells) were found to
support a
low rate of development to the blastocyst stage with some offspring obtained.
See
25 Collas and Barnes, 1994, "Nuclear transplantation by microinjection of
inner cell
mass and granulosa cell nuclei," Mol. Reprod. Devel. 38: 264-67; see also Sims
et al.,
1994, "Production of calves by transfer of nuclei from cultured inner cell
mass cells,"
Proc. Natl. Acad. Sci. USA 91: 6143-47. However, it was found that
trophectodermal
cells (the cells from the blastocyst that form the placenta) did not support
the
30 development of the nuclear fusion to the blastocyst stage. Collas and Robl,
1991.
Based on these observations, as well as early experiments with amphibian
nuclear
transplantation, it was the overwhelming opinion of those skilled in the art
at the time
that once a cell becomes committed to a particular somatic cell lineage, its
nucleus
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irreversibly loses its ability to be "reprogrammed," i.e. to support full term
development when used as a nuclear donor for nuclear transfer.
Therefore, it was quite astounding in 1998 when researchers at the Roslin
Institute reported that cells committed to somatic cell lineage could support
embryo
development when used as nuclear transfer donors. Equally astounding, and more
commercially significant, scientists at the University of Massachusetts and
Advanced
Cell Technology then showed the production of transgenic cattle by nuclear
transfer
using transgenic fibroblast donor cells. See also, Wells, 1998, "Cloning
symposium:
Reprogramming cell fate - transgenesis and cloning," Monash Medical Center,
Melbourne, Australia, April 15-16 (reporting the production of a calf using
fibroblast
cells). Differentiated cells have also been successfully used as nuclear
transfer donors
to produce cloned mice. See Wakayama et al., 1998, "Full-term development of
mice
from enucleated oocytes injected with cumulus cell nuclei," Nature, 394: 369-
74.
Still further, an experiment by researchers at the University of Massachusetts
and Advanced Cell Technology was recently reported in a lead story in the New
York
Times, January 1999, wherein a nuclear transfer fusion embryo was produced by
the
insertion of an adult human differentiated cell (obtained from the cheek of an
adult
human donor) into an enucleated bovine oocyte. Thus, it would appear, based on
these results, that at least under some conditions differentiated somatic
cells can be
reprogrammed or de-differentiated through the process of nuclear transfer.
It would be advantageous, therefore, if somatic cells to be used for nuclear
transfer could be used to facilitate complex genetic manipulations of donor
cells, and
particularly the replacement of chromosomes in cloned animals.
Summary of Invention
The present invention makes use of somatic cell donor cells for nuclear
transfer to create complex chromosomal arrangements, and particularly
chromosomal
replacements, in cloned and transgenic animals. This technology may be used to
produce cloned cells, embryos, blastocysts, fetuses and animals that are
autosomally
isogenic and sexually non-isogenic, in order to make a population more uniform
or
improve quality control in xenotransplantation. Such chromosome shuffling
techniques can also be used to eliminate chromosomal abnormalities, such as
inversions or translocations from the clone of an animal, produce a sexual
mate for an
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extinct animal where the genome of only one animal is extant, or to produce
the
opposite sex of an existing animal or embryo where the genome of only one sex
is
available or desired.
For instance, when the object is to produce a female animal having desired
traits, somatic cells from the desired animal are isolated, and one X
chromosome is
removed and replaced with a Y chromosome from another animal. Alternatively,
both X chromosomes may be replaced with the sex chromosomes - one X and one Y
- from another mammal. Multiple females may be cloned from the original
somatic
cells, and males would be produced from the autosomally isogenic sexually non-
isogenic (AISN) cells. The cloned bulls could then be used to breed females by
sexual reproduction rather than by cloning. Furthermore, semen from these
males can
be frozen for use in artificial insemination in order to produce more females
having
the desired trait.
To improve the effectiveness of this business model, the male AISN animals
may be genetically modified to produce only female animals using the
technology
described in Application Serial No. 60/184,830, now PCT/L1S01/05932, herein
incorporated by reference in its entirety. This would allow the marketing of
semen
and the ready propagation of female animals having a desirable genetic make-up
while simultaneously preventing customers from breeding their the AISN animals
on
their own without exchange of compensation for the technology. In the case of
the
beef and pig industries, single sex technology can also be used to produce all
male
offspring, and female AISN animals could be marketed as a business strategy.
Normal sexual reproduction, however, results in crossing over of
chromosomes and random segregation of alleles in the haploid gametes, and can
lead
to genetic diversity even in the offspring of autosomally isogenic cloned
animals
because these cloned animals still have two different chromosomal alleles in
each pair
of cloned chromosomes. Therefore, the chromosome shuffling techniques of the
present invention may also be combined with nuclear transfer techniques
designed to
create homozygous diploids of desirable haploid genomes, in order to achieve
allelically isogenic breeding pairs of animals that differ only as to their
sex
chromosomes, i.e., each is a complete autosomal homozygous diploid. Breeding
autosomally and allelically isogenic animals results in isogenic male and
female
offspring without the need for years of inbreeding or successive cloning in
order to
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generate animals. Further, such breeding avoids the potential genetic
diversity
associated with sexual reproduction between cloned breeding pairs where
crossing
over and chromosomal segregation can result in the appearance of undesirable
recessive traits in the progeny.
Such allelically isogenic breeding pairs will have significant utility in the
agricultural field where it is often desirable to propagate animals with
specific traits
such as high milk output, milk with specific lipid or protein profiles, or
animals which
produce meat, leather, wool or fiber having a desired characteristic. Such
breeding
pairs would also find utility in laboratory settings as well as
xenotransplantation
I 0 studies, where lowering the statistical "noise" from genetic diversity, or
eliminating
the risk of introducing viral contaminants is desirable. Autosomally and
allelically
isogenic breeding pairs provide the ultimate business model, whereby
purchasers and
handlers can be assured that desirable animals may be easily maintained via
sexual
reproduction or artificial insemination without the need for nuclear transfer
15 techniques.
Thus, it is an object of the present invention to provide methods of altering
the
sex of a cloned animal, embryo, fetus or cell by removing or replacing one sex
chromosome with the alternative sex chromosome from another animal.
It is also an object of the present invention to provide methods for producing
a
20 sexual mate for an extinct or endangered animal, where the alternative
chromosome
that is inserted may be from either a non-isogenic allogeneic animal or cell,
or a
xenogeneic animal cell, i.e., from a species closely related to the extinct
animal, if
there are no existing allogeneic mates.
Also provided are methods for eliminating chromosomal abnormalities from
25 the clone of an animal, whereby damaged autosomes are removed and replaced
with
non-damaged autosomes from a non-isogenic animal.
Also provided are methods of making autosomally and allelically isogenic
breeding pairs, whereby chromosomal shuffling and nuclear transfer are used to
make
haploid cells that can be combined or used in the G2 stage of the cell cycle
to produce
30 completely homozygous diploids that are sexually non-isogenic. Methods of
making
autosomally isogenic, allelically isogenic diploid nuclear transfer units are
also
encompassed, as are methods of making cells, oocytes, balstocysts, inner cell
masses,
ES cells, embryos, and individual animals having the same characteristics.
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An object of the invention is also to provide business methods for using the
breeding pairs to mass produce animals that have been genetically modified,
bred or
selected to provide an advantage in a desired market, as well as business
methods to
maintain control over the breeding of such animals by marketing animals and/or
semen that can only be used by purchasers and handlers to produce animals of a
single sex.
Brief Description of the Drawings
Figure 1. In protocol for Percoll separation of somatic cells from semen,
diagram depicting Percoll layers prior to (A) and following (B)
centrifugation.
Detailed Description of the Invention
The present invention concerns the use of chromosomal replacement
techniques in the context of producing cloned and transgenic animals, in order
to
correct chromosome abnormalities or alter autosomal genotypes, and provide for
novel breeding pairs by replacing the sex chromosome in animals to be cloned.
Replacement of a sex chromosome, or an X or Y chromosome, will result in
animals
that are autosomally isogenic and sexually non=isogenic (AISN), with
"autosomally
isogenic" meaning that the paired sets of autosomes (non-sex chromosomes) in
each
animal are isogenic or identical. Also included in the invention are animals
that are
both "autosomally" and "allelically" isogenic whereby each particular pair of
chromosomes is internally isogenic or identical within a single animal as well
as
between animals.
The invention therefore encompasses methods of altering the sex of a cloned
animal, or an animal to be cloned, or an embryo, blastocyst, fetus or cell
comprising:
(1) isolating a somatic cell from an animal to be cloned;
(2) removing or programming for removal one sex chromosome from said
somatic cell;
(3) inserting the alternative sex chromosome from a non-isogenic animal;
and
(4) using nuclear transfer to create an autosomally isogenic, sexually non-
isogenic animal, embryo, blastocyst, fetus or cell.
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Such methods may also be used in instances whereby an offspring of a
particular sex
is desired as a result of sexual reproduction, where the method includes:
( 1 ) isolating a fertilized ovum, embryo or blastocyst;
(2) testing the sex of said ovum, embryo or blastocyst;
(3) removing or programming for removal the sex chromosome from one
cell of said ovum, embryo or blastocyst if it is not of the desired sex;
(4) inserting the alternative sex chromosome isolated from an allogeneic
animal;
(5) using nuclear transfer to create an autosomally isogenic, sexually non-
isogenic embryo or blastocyst; and
(6) implanting said embryo or blastocyst into a surrogate female to isolate
an animal having a desired sex.
When a sex chromosome is removed according to the present invention, it
may be either an X or a Y chromosome, and it may be replaced by the
alternative sex
chromosome from a non-isogenic allogeneic animal, or even a non-isogenic,
xenogeneic animal. In the case where the somatic cell of interest is from a
male
animal, the Y chromosome may be replaced by the X chromosome from another copy
of the somatic cell to yield a cell with two X chromosomes.
Also encompassed are methods of producing a sexual mate for an extinct or
endangered animal by removing or programming for removal one sex chromosome
from said somatic cell and inserting the alternative sex chromosome from a non-
isogenic animal, and using nuclear transfer to create an autosomally isogenic,
sexually
non-isogenic animal mate for an extinct or endangered animal. In this
embodiment,
particularly for extinct animals, the somatic cell may need to be isolated
from a
sample of frozen cells. In cases where an animal is endangered or nearing
endangered
levels, somatic cells, preferably semen cells, may be frozen in preparation
for the
methodology of the invention. Where the animal is extinct and frozen cells for
replacement chromosomes do not exist, the alternative chromosome may be taken
from a xenogeneic animal, preferably one that is closely related to the
extinct animal.
In this regard, Application Serial No. pertains specifically to the cloning of
endangered species, which material is hereby incorporated in its entirety.
Also encompassed are methods of eliminating chromosomal abnormalities
from the clone of an animal a damaged chromosome from a somatic cell is
removed
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or programmed for removal, and a non-damaged chromosome from a non-isogenic
animal is inserted. Nuclear transfer is then used to create an animal, embryo,
blastocyst, fetus or cell from said chromosomally corrected somatic cell.
The chromosome to be replaced may be removed by any feasible technique.
For instance, the unwanted chromosome may be removed by targeting by
homologous
recombination a gene or DNA sequence that results in loss of the chromosome
upon
mitosis or meiosis. As discussed in U.S. Patents 5,270,201 and 6,077,697,
clu-omosomal instability results when sequences are introduced which function
as a
centromere. Such sequences cause a dicentric chromosome to be created, which
undergoes breakage potentially leading to loss of the chromosome during cell
division. Loss of chromosomes that have been genetically modified with
additional
centromeric sequences can be detected by karyotype analysis. Cells which lose
the
targeted chromosome may be also be selected by including a negative selectable
marker such as thymidine kinase whereby cells retaining the chromosome or
pieces of
the chromosome will not survive under selective conditions (i.e., gancyclovir
in the
case of thymidine kinase).
As noted above, an advantage of using somatic cells as nuclear donors is that
they may be expanded readily in culture prior to chromosome shuffling
techniques.
However, embryonic cells may also be used, as may the nuclei of somatic cells,
which
are advantageous in that they may be preserved in a preservative (such as
alcohol)
prior to nuclear transfer, i.e., stored for future use. Preferred somatic
cells will be
proliferating, i.e., in a proliferative state, but need not necessarily be
expanded in
culture. The somatic cells may be genetically altered in other ways prior to
or
subsequent to chromosome exchange. For instance, said cells may be modified
with a
chromosomal insertion or deletion, where a transgenic animal is desired that
produces
specific proteins in its bodily fluids or mammary glands, or where it is
desirable to
remove or mutate genes involved in xenotransplantation rejection. The
alternative sex
chromosome to be introduced may also be genetically altered from its native
state.
The methods of the present invention may be performed with a wide variety of
animals, including mammals, fish, reptiles or birds. Preferred animals for
agricultural
and xenotransplantation uses to be made by the present invention are ungulates
selected from the group consisting of bovine, porcine, sheep and goat.
Preferred
extinct or endangered animals to be reconstituted by the methods of the
present
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invention include the gaur, bucardo, giant panda, cheetah, African bongo
antelope,
Sumarran tiger, Giant panda, Indian desert cat, mouflon sheep and rare red
deer.
Preferred animals to be generated for laboratory use include mouse, hamster,
guinea
pig and primates. The methods may also be used to clone cats, dogs, horses or
other
companion animal, or breed champion lines of such mammals.
The chromosomes to be inserted according to the claimed methods may be
inserted via microcell-mediated chromsome transfer, or any other suitable
technique
known in the art, e.g., via injection. Methods for the preparation and fusion
of
microcells containing single chromosomes are well known. See, e.g., U.S.
Patent Nos
5,240,840; 4,806,476; 5,298,429 (herein incorporated by reference in their
entirety;
see also Fournier, 1981, Proc. Natl. Acad. Sci. USA 78: 6349-53; Lambert et
al.,
1991, Proc. Natl. Acad. Sci. USA 88: 5907-59; Yoshida et al., 1994, J. Surg.
Oncol.
55:170-74; Dong et al., 1996, World J. Urol. 14: 182-89. Chromosomes to be
introduced into cloned cells or cells to be cloned will preferably include a
selectable
marker, such as aminoglycoside phosphotransferase, for example, so that cells
receiving the chromosome via microcell fusion may be readily selected from
those
that do not. In this regard, Siden and colleagues describe the construction of
a panel
of four microcell hybrids containing four separate insertions of the exogenous
neomycin resistance gene into mouse chromosome 17. See Siden et al., 1989,
Somat.
Cell Mol. Genet. 15(3): 245-53.
U.S. Patent No. 6,133,503 also describes methodology for producing
microcells by treating a host donor cell with a mitotic spindle inhibitor such
as
colchicine, which results in the formation of micronuclei, then with
cytochalasin B,
which results in the extrusion of microcells which contain one or a few
chromosomes.
The methods of U.S. Patent No. 5,635,376 are also helpful in the context of
the
present invention, in that this patent provides for female muntjac cell lines
in which
there is, for example, a ten-fold difference in chromosomal size between the
diploid
muntjac chromosomes and human chromosome 11. Thus, these female muntjac cell
lines are useful for the amplification of desired chromosomes prior to use in
cells to
be cloned because desired chromosomes may be purified to apparent homogeneity
from the resulting hybrids using conventional equipment given the large size
difference between the chromosome of interest and the muntjac chromosomes.
These
patents are herein incorporated by reference in their entirety.
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The cloned animals, embryos, blastocysts, fetuses and cells produced by the
methods described herein are also part of the invention, as are the sexual
mates and
breeding pairs produced and their offspring. Also included are the individual
replacement chromosomes used for the present invention and any DNAs used to
make
genetic modifications, as well as any intermediary cell lines such as muntjac
cell lines
used to amplify the desired replacement chromosomes.
As described briefly above, in certain embodiments, particularly business
models where isogenic animals are to be produced via sexual reproduction or
artificial
insemination, it is desirable that the animals be allelically isogenic as well
as
autosomally isogenic. Accordingly, the present invention includes methods of
making an autosomally isogenic, allelically isogenic breeding pair of animals
comprising:
( 1 ) isolating a somatic cell from a preferred animal;
(2) inducing meiosis to produce a haploid cell from said somatic cell;
(3) making a diploid cell from said haploid cell which contains isogenic
alleles;
(4) expanding said diploid cell;
(5) isolating a copy of said diploid cell or the nucleus therefrom;
(6) removing one sex chromosome from said copy of said isolated diploid
cell;
(7) inserting the alternative sex chromosome from a non-isogenic animal;
(8) using nuclear transfer to create a first animal that is autosomally
isogenic, allelically isogenic and sexually non-isogenic to said
allelically isogenic diploid cell; and
(9) using nuclear transfer to create a second animal that is autosomally
isogenic, allelically isogenic and sexually isogenic to said allelically
isogenic diploid cell, wherein sexual reproduction between said first
animal and said second animal produces offspring that are autosomally
isogenic and allelically isogenic to said first and second animal.
Such methods may be further supplemented by ensuring that the breeding pair of
animals only produces animals of a single sex, by also including a step or
steps
whereby a nucleic acid construct is introduced into at least one sex
chromosome of
the germ line of said male animal, wherein said nucleic acid construct encodes
a
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transgene which is expressed post-meiotically in developing spermatids, and
wherein
expression of said transgene alters the fertility of sperm resulting from said
developing spermatids, such that said male produces progeny of a single sex.
Such
methods are described in copending Application Serial No. 60/184,830, which is
herein incorporated by reference in its entirety.
Inducing meiosis to produce a haploid cell from a somatic cell may be
accomplished by any successful method. Preferably, meiosis is accomplished by
nuclear transfer of said somatic cell or the nucleus from said somatic cell
(2n) into a
metaphase II enucleated oocyte, and activating said nuclear transfer unit to
extrude a
polar body (n), thereby resulting in a haploid activated nuclear transfer
unit.
Activation may be accomplished by exposing said nuclear transfer unit to one
or more
treatments selected from the group consisting of hyaluronidase, ethanol,
cytochalasin
B, Ca2+ ions, change in osmolarity, electrical pulse, bohemine, ionomycin and
sperm
factor. The fact that haploid oocytes, when activated, form morphologically
normal
blastocysts has been documented by several researchers. See Kaufman, 1982, J.
Embryol. Exp. Morphol. 71: 139-54 (reporting activation with 7% ethanol); Mann
and
Lovell-Badge, 1984, Nature 310(5972): 66-7; O'Neill and Kaufman, 1988, 248(1):
125-31 (reporting activation with hyaluronidase); De Suffer et al., 1992, J.
Assist.
Reprod. Genet. 9(4): 328-37 (activation using puromycin); Henery and Kaufman,
1992, Mol. Reprod. Dev. 31(4): 258-63 (activation in 7% ethanol); Kim et al.,
1997,
Zygote S(4): 365-70 (activation by ethanol plus cytochalasin B); Escriba and
Garcia-
Ximenez, 1999, Theriogenology 51 (5): 963-73, and 2000, Anim. Reprod. Sci. 28:
59(1-2): 99-107 (activation by altering the osmolarity and Ca2+ concentration
with
electrical pulses in mannitol medium); and Alberio et al., 2000, Mol. Reprod.
Dev.
55(4): 422-32 (reporting that bohemine with or without ionomycin produces
activated
haploid oocytes).
Diploid cells containing isogenic alleles may be made by allowing the
activated haploid oocyte to develop to at least the two cell stage, isolating
and/or
separating the cells, and fusing two allelically isogenic haploid cells from
said
developing activated oocyte into an enucleated metaphase II oocyte.
Alternatively,
the homozygous diploid may be made by isolating one haploid cell and allowing
it to
advance to the G2 phase of the cell cycle, at which point it is 2n or
transiently diploid,
and may be used as the donor nucleus for nuclear transfer. Some researchers
have
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used cytochalasin B to induce diploidization of a female pronucleus following
removal of the male pronucleus from a fertilized egg. See Markert and Petters,
1977,
"Homozygous mouse embryos produced by microsurgery," J. Exp. Zool. 201 (2):
295-
302; see also Anderegg and Markert, 1986, "Successful rescue of
microsurgically
produced humozygous uniparental mouse embryos via production of aggregation
chimeras," Proc. Natl. Acad. Sci. USA 83(17): 6509-13. Kono and colleagues
have
also shown that heterozygous bispermic androgenones (eggs with two Y
chromosomes made by fertilizing enucleated oocytes in vitro), also develop to
the
blastoyst stage. See Kono et al., 1993, Mol. Reprod. Dev. 34(1): 43-6.
Thus, specific methods of making allelically isogenic AISN breeding pairs
according to the present invention include several embodiments. For instance,
included are methods of making an autosomally isogenic, allelically isogenic
breeding
pair of animals comprising:
( 1 ) isolating a somatic cell from a preferred female animal;
(2) inducing meiosis to produce a haploid cell from said somatic cell;
(3) expanding said haploid cell;
(4) isolating a copy of said haploid cell or the nucleus therefrom;
(5) removing the X chromosome from said copy of said isolated haploid
cell;
(6) inserting a Y chromosome isolated from a male animal;
(7) using nuclear transfer to create a first male animal that is autosomally
isogenic, allelically isogenic and sexually non-isogenic to said haploid
cell by fusing an isolated haploid cell or the nucleus therefrom selected
from the expanded haploid cells of step (3) and the haploid cell or the
nucleus therefrom from the haploid cell of step (7) with an enucleated
metaphase II oocyte;
(8) using nuclear transfer to create a second animal that is autosomally
isogenic, allelically isogenic arid sexually isogenic to said haploid cell,
by fusing two isolated haploid cells or the nuclei therefrom selected
from the expanded haploid cells of step (3) with an enucleated
metaphase II oocyte;
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wherein sexual reproduction between said first animal and said second animal
produces offspring that are autosomally isogenic and allelicall~y isogenic to
said first
and second animal.
Also included are methods of making an autosomally isogenic, allelically
isogenic breeding pair of animals comprising:
( 1 ) isolating a somatic cell from a preferred male animal;
(2) inducing meiosis to produce a haploid cell from said somatic cell;
(3) selecting a single haploid cell and determining whether it contains an
X or Y chromosome;
(4) expanding said haploid cell;
(5) isolating a copy of said haploid cell or the nucleus therefrom;
(6) removing the sex chromosome from said copy of said isolated haploid
cell;
(7) inserting the alternative sex chromosome into said copy of said haploid
cell wherein the alternative sex chromosome is isolated from either a
non-isogenic animal or the original preferred animal or another haploid
cell produced from said somatic cell and optionally expanding said
haploid copy if an X chromosome is inserted;
(8) using nuclear transfer to create two animals that are autosomally
isogenic, allelically isogenic and sexually non-isogenic by fusing
isolated haploid cells or the nuclei therefrom from the expanded
haploid cells of step (4) and/or the haploid cell or cells or the nuclei
therefrom from the haploid cell or cells of step (7) with an enucleated
metaphase II oocyte in order to create one animal that has two X
chromosomes and one animal that has an X and a Y chromosome;
wherein sexual reproduction between said first animal and said second animal
produces offspring that are autosomally isogenic and allelically isogenic to
said first
and second animal.
Also included are methods of making an autosomally isogenic, allelically
isogenic breeding pair of animals comprising:
( 1 ) isolating a somatic cell from a preferred female animal;
4
(2) inducing meiosis to produce a haploid cell from said somatic cell;
(3) expanding said haploid cell;
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(4) isolating a copy of said haploid cell or the nucleus therefrom;
(5) removing the X chromosome from said copy of said isolated haploid
cell:
(6) inserting a Y chromosome isolated from a male animal;
(7) using nuclear transfer to create a first male animal that is autosomally
isogenic, allelically isogenic and sexually non-isogenic to said haploid
cell by fusing an isolated haploid cell or the nucleus therefrom selected
from the expanded haploid cells of step (3) and the haploid cell or the
nucleus therefrom from the haploid cell of step (7) with an enucleated
metaphase II oocyte;
(8) using nuclear transfer to create a second animal that is autosomally
isogenic, allelically isogenic and sexually isogenic to said haploid cell,
by fusing an isolated cell in the G2 cell cycle phase (2n) or the nucleus
therefrom selected from the expanded haploid cells of step (3) with an
enucleated metaphase II oocyte;
wherein sexual reproduction between said first animal and said second animal
produces offspring that are autosomally isogenic and allelically isogenic to
said first
and second animal.
Also included are methods of making an autosomally isogenic, allelically
isogenic breeding pair of animals comprising:
(1) isolating a somatic cell from a preferred male animal;
(2) inducing meiosis to produce a haploid cell from said somatic cell;
(3) selecting a single haploid cell and determining whether it contains an
X or Y chromosome;
(4) expanding said haploid cell;
(5) isolating a copy of said haploid cell or the nucleus therefrom;
(6) removing or the sex chromosome from said copy of said isolated
haploid cell;
(7) inserting the alternative sex chromosome isolated from a non-isogenic
animal or the original preferred animal or another haploid cell
produced from said somatic cell and optionally expanding said haploid
copy if an X chromosome is inserted;
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(8) using nuclear transfer to create two animals that are autosomally
isogenic, allelically isogenic and sexually non-isogenic, wherein
(a) the female animal is made by fusing two isolated
haploid cells or the nuclei therefrom containing X
chromosomes selected from the expanded haploid cells of step
(4) or the expanded haploid cells of step (7) with an enucleated
metaphase II oocyte in order to create one animal that has two
X chromosomes OR by fusing one isolated haploid cell at the
G2 cell cycle stage containing an X chromosome with an
enucleated metaphase II oocyte in order to create one animal
that has two X chromosomes; and
(b) the male animal is made by fusing one isolated haploid
cell having an X chromosome with one isolated haploid cell
having a Y chromosome with an enucleated metaphase II
oocyte in order to create one animal that has an X and a Y
chromosome;
wherein sexual reproduction between said first animal and said second animal
produces offspring that are autosomally isogenic and allelically isogenic to
said first
and second animal.
The nuclear transfer units made by the methods of the present
invention are also included. For instance, a female allelically isogenic
diploid nuclear
transfer unit may be made by a method comprising:
(1) isolating a somatic cell from a preferred female animal;
(2) inducing meiosis of said somatic cell by nuclear transfer of said
somatic cell or the nucleus from said somatic cell (2n) into a
metaphase II enucleated oocyte and activating said nuclear transfer
unit to extrude a polar body (n), thereby resulting in a haploid activated
nuclear transfer unit;
(3) allowing said haploid activated nuclear transfer unit to differentiate
and expand to at least the two cell stage; and
(4) fusing either
(a) two haploid cells from step (3); or
(b) one haploid cell from step (3) at the G2 cell cycle stage;
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with an enucleated metaphase II oocyte in order to create a female allelically
isogenic diploid nuclear transfer unit. The method is performed such that the
diploid nuclear transfer unit created at step (4) is activated such that there
is no
extrusion of a polar body. Activated diploid nuclear transfer units may
further
develop into an allelically isogenic cells, blastocysts, inner cell masses, ES
cells, embryos, fetuses or animals.
Methods of making male autosomally isogenic, allelically isogenic diploid
nuclear transfer units are also included, and such methods may be performed
by:
( 1 ) isolating a somatic cell from a preferred animal;
(2) inducing meiosis of said somatic cell by nuclear transfer of said
somatic cell or the nucleus from said somatic cell (2n) into a
metaphase II enucleated oocyte and activating said nuclear transfer
unit to extrude a polar body (n), thereby resulting in a haploid activated
nuclear transfer unit;
(3) allowing said haploid activated nuclear transfer unit to differentiate
and expand to at least the two cell stage;
(4) replacing the sex chromosome in one cell from taken from said
differentiated and expanded haploid cells using microcell-mediated
chromosome transfer from the sex chromosome from a non-isogenic
animal or from another haploid or somatic cell from said preferred
animal if said preferred animal was a male;
(5) fusing two haploid cells:
(a) one from the expanded cells of step (3); and
(b) the cell made in step (4);
with an enucleated metaphase II oocyte in order to create a male
autosomally isogenic, allelically isogenic diploid nuclear transfer unit.
The allelically isogenic diploid nuclear transfer unit made by the
methods of the invention are also encompassed.
Because the methods described herein enable one to pass the advantages of the
cloning technology to the agricultural and other industries while at the same
time
enable the control over the dissemination of genetically engineered molecules
to
remain with the inventor or the assignee, the methods described herein are
particularly
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useful business models. Accordingly, the invention also includes business
methods
for producing uniform, isogenic animals, comprising:
( 1 ) producing autosomally isogenic and allelically isogenic male and
female animals according to the methods described herein; and
(2) breeding said male and female animals to produce uniform, isogenic
animals.
Female animals and/or said male animals may be genetically modified, bred or
selected to provide an advantage in a desired market. For instance, in the
agricultural
market, female animals may be genetically modified, bred or selected to
produce a
high milk output, milk with specified lipid or protein profile, milk that
contains a
therapeutic protein, or milk with superior nutritional value. Alternatively,
female
and/or male animals may be genetically modified, bred or selected to produce
meat,
leather, wool or fiber having a desired characteristic.
Other target markets include laboratories, where there is a need for isogenic
animals including rats, monkeys, rabbits, mice, guinea pigs to remove the
statistical
noise from experimentation and trials for the development of therapeutic
drugs. A
target market would also include a xenotransplantation facility, where animals
such as
cows, pigs and primates are developed to provide compatible organs for human
transplantation. For instance, female animals and/or male animals may be
genetically
modified with a specific human HLA type profile, or modified such that native
proteins that cause graft rejection are deleted, modified or replaced with
proteins that
do not cause graft rejection in humans.
One of the most effective business models is where the male animal has been
genetically modified such that it only produces offspring of a single sex,
i.e., such that
it only produces female offspring. Such a model is useful where only female
uniform,
isogenic animals are sold commercially. Frozen semen from a male isogenic
animal
may also be isolated and sold to purchasers of female uniform, isogenic
animals such
that artificial insemination may be used to create further uniform, isogenic
animals.
Male animals according to the invention may also be genetically modified
such that they only produce male offspring, or such that they produce no
offspring.
This would be useful where only male uniform, isogenic animals are sold
commercially. A single female isogenic animal could then sold or leased by
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purchasers of male uniform, isogenic animals such that purchasers may breed
said
female with a male in order to create further male uniform, isogenic animals.
The uniform, isogenic animals produced in the business methods described
herein are also included in the invention, as is semen, and kits containing
frozen
semen for artificial insemination.
The skilled artisan will envision variations to the methods disclosed herein
without departing from the scope of the invention.
Example 1
Isolation of Somatic Cells from Semen
The cloning of animals by nuclear transfer has many applications in such
diverse fields as agriculture, medicine and the preservation of endangered
species.
One difficulty commonly faced, however, is an adequate source of somatic
cells. In
the case of agricultural species such as cattle, highly-valued studs are often
lost with
no known preservation of the genome for cloning. This invention describes a
technique to isolate viable somatic cells from semen, urine, milk and other
sources
where the isolation of somatic cells is problematic.
While semen is often thought of as being largely a solution of spermatozoa
that are haploid, somatic diploid cells may occasionally be shed as well. We
centrifuged 0.75 ml of bovine semen at 700x g (45%-90% percoll gradient for 30
minutes), aspirated the supernatant, and resuspended the pellet of 500 ml in
DMEM
medium with 15 FCS. The resulting cell suspension was then plated in 35 mm2
tissue
culture plate. The culture dishes were aspirated, washed and refed 24 hours
(after and
every other day following). After five days of culture, fibroblastic cells
were
observed attached to the tissue culture dish. These somatic cells can then be
propagated, cryopreserved, or used as somatic cell donors for the production
of
nuclear transfer embryos and calves. An alternative approach would be to use a
Fluorescence Cell Sorter machine, which can separate sperm from somatic cells
based
upon DNA content.
To reduce the chance of spontaneous abortion, fetuses may be extracted at 40
days, and fetal fibroblasts isolated and frozen. From these fetal fibroblasts,
the final
animals can be cloned. Cells can be isolated in a similar manner from other
fluids
such as milk, blood or urine where such samples have been saved. In addition,
such
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cells can be cultured from frozen tissue such as skin biopsy, skeletal muscle,
or whole
frozen animals.
The success of this method can be explained perhaps by analyzing the method
of semen processing for the purpose of freezing and later use in artificial
insemination. During extraction, an artificial vagina is used to collect the
ejaculate
and perhaps some of the cells that are around the penis along with free
somatic cells
originating in the accessory glands, ducts and testicle themselves will be
mingled with
the ejaculate. This technique will allow bulls to be "resurrected" in
instances where
the bulls are no longer alive but their frozen semen is available. The method
is
reproduced in detail below:
A. Establishment of Cell Lines from Cr~preserved Semen
NOTE: , Please wear gloves for every step of the procedure to prevent cross
contamination of samples
Percoll separation of sperm (performed at room temperature)
Step 1. In a sterile 1 S ml conical centrifuge tube, layer 2 ml 90% Percoll
then
carefully layer 2 ml of 45% Percoll on top of the 2 ml of 90% Percoll layer as
shown
in the diagram below. It is best to use either a 1000 u1 pipette or a 9 ml
pastuer
pippete. It is very critical to have a very defined interface between the two
layers.
This will be observed clearly because the 45% Percoll is pinkish in hue and
the 90%
Percoll is clear. A very defined interface will be observed if layered
correctly (see
Figure 1 A).
Step 2: Thaw semen in 35°C water for 1 min. Record all information
from semen
straw, including bull name and registration numbers and collection date into
your
laboratory notebook. Step 3: Thoroughly dry the straw of semen with a KemWipe
wet with ethanol and then snip end of semen straw with a clean scissor. Place
the
open end into a clean 15 ml conical tube. Then carefully snip off the plug end
of the
straw and deposit all semen into tube.
Step 4: With a 500 u1 pipette, carefully layer all of the semen onto the top
of the Percoll .
layers.
Step 5: Centrifuge at 700 x g (2000 rpm using a 6.37 inch tip radius) for 30
minutes.
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Step 6: After centrifugation, a sperm pellet will be observed at the bottom of
the 90%
Percoll layer as shown in,diagram below (Figure 1B).
Step 7: Aspirate off the Percoll gradients leaving the sperm pellet in the tip
of the tube.
This is usually about only 200 u1 of pellet (this will vary depending on the
number of
semen straws thawed).
Step 8: With a clean pipette tip, move the pellet into either a 35 mm tissue
culture
treated plate or a 4 well Nunc plate with complete DMEM medium.
Step 9: Remove the medium the following day and add fresh medium to the
plates.
Step 10: Carefully observe the plates for the presence of cells - this will
depend on the
semen, usually 7-14 days after the initial plating.
Step 11: Follow standard Cell Culture Techniques once a cell line is observed.
Stock Solutions
45% Percoll Solution
I S A. Ingredients
1. 1.5 ml 90% Percoll Stock Solution.
2. 1.5 ml Sperm TL with BSA.
B. Procedure
1. Use aseptic techniques.
2. Transfer ingredients to a sterile tube.
3. Invert to mix.
4. Do not attempt to filter.
germ TL Without BSA
A. Ingredients
1. 25 ml sperm TL stock.
2. Adjust pH to 7.4 with 1 M HCI.
3. Filter sterilize
4. Prepare daily.
Modified Sperm TL (1 Ox stock used to prepare 90% Percoll)
A. Ingredients
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1. 3.09 ml 1 M KCI.
2. 2.92 ml 0.1 M NaH2P04
3. 4.675 gm NaCI
4. 2.380 gm Hepes
B. Procedure
1. Add prescribed amounts of KC1 and NaH2P04 solutions
to ~ 50 ml Hz0
in volurrietric flask.
2. Add NaCI and Hepes.
3. Adjust water to 100 ml.
4. Adjust pH to 7.3.
5. Filter sterilize and transfer to a glass bottle.
6. Store refrigerated indefinitely.
7. Readjust pH as needed.
1 M CaCh - used in making 90% Percoll
A. Ingredients
1. 735 mg CaCl2*2H20.
2. Reagent grade water.
B. Preparation
1. Weigh CaClz.
2. Add 5 ml H20.
3. Filter sterilize or autoclave.
4. Store in glass bottle indefinitely.
0.1 M Mg-C12 - used in making 90% Percoll
A. Ingredients
1. 20.3 mg MgClz*6H20.
2. Reagent grade water.
B. Preparation
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1. Weigh MgClz.
2. Add 10 ml water.
3. Filter sterilize or autoclave.
4. Store in glass bottle indefirlltely.
90% Percoll
Solution
A. Ingredients
1. 45.0 ml Percoll
2. 5.0 ml Modified Sperm Tl
(1 Ox stock)
3. .0985 ml 1M CaCl2
4. .197 ml 0.1 M MgCl2
5. .184 ml Lactic Acid (60%
syrup)
6. 104.5 mg NaHC03
B. Procedure
1. Combine ingredients while stirnng.
2. Store refrigerated.
3. Do not attempt to filter.
1. SPERM TL STOCK
Compound Final mg/100m1
mg/SOOmI
mM
NaCI 100 582 2910
KC1 3.1 23 115
NaHC03 25 209 1045
NaH2P04H20 0.29 4.1 20.5
Hepes 10 238 1190
Na Lactate 21.6 368 u1 1840 u1
(60% syrup)
Phenol Red lul/ml 100 u1 500 u1
CaC122H20* I 2.10 I 29 I 145
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MgClz6Hz0* 1.5 [ 31 155
*Add last.
Check osmolarity (290-310 mOSM).
Filter into sterile bottle.
Store at 4°C.
Media components derived from : Parrish, J.J., J.L. Susko-Parrish and N.L.
First. 1985. Theriogenology 24:537.
Chemical Components
C7902 CaClz*2Hz0 Calcium Chloride-HZO
H3375 Hepes
M2393 MgClz-6Hz0 Magnesium Chloride-6H20
P1644 Percoll
P0290 Phenol Red
P5405 KCl Potassium Chloride
55761 NaHC03 Sodium Bicarbonate
55886 NaCI Sodium Chloride
L4263 Sodium Lactate (60%
syrup)
59638 NazHP04*H20 Sodium Phosphate
B. Nuclear transfer using~somatic cells isolated from semen
Using the above techniques, we have found that when a single straw of semen
is thawed and put in culture under conditions that will favor the growth of
epithelial/fibroblast-like cells, colonies can be detected. Using this
protocol, we were
able to obtain somatic cells from a straw of bull semen, and use those somatic
cells to
generate embryos by nuclear transfer.
Three replicates of nuclear transfer were performed with three separate
Londondale Sperm Cell Lines:
Cultured Cleaved % Cleaved Blastocysts % Blastocysts
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51 26 51% 9 18%
191 73 38% 37 19%
49 28 57% 10 20%
I I I I I
6 embryos were transferred into three recipients, but no pregnancy was
detected.
One replicate of nuclear transfer was performed with a Whiteleather Mark
Sperm Cell Line.
Cultured Cleaved % Cleaved Blastoc sts % Blastoc sts
53 18 33% 8 15%
6 Embryos were transferred into 3 recipients - 1 pregnancy was detected and is
still
ongoing (approx 67 days - sexed as male).
C. Characterization of Sperm Cell Lines
Karyotypin~
Karyotypes were done on both sperm cell lines; images taken and saved.
Results indicate that the cells are of bovine origin and have 60 chromosomes.
Samples of NT embryos, cell line, semen and extracted DNA were sent to Celera
AgGen for DNA analysis.
Staining of Semen Cell Line
Initial staining of cell lines was performed using alpha tubulin as a general
(positive control) marker and Pan Cytokeratin as epithelium marker. Results
indicated that there was no staining for the Pan Cytokeratin marker for both
concentrations used. Alpha tubulin positive control worked (images not shown).
This
suggests that the cells are not of epithelial nor endothelial origin, and are
probably
fibroblasts.
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