U.S. patent application number 11/439788 was filed with the patent office on 2006-09-21 for methods for cloning mammals using reprogrammed donor chromatin or donor cells.
Invention is credited to Philippe Collas, P. Kasinathan, James M. Robl, Eddie Sullivan.
Application Number | 20060212952 11/439788 |
Document ID | / |
Family ID | 22979301 |
Filed Date | 2006-09-21 |
United States Patent
Application |
20060212952 |
Kind Code |
A1 |
Collas; Philippe ; et
al. |
September 21, 2006 |
Methods for cloning mammals using reprogrammed donor chromatin or
donor cells
Abstract
The invention provides methods for cloning mammals that allow
the donor chromosomes or donor cells to be reprogrammed prior to
insertion into an enucleated oocyte. The invention also features
methods of inserting chromosomes or nuclei into recipient
cells.
Inventors: |
Collas; Philippe; (Oslo,
NO) ; Robl; James M.; (Brandon, SD) ;
Sullivan; Eddie; (Tea, SD) ; Kasinathan; P.;
(Brandon, SD) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
22979301 |
Appl. No.: |
11/439788 |
Filed: |
May 24, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10032191 |
Dec 21, 2001 |
|
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11439788 |
May 24, 2006 |
|
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60258151 |
Dec 22, 2000 |
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Current U.S.
Class: |
800/15 ; 800/14;
800/16; 800/17; 800/18; 800/21 |
Current CPC
Class: |
A01K 67/0273 20130101;
C12N 2517/10 20130101; A01K 2227/103 20130101; C12N 2501/999
20130101; A01K 2227/105 20130101; A01K 2227/108 20130101; A01K
2227/102 20130101; C12N 15/873 20130101; A01K 2227/101 20130101;
A01K 2267/02 20130101; C12N 15/8771 20130101; A01K 2227/107
20130101 |
Class at
Publication: |
800/015 ;
800/014; 800/016; 800/017; 800/018; 800/021 |
International
Class: |
A01K 67/027 20060101
A01K067/027 |
Claims
1. A method of cloning a non-primate mammal, said method comprising
the steps of: (a) permeabilizing a non-primate mammalian cell,
thereby generating a permeabilized cell having pores in its plasma
membrane or a partial plasma membrane; (b) incubating said
permeabilized cell in an extract from an oocyte under conditions
that allow chromatin condensation and nuclear envelope breakdown of
said permeabilized cell; (c) inserting said cell formed in step (b)
into a nucleated or enucleated oocyte, thereby forming a
reconstituted oocyte; and (d) transferring said reconstituted
oocyte or an embryo formed from said reconstituted oocyte into the
uterus of a host mammal under conditions that allow said
reconstituted oocyte or said embryo to develop into a fetus.
2. The method of claim 1, wherein a chromatin mass is formed from
incubation of said permeabilized cell in said extract from said
oocyte.
3. The method of claim 1, wherein, said cell formed in step (b) is
incubated under conditions that allow the plasma membrane of said
cell to reseal.
4. The method of claim 1, wherein said cell formed in step (b) is
purified from said extract from said oocyte prior to insertion into
said nucleated or enucleated oocyte.
5. The method of claim 1, wherein said fetus develops into a viable
offspring.
6. The method of claim 1, wherein said reconstituted oocyte from
step (c) is cultured under conditions that allow cell division and
one of the resulting cells is recloned one or more times.
7. The method of claim 1, wherein said permeabilized cell and said
nucleated or enucleated oocyte are from the same species.
8. The method of claim 1, wherein said non-primate mammal is a cow,
sheep, rabbit, pig, mouse, rat, goat, or buffalo.
9. The method of claim 8, wherein said non-primate mammal is a
cow.
10. The method of claim 1, wherein said permeabilized cell is a
fibroblast, epithelial cell, neural cell, epidermal cell,
keratinocyte, hematopoietic cell, melanocyte, chondrocyte,
B-lymphocyte, T-lymphocyte, erythrocyte, macrophage, monocyte,
muscle cell, embryonic stem cell, embryonic germ cell, fetal cell,
placental cell, or embryonic cell.
11. The method of claim 1, wherein said permeabilized cell is a
cell of the female reproductive system.
12. The method of claim 11, wherein said permeabilized cell is a
mammary gland, ovarian cumulus, granulosa, or oviductal cell.
13. The method of claim 1, wherein said reconstituted oocyte from
step (b) expresses lamin A, lamin C, or NuMA protein at a level
that is less than 5-fold greater than the corresponding level
expressed by a control oocyte from the same species.
14. The method of claim 1, wherein said extract from said oocyte is
an extract from a bovine oocyte.
15. The method of claim 1, wherein said extract from said oocyte is
an extract from a sea urchin oocyte.
16. The method of claim 1, wherein said permeabilized cell is
generated by incubating a somatic cell from a non-primate mammal
with streptolysin O.
17. The method of claim 16, wherein said streptolysin O
concentration is between 100-4000 ng/ml.
18. The method of claim 17, wherein said streptolysin O
concentration is 500 ng/ml.
19. The method of claim 16, wherein said incubating with
streptolysin O is carried out for 15-60 minutes.
20. The method of claim 19, wherein said incubating with
streptolysin O is carried out for between 25-30 minutes.
21. The method of claim 16, wherein said incubating with
streptolysin O is carried out at between 25-38.degree. C.
22. The method of claim 21, wherein said incubating with
streptolysin O is carried out at 38.degree. C.
23. The method of claim 1, wherein said inserting in step (c) is
carried out by fusion of said permeabilized cell with said
nucleated or enucleated oocyte.
24. The method of claim 1, wherein said oocyte of step (c) is
enucleated.
25. The method of claim 1, wherein said reconstituted oocyte is
activated prior to transfer into the uterus of said host mammal.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional application of and claims
priority from U.S. patent application Ser. No. 10/032,191, filed
Dec. 21, 2001, which claims benefit of the filing date of U.S.
Provisional Application No. 60/258,151, filed Dec. 22, 2000, hereby
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] In general, the invention features improved methods for
cloning mammals and methods for inserting chromosomes, nuclei, or
chromatin masses into recipient cells.
[0003] The cloning of mammals allows the production of multiple
mammals with an identical DNA content. The donor genetic material
used to generate these mammals may be selected or engineered such
that the cloned mammals have desirable properties, such as
increased resistance to disease. Unfortunately, the efficiency of
cloning mammals using donor somatic cells is generally low,
resulting in only about 1-2% of nuclear transplant embryos
developing to term (Polejaeva et al., Nature 407:86-90, 2000). A
significant problem with cloning is the loss of mid to late term
pregnancies and the low viability of the offspring. Thus, more
efficient methods are needed for cloning mammals. These improved
methods may reduce the cost and time required to generate multiple
viable offspring.
SUMMARY OF THE INVENTION
[0004] The purpose of the present invention is to provide improved
methods for cloning mammals. In particular, these methods involve
the condensation of a donor nucleus into a chromatin mass to allow
the release of nuclear components such as transcription factors
that may promote the transcription of genes that are undesirable
for the development of the nuclear transplant embryo into a viable
offspring. In a related method, a permeabilized cell is incubated
with a reprogramming media (e.g., a cell extract) to allow the
addition or removal of factors from the cell, and then the plasma
membrane of the permeabilized cell is resealed to enclose the
desired factors and restore the membrane integrity of the cell. If
desired, the steps of any of these methods may be repeated one or
more times or different reprogramming methods may be performed
sequentially to increase the extent of reprogramming, resulting in
greater viability of the cloned fetuses. The invention also
provides methods for generating chimeric embryos in which some or
all of the placental tissue is from one genetic source and the
majority of the fetal tissue is from another genetic source. These
chimeric embryos may have fewer placental abnormalities and thus
may have an increased survival rate. In addition, a novel method
has been developed for the insertion of the chromatin mass or a
nucleus into the recipient ooctye that involves the use of
fusigenic compounds.
[0005] Accordingly, in a first aspect, the invention provides a
method of cloning a mammal. This method involves (a) incubating a
donor nucleus that has less than four sets of homologous
chromosomes (i.e., has fewer than two pairs of complete chromatids)
under conditions that allow formation of a chromatin mass without
causing DNA replication, (b) inserting the chromatin mass into an
enucleated oocyte, thereby forming a nuclear transfer oocyte and
(c) transferring the nuclear transfer oocyte or an embryo formed
from the nuclear transfer oocyte into the uterus of a host mammal
under conditions that allow the nuclear transfer oocyte or embryo
to develop into a fetus. In a preferred embodiment, the donor
nucleus is incubated with a reprogramming media (e.g., a cell
extract) under conditions that allow nuclear or cytoplasmic
components such as transcription factors, repressor proteins, or
chromatin remodeling proteins to be added to, or removed from, the
nucleus or resulting chromatin mass. Preferably, the donor nucleus
is contacted with one or more of the following under conditions
that allow formation of a chromatin mass: a mitotic extract in the
presence or absence of an anti-NuMA antibody, a detergent and/or
salt solution, or a protein kinase solution. In other preferred
embodiments, the reconstituted oocyte or the resulting embryo
expresses lamin A, lamin C, or NuMA protein at a level that is less
than 5 fold greater than the corresponding level expressed by a
control oocyte or a control embryo with the same number of cells
and from the same species.
[0006] In a related aspect, the invention provides another method
of cloning a mammal. This method involves incubating a
permeabilized cell with a reprogramming media (e.g., a cell
extract) under conditions that allow the removal of a factor (e.g.,
a nuclear or cytoplasmic component such as a transcription factor)
from a nucleus, chromatin mass, or chromosome of the permeabilized
cell or the addition of a factor to the nucleus, chromatin mass, or
chromosome, thereby forming a reprogrammed cell. The reprogrammed
cell is inserted into an enucleated oocyte, and the resulting
oocyte or an embryo formed from the oocyte is transferred into the
uterus of a host mammal under conditions that allow the oocyte or
embryo to develop into a fetus. In preferred embodiments, the
permeabilized cell is contacted with one or more of the following
under conditions that allow formation of a chromatin mass: a
mitotic extract in the presence or absence of an anti-NuMA
antibody, a detergent and/or salt solution, or a protein kinase
solution. In yet another preferred embodiment, the permeabilized
cell is incubated with an interphase reprogramming media (e.g., an
interphase cell extract). In still another preferred embodiment,
the nucleus in the permeabilized cell remains membrane-bounded, and
the chromosomes in the nucleus do not condense during incubation
with this interphase reprogramming media. In certain embodiments,
incubating the permeabilized cell in the reprogramming media does
not cause DNA replication or only causes DNA replication in less
than 50, 40, 30, 20, 10, or 5% of the cells. In other embodiments,
incubating the permeabilized cell in the reprogramming media causes
DNA replication in at least 60, 70, 80, 90, 95, or 100% of the
cells. In various embodiments, the permeabilized cell is formed by
incubating an intact cell with a detergent, such as digitonin, or a
bacterial toxin, such as Streptolysin O. In yet another preferred
embodiment, the reprogrammed cell is incubated under conditions
that allow the membrane of the reprogrammed cell to reseal prior to
insertion into the oocyte. In other preferred embodiments, the
reconstituted oocyte or the resulting embryo expresses lamin A,
lamin C, or NuMA protein at a level that is less than 5 fold
greater than the corresponding level expressed by a control oocyte
or a control embryo with the same number of cells and from the same
species.
[0007] The invention also provides methods for cloning a mammal
that involve the use of cells from two different embryos. For
example, cells from a nuclear transfer embryo (e.g., an embryo
formed by inserting a cell, nucleus, or chromatin mass into an
enucleated oocyte) can be combined with cells from an in vitro
fertilized, naturally-occurring, or parthenogenetically activated
embryo. Preferably, the majority of the cells and their progeny
from the nuclear transfer embryo are incorporated into fetal tissue
of the resulting chimeric embryo. At least some of the cells and
their progeny from the second embryo are preferably incorporated
into placental tissue and promote the viability of the resulting
chimeric embryo.
[0008] Accordingly, in one such aspect, the invention features a
method of cloning a mammal that involves inserting a cell, nucleus,
or chromatin mass into an enucleated oocyte, thereby forming a
first embryo. One or more cells from the first embryo are contacted
with one or more cells from an in vitro fertilized,
naturally-occurring, or parthenogenetically activated second
embryo, forming a third embryo. The third embryo is transferred
into the uterus of a host mammal under conditions that allow the
third embryo to develop into a fetus. In one embodiment, at least
one of the first embryo and the second embryo is a compaction
embryo. In another embodiment, the first embryo and the second
embryo are at different cell-stages. The first embryo and the donor
cell used to produce the second embryo can be from the same species
or from different genuses or species. Preferably, at least 10, 20,
30, 40, 50, 60, 70, 80, 90, 95, or 100% cells in the trophectoderm
or placental tissue of the fetus are derived from the second
embryo, or at least 30, 40, 50, 60, 70, 80, 90, 95, or 100% cells
in the inner cell mass or fetal tissue of the fetus are derived
from the first embryo. In other preferred embodiments, the first
embryo or the third embryo expresses lamin A, lamin C, or NuMA
protein at a level that is less than 5 fold greater than the
corresponding level expressed by a control embryo with the same
number of cells and from the same species.
[0009] In a related aspect, the invention features another method
of cloning a mammal. This method involves contacting a donor
nucleus with a reprogramming media (e.g., cell extract) under
conditions that allow formation of a chromatin mass, and inserting
the chromatin mass into an enucleated oocyte, thereby forming a
first embryo. One or more cells from the first embryo are contacted
with one or more cells from an in vitro fertilized,
naturally-occurring, or parthenogenetically activated second
embryo, forming a third embryo. The third embryo is transferred
into the uterus of a host mammal under conditions that allow the
third embryo to develop into a fetus. In a preferred embodiment,
the chromatin mass is formed by contacting a donor nucleus that has
less than four sets of homologous chromosomes with a reprogramming
media under conditions that allow formation of a chromatin mass
without causing DNA replication. Preferably, the donor nucleus is
contacted with one or more of the following under conditions that
allow formation of a chromatin mass: a mitotic extract in the
presence or absence of an anti-NuMA antibody, a detergent and/or
salt solution, or a protein kinase solution. In various
embodiments, both the first embryo and the second embryo are
compaction embryos; both the first embryo and the second embryo are
precompaction embryos, or one of the embryos is a compaction embryo
and the other embryo is a precompaction embryo. The first embryo
and the second embryo can be at different cell-stages or at the
same cell-stage. The first embryo and the donor nucleus used to
produce the second embryo can be from the same species or from
different genuses or species. Preferably, at least 10, 20, 30, 40,
50, 60, 70, 80, 90, 95, or 100% cells in the trophectoderm or
placental tissue of the fetus are derived from the second embryo,
or at least 30, 40, 50, 60, 70, 80, 90, 95, or 100% cells in the
inner cell mass or fetal tissue of the fetus are derived from the
first embryo. In other preferred embodiments, the first embryo or
the third embryo expresses lamin A, lamin C, or NuMA protein at a
level that is less than 5 fold greater than the corresponding level
expressed by a control embryo with the same number of cells and
from the same species.
[0010] In another related aspect, the invention features yet
another method of cloning a mammal. This method involves incubating
a permeabilized cell in a reprogramming media (e.g., cell extract)
under conditions that allow the removal of a factor from a nucleus,
chromatin mass, or chromosome of the permeabilized cell or the
addition of a factor from the reprogramming media to the nucleus,
chromatin mass, or chromosome, thereby forming a reprogrammed cell.
The reprogrammed cell is inserted into an enucleated oocyte,
thereby forming a first embryo. One or more cells from the first
embryo are contacted with one or more cells from an in vitro
fertilized, naturally-occurring, or parthenogenetically activated
second embryo, forming a third embryo. The third embryo is
transferred into the uterus of a host mammal under conditions that
allow the third embryo to develop into a fetus. In a preferred
embodiment, the permeabilized cell is incubated with a
reprogramming media (e.g., a cell extract) under conditions that
allow nuclear or cytoplasmic components such as transcription
factors to be added to, or removed from, the nucleus or resulting
chromatin mass. In other preferred embodiments, the permeabilized
cell is contacted with one or more of the following under
conditions that allow formation of a chromatin mass: a mitotic
extract in the presence or absence of an anti-NuMA antibody, a
detergent and/or salt solution, or a protein kinase solution. In
yet another preferred embodiment, the permeabilized cell is
incubated with an interphase reprogramming media (e.g., an
interphase cell extract). In still another preferred embodiment,
the nucleus in the permeabilized cell remains membrane-bounded, and
the chromosomes in the nucleus do not condense during incubation
with this interphase reprogramming media. In some embodiments,
incubating the permeabilized cell in the reprogramming media does
not cause DNA replication or only causes DNA replication in less
than 50, 40, 30, 20, 10, or 5% of the cells. In other embodiments,
incubating the permeabilized cell in the reprogramming media causes
DNA replication in at least 60, 70, 80, 90. 95, or 100% of the
cells. In various embodiments, the permeabilized cell is formed by
incubating an intact cell with a detergent, such as digitonin, or a
bacterial toxin, such as Streptolysin O. In yet another preferred
embodiment, the reprogrammed cell is incubated under conditions
that allow the membrane of the reprogrammed cell to reseal prior to
insertion into the oocyte. In various embodiments, both the first
embryo and the second embryo are compaction embryos; both the first
embryo and the second embryo are precompaction embryos, or one of
the embryos is a compaction embryo and the other embryo is a
precompaction embryo. The first embryo and the second embryo can be
at different cell-stages or at the same cell-stage. The first
embryo and the donor cell used to produce the second embryo can be
from the same species or from different genuses or species.
Preferably, at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or
100% cells in the trophectoderm or placental tissue of the fetus
are derived from the second embryo, or at least 30, 40, 50, 60, 70,
80, 90, 95, or 100% cells in the inner cell mass or fetal tissue of
the fetus are derived from the first embryo. In other preferred
embodiments, the first embryo or the third embryo expresses lamin
A, lamin C, or NuMA protein at a level that is less than 5 fold
greater than the corresponding level expressed by a control embryo
with the same number of cells and from the same species.
[0011] In preferred embodiments of any of the above methods for
cloning a mammal using cells from two embryos, part or all of the
zona pellucida of the first embryo or second embryo is removed
before the cells from each embryo are contacted. In one embodiment,
the cells from the first and second embryos are contacted by being
placed adjacent to each other in solution or on a solid support. In
another embodiment, standard techniques are used to inject cells
from the first embryo into the second embryo. The cells can be
injected into any region of the second embryo, such as the
periphery of the embryo between the zona pellucida and the embryo
itself. Exemplary naturally occurring embryos include embryos that
are surgically or nonsurgically removed from a pregnant mammal
(e.g., a bovine) using standard methods. Exemplary in vitro
fertilized embryos include intra-cytoplasmic sperm injection
embryos generated using standard methods. It is also contemplated
that cells from more than two embryos (e.g., cells from 3, 4, 5, 6,
or more embryos) can be combined to form a chimeric embryo for
generation of a cloned mammal.
[0012] In preferred embodiments of any of the above aspects, the
reprogramming media (e.g., a cell extract) is modified by the
enrichment or depletion of a factor, such as a DNA
methyltransferase, histone deacetylase, histone, protamine, nuclear
lamin, transcription factor, activator, or repressor. In other
preferred embodiments, the level of expression of NuMA or AKAP95
protein in the oocyte or chimeric embryo is at least 2, 5, 10, or
20-fold greater in the nucleus than in the cytoplasm. In yet other
embodiments, at least 30, 40, 50, 60, 70, 80, 90, or 100% of the
AKAP95 protein in the oocyte or chimeric embryo is extracted with a
solution of 0.1% Triton X-100, 1 mg/ml DNase I, and either 100 mM
or 300 mM NaCl. Preferably, the chromatin mass is purified from the
reprogramming media (e.g., extract) prior to insertion into the
enucleated oocyte. In another preferred embodiment, inserting the
chromatin mass into the enucleated oocyte involves contacting the
chromatin mass and the oocyte with a fusigenic compound under
conditions that allow the chromatin mass to enter the ooctye. In
yet another preferred embodiment, the fetus develops into a viable
offspring. Preferably, at least 1, 3, 5, 10, 20, 30, 40, 50, 60,
70, 80, or 90% of the nuclear transfer oocytes or embryos develop
into viable offspring. In this method, the oocyte containing the
chromatin mass or reprogrammed cell may be cultured under
conditions that allow cell division and one of the resulting cells
may be recloned one or more times. The donor nucleus, donor
chromatin mass, or donor cell and the oocyte used in the method may
be from the same species, or they may be from different species or
genuses. The mammal may be a human or non-human mammal, and the
oocyte may be fertilized or unfertilized. Preferably the donor
nucleus, chromatin mass, or permeabilized cell is from a G.sub.1 or
G.sub.0 phase cell. In addition, the genomic DNA of the cloned
embryo, fetus, or mammal is preferably substantially identical to
that of the donor cell. It is also contemplated that the chromatin
mass or reprogrammed cell may be inserted into an embryo for the
production of a chimeric embryo, fetus, or mammal containing a
mixture of cells with DNA substantially identical to that of the
chromatin mass or reprogrammed cell and cells with DNA
substantially identical to that of the naturally-occurring cells in
the embryo. It is also contemplated that a nucleated oocyte may be
used in the methods of the invention.
[0013] The reprogramming media used in any of the aspects of the
invention may or may not contain exogenous nucleotides. In other
preferred embodiments, a chromatin mass in a reprogramming media or
formed in a permeabilized cell is contacted with a vector having a
nucleic acid encoding a gene of interest under conditions that
allow random integration or homologous recombination between the
nucleic acid in the vector and the corresponding nucleic acid in
the genome of the chromatin mass, resulting in the alteration of
the genome of the chromatin mass. Due to the lack of an intact
plasma membrane and the lack of a nuclear membrane, a chromatin
mass in a permeabilized cell or in solution may be easier to
genetically modify than a naturally-occurring cell. Examples of
cells that may be used to generate reprogramming extracts include
embryonic stem cells and adult stem cells from brain, blood, bone
marrow, pancreas, liver, skin, or any other organ or tissue. Other
exemplary reprogramming cell extracts include oocyte extracts
(e.g., bovine or sea urchin oocyte extracts) and male germ cell
extracts (e.g., spermatogonia, spermatocyte, spermatid, or sperm
extracts from vertebrates, invertebrates, or mammals such as
bovine). The donor or permeabilized cell can be non-immortalized or
naturally, spontaneously, or genetically immortalized. The donor
cell, permeabilized cell, recipient cell, or cytoplast can be from
a source of any age, such as an embryo, fetus, youth, or adult
mammal. Cells from younger sources may have acquired fewer
spontaneous mutations and may have a longer life-span after
insertion into an oocyte.
[0014] The invention also provides methods of inserting
chromosomes, chromatin masses, or nuclei into recipient cells.
These methods are useful for transferring donor genetic material
into a recipient oocyte for the cloning of a mammal. These methods
may also be used to replace the genetic material of one cell with
that of another cell.
[0015] According to this aspect of the invention, a technique is
provided for inserting chromosomes or a chromatin mass into a
recipient cell that involves contacting the chromosomes or
chromatin mass and the cell with a fusigenic compound under
conditions that allow the chromosomes or chromatin mass to enter
the recipient cell. In one preferred embodiment, the chromosomes or
the chromatin mass are incubated with the fusigenic compound prior
to being contacted with the recipient cell. The chromosomes or
chromatin mass may be condensed or not condensed, and the
chromosomes or chromatin mass and the recipient cell may be from
the same species or may be from different species or genuses. In
another preferred embodiment, the recipient cell is a fertilized or
unfertilized oocyte. Preferably, the recipient cell or the
chromosomes are from a human or non-human mammal. In various
embodiments, the recipient cell is an adult, fetal, or embryonic
cell. In one particular preferred embodiment, all of the
chromosomes of a donor cell are inserted into the recipient cell.
Preferably, the donor cell is haploid (DNA content of n), diploid
(2n), or tetraploid (4n), and the recipient cell is hypodiploid
(DNA content of less than 2n), haploid, or enucleated. In another
embodiment, the chromosomes are from more than one donor cell, such
as two haploid cells. In yet another preferred embodiment, the
chromosomes are obtained by contacting a donor nucleus that has
less than four sets of homologous chromosomes with a mitotic
extract, a detergent and/or salt, or a protein kinase under
conditions that allow formation of a chromatin mass without causing
DNA replication. Preferred fusigenic compounds include polyethylene
glycol (PEG), and lipids such as Lipofectin.RTM.,
Lipofectamin.RTM., DOTAP.RTM.
{N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylamonium
methylsulfate; C.sub.43H.sub.83NO.sub.8S},
DOSPA.RTM.{2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1--
propanaminium trifuoroacetate}, and DOPE.RTM. (dioleoyl
phosphatidylethanolamine). Other preferred lipids include neutral
and monovalent or multivalent cationic lipids, such as those
containing quaternary ammonium groups. Additional preferred lipids
have a cholesterol moiety such as that formed from the reaction of
the hydroxyl group in cholesterol with a group in the lipid. Still
other preferred lipids have a saturated or unsaturated fatty acid
that preferably contains between 5 and 10, 10 and 15, 15 and 20, or
20 and 30 carbon atoms, inclusive. These lipids may be synthesized
using standard chemical synthesis techniques, obtained from
naturally-occurring sources, or purchased from commercially
available source (Summers et al., Biophys J. 71(6):3199-206, 1996;
Nabekura et al., Pharm Res.13(7):1069-72, 1996; Walter et al.,
Biophys J. 66(2 Pt 1):366-376, 1994; Yang et al., Biosci Rep.
13(3):143-157, 1993; Walter and Siegel, Biochemistry.
6:32(13):3271-3281, 1993). Other preferred fusigenic compounds are
phospholipids such as membrane vesicle fractions from sea urchin
eggs or any other source (Collas and Poccia, J. of Cell Science
109, 1275:1283, 1996). Preferably, contacting chromosomes with the
membrane vesicle fraction does not result in the chromosomes being
encapsulated by an intact membrane.
[0016] In a related aspect, the invention provides a method of
inserting a nucleus into a recipient cell that includes contacting
the nucleus and the cell with a fusigenic compound under conditions
that allow the nucleus to enter the recipient cell. The fusigenic
compound is either a lipid or is not a polymer consisting of
identical monomers. Preferably, the nucleus is incubated with the
fusigenic compound prior to being contacted with the recipient
cell. In various embodiments, the nucleus and the recipient cell
are from the same species or are from different species or
different genuses. Preferably, the nucleus is haploid, diploid, or
tetraploid, and the recipient cell is hypodiploid, haploid, or
enucleated. In one preferred embodiment, the recipient cell is a
fertilized or unfertilized oocyte. Preferably, the recipient cell
or the nucleus is from a human or a non-human mammal. In other
embodiments, the recipient cell is an adult, fetal, or embryonic
cell. Preferred fusigenic compounds are lipids such as
Lipofectin.RTM., Lipofectamin.RTM., DOTAP.RTM., DOSPA.RTM., and
DOPE.RTM.. Other preferred lipids include neutral lipids and
monovalent or multivalent cationic lipids, such as those containing
quaternary ammonium groups. Additional preferred lipids have a
cholesterol moiety or a saturated or unsaturated fatty acid that
preferably contains between 5 and 10, 10 and 1 5, 1 5 and 20, or 20
and 30 carbon atoms, inclusive. Other preferred fusigenic compounds
are phospholipids such as membrane vesicle fractions from sea
urchin eggs or any other source (Collas and Poccia, supra).
Preferably, contacting a nucleus with the membrane vesicle fraction
does not result in the nucleus being encapsulated by an intact
membrane.
[0017] In preferred embodiments of various aspects of the
invention, the nucleus or chromosomes are from an adult, fetal, or
embryonic cell. The nucleus or chromosomes may also be obtained
from any of the following preferred donor cells, or they may be
inserted into any of the following preferred recipient cells.
Examples of preferred cells include differentiated cells such as
epithelial cells, neural cells, epidermal cells, keratinocytes,
hematopoietic cells, melanocytes, chondrocytes, B-lymphocytes,
T-lymphocytes, erythrocytes, macrophages, monocytes, fibroblasts,
and muscle cells; and undifferentiated cells such as embryonic
cells (e.g., stem cells and embryonic germ cells). In another
preferred embodiment, the cell is from the female reproductive
system, such as a mammary gland, ovarian cumulus, granulosa, or
oviductal cell. Other preferred cells include fetal cells and
placental cells. Preferred cells also include those from any organ,
such as the bladder, brain, esophagus, fallopian tube, heart,
intestines, gallbladder, kidney, liver, lung, ovaries, pancreas,
prostate, spinal cord, spleen, stomach, testes, thymus, thyroid,
trachea, ureter, urethra, and uterus. Preferred non-human mammals
include members of the genus Bos. Examples of other preferred
mammals include cows, sheep, big-horn sheep, goats, buffalos,
antelopes, oxen, horses, donkeys, mule, deer, elk, caribou, water
buffalo, camels, llama, alpaca, rabbits, pigs, mice, rats, guinea
pigs, hamsters, and primates such as monkeys. In yet another
preferred embodiment, the nucleus, permeabilized cell, or
chromosomes are from a transgenic cell or mammal or contain a
mutation not found in the donor cell or not found in a
naturally-occurring cell.
[0018] Preferred transgenic donor nuclei and donor cells encode
proteins that confer improved resistance to disease or parasites in
the cloned mammal. Alternatively, the donor nuclei or donor cells
may be engineered so that the cloned mammal produces a recombinant
product, such as the production of a human protein in the urine,
blood, or milk of a bovine. For example, proteins may be expressed
in the urine of cattle by inserting a polynucleotide sequence
encoding a human protein under the control of an uroplakin
promoter. Examples of therapeutic proteins that made be produced in
the milk of cloned bovines include human monoclonal antibodies and
human clotting factors such as any of factors I to XIII (Voet and
Voet, Biochemistry, John Wiley & Sons, New York, 1990). These
heterologous proteins may be expressed under the control of a
prolactin promoter or any other promoter suitable for expression in
the milk of a bovine. For the production of human antibodies in the
milk, blood, or other fluids of cloned mammals, standard methods
may be used to inactivate or "knockout" the endogenous genes for
antibody heavy or light chains so that functional antibodies are no
longer encoded by a donor nucleus and to insert genes encoding the
heavy and light chains of human IgA, IgD, IgE, IgG, or IgM into the
genome of the donor nucleus. Recombinant proteins from these or
other tissues or fluids may be purified using standard purification
methods (see, for example, Ausubel et al., supra).
[0019] It is also contemplated that cells, tissues, or organs from
an embryo, fetus, or adult mammal produced using the methods of the
invention may be used as a source of material for medical
applications, such as the treatment or prevention of disease in
humans. For example, cells, tissues, or organs may be developed in
vitro from a cloned embryo and then transferred to a mammal (e.g.,
a human), removed from a cloned mammal and transferred to another
mammal of a different species, or removed from a cloned mammal and
transferred to another mammal of the same species. For example,
neuronal tissue from a cloned mammal may be grafted into an
appropriate area in the human nervous system to treat, prevent, or
stabilize a neurological disease such as Alzheimer's disease,
Parkinson's disease, Huntington's disease, or ALS; or a spinal cord
injury. In particular, degenerating or injured neuronal cells may
be replaced by the corresponding cells from a cloned mammal. This
transplantation method may also be used to treat, prevent, or
stabilize autoimmune diseases including, but not limited to,
insulin dependent diabetes mellitus, rheumatoid arthritis,
pemphigus vulgaris, multiple sclerosis, and myasthenia gravis. In
these procedures, the cells that are attacked by the recipient's
own immune system may be replaced by transplanted cells. The cloned
mammals may also be used as a source of cartilage, bone marrow, or
any other tissue or organ.
[0020] For the production of a cloned mammal as a source of donor
transplant material, the donor nucleus or donor cell used to
generate the cloned mammal is preferably modified to encode a
heterologous MHC Class I protein having an amino acid sequence
substantially identical to the sequence of a MHC Class I protein
found in the recipient mammal that will be administered the donor
material. Alternatively, the donor nucleus encodes a heterologous
MHC Class I protein having an amino acid sequence substantially
identical to the sequence of an MHC Class I protein found in
another mammal of the same genus or species as the recipient
mammal. These donor cells, tissues, or organs from cloned mammals
that express heterologous MHC proteins are less likely to elicit an
adverse immune response when administered to the recipient mammal.
Other preferred donor transplant material is obtained from a cloned
mammal that was generated using a donor nucleus or donor cell which
was modified to express a heterologous protein that inhibits the
complement pathway of the recipient mammal, such as the human
complement inhibitor CD59 or the human complement regulator decay
accelerating factor (h-DAF) (see, for example, Ramirez et al.,
Transplantation 15:989-998, 2000; Costa et al., Xenotransplantation
6:6-16, 1999). In yet another preferred embodiment, the donor
nucleus or donor cell has a mutation that reduces or eliminates the
expression or activity of a galactosyltransferase, such as
alpha(1,3)-galactosyltransferase (Tearle et al., Transplantation
61:13-19, 1996; Sandrin, Immunol. Rev. 141:169-190, 1994; Costa et
al., Xenotransplantation 6:6-16, 1999). This enzyme modifies cell
surface molecules with a carbohydrate that elicits an adverse
immune response when cells expressing this galactose
alpha(1,3)-galactose epitope are administered to humans. Thus,
donor transplant material that has a lower level of expression of
this epitope may have a lower incidence of rejection by the
recipient mammal.
[0021] As used herein, by "chromatin mass" is meant more than one
chromosome not enclosed by a membrane. Preferably, the chromatin
mass contains all of the chromosomes of a cell. An artificially
induced chromatin mass containing condensed chromosomes may be
formed by exposure of a nucleus to a mitotic reprogramming media
(e.g., a mitotic extract) as described herein. Alternatively, an
artificially induced chromatin mass containing decondensed or
partially condensed chromosomes may be generated by exposure of a
nucleus to one of the following, as described herein: a mitotic
extract containing an anti-NuMA antibody, a detergent and/or salt
solution, or a protein kinase solution. A chromatin mass may
contain discrete chromosomes that are not physically touching each
other or may contain two or more chromosomes that are in physical
contact.
[0022] If desired, the level of chromosome condensation may be
determined using standard methods by measuring the intensity of
staining with the DNA stain, DAPI. As chromosomes condense, this
staining intensity increases. Thus, the staining intensity of the
chromosomes may be compared to the staining intensity for
decondensed chromosomes in interphase (designated 0% condensed) and
maximally condensed chromosomes in mitosis (designated 100%
condensed). Based on this comparison, the percent of maximal
condensation may be determined. Preferred condensed chromatin
masses are at least 50, 60, 70, 80, 90, or 100% condensed.
Preferred decondensed or partially condensed chromatin masses are
less than 50, 40, 30, 20, or 10% condensed.
[0023] By "nucleus" is meant a membrane-bounded organelle
containing most or all of the DNA of a cell. The DNA is packaged
into chromosomes in a decondensed form. Preferably, the membrane
encapsulating the DNA includes one or two lipid bilayers or has
nucleoporins.
[0024] By "nucleus that has less than four sets of homologous
chromosomes" is meant a nucleus that has a DNA content of less than
4n, where "n" is the number of chromosomes found in the normal
haploid chromosome set of a mammal of a particular genus or
species. Such a nucleus does not have four copies of each gene or
genetic locus. Preferably, the nucleus is diploid and thus has two
sets of homologous chromosomes but has less than two complete pairs
of chromatids.
[0025] By "pronucleus" is meant a haploid nucleus resulting from
meiosis or a nuclear transfer pronucleus. The female pronucleus is
the nucleus of the oocyte or ovum before fusion with the male
pronucleus. The male pronucleus is the sperm nucleus after it has
entered the oocyte or ovum at fertilization but before fusion with
the female pronucleus. A nuclear transfer pronucleus is a
pronucleus (e.g., a diploid pronucleus) that forms after
introduction of a donor cell, nucleus, or chromatin mass into an
oocyte. The nuclear transfer pronucleus has less than four sets of
homologous chromosomes.
[0026] By "donor cell" is meant a cell from which a nucleus or
chromatin mass is derived, or a permeabilized cell.
[0027] By "permeabilization" is meant the formation of pores in the
plasma membrane or the partial or complete removal of the plasma
membrane.
[0028] By "reprogramming media" is meant a solution that allows the
removal of a factor from a cell, nucleus, chromatin mass, or
chromosome or the addition of a factor from the solution to the
cell, nucleus, chromatin mass, or chromosome. Preferably, the
addition or removal of a factor increases or decreases the level of
expression of an mRNA or protein in the donor cell, chromatin mass,
or nucleus or in a cell containing the reprogrammed chromatin mass
or nucleus. In another embodiment, incubating a permeabilized cell,
chromatin mass, or nucleus in the reprogramming media alters a
phenotype of the permeabilized cell or a cell containing the
reprogrammed chromatin mass or nucleus relative to the phenotype of
the donor cell. In yet another embodiment, incubating a
permeabilized cell, chromatin mass, or nucleus in the reprogramming
media causes the permeabilized cell or a cell containing the
reprogrammed chromatin mass or nucleus to gain or lose an activity
relative to the donor cell.
[0029] Exemplary reprogramming media include solutions, such as
buffers, that do not contain biological molecules such as proteins
or nucleic acids. Such solutions are useful for the removal of one
or more factors from a nucleus, chromatin mass, or chromosome.
Other preferred reprogramming medias are extracts, such as cellular
extracts from cell nuclei, cell cytoplasm, or a combination
thereof. Exemplary cell extracts include extracts from oocytes
(e.g., mammalian, vertebrate, or invertebrate oocytes), male germ
cells (mammalian, vertebrate, or invertebrate germ cells such as
spermatogonia, spermatocyte, spermatid, or sperm), and stem cells
(e.g., adult or embryonic stem cells). Yet other reprogramming
media are solutions or extracts to which one or more
naturally-occurring or recombinant factors (e.g., nucleic acids or
proteins such as DNA methyltransferases, histone deacetylases,
histones, protamines, nuclear lamins, transcription factors,
activators, repressors, chromatin remodeling proteins, growth
factors, interleukins, cytokines, or other hormones) have been
added, or extracts from which one or more factors have been
removed. Still other reprogramming media include solutions of
detergent (e.g., 0.01% to 0.1%, 0.1% to 0.5%, or 0.5% to 2% ionic
or non-ionic detergent such as one or more of the following
detergents: SDS, Triton X-100, Triton X-114, CHAPS,
Na-deoxycholate, n-octyl glucoside, Nonidet P40, IGEPAL, Tween 20,
Tween 40, or Tween 80), salt (e.g., .about.0.1, 0.15, 0.25, 0.5,
0.75, 1, 1.5, or 2 M NaCl or KCl), polyamine (e.g., .about.1 .mu.M,
10 .mu.M, 100 .mu.M, 1 mM or 10 mM spermine, spermidine, protamine,
or poly-L-lysine), a protein kinase (e.g., cyclin-dependent kinase
1, protein kinase C, protein kinase A, MAP kinase,
calcium/calmodulin-dependent kinase, CK1 casein kinase, or CK2
casein kinase), and/or a phosphatase inhibitor (e.g., .about.10
.mu.M, 100 .mu.M, 1 mM, 10 mM, 50 mM, 100 mM of one or more of the
following inhibitors: Na-orthovanadate, Na-pyrophosphate,
Na-fluoride, NIPP1, inhibitor 2, PNUTS, SDS22, AKAP149, or ocadaic
acid). In some embodiments, the reprogramming medium contains an
anti-NuMA antibody. If desired, multiple reprogramming media may be
used simultaneously or sequentially to reprogram a donor cell,
nucleus, or chromatin mass.
[0030] By "interphase reprogramming media" is meant a media (e.g.,
an interphase cell extract) that induces chromatin decondensation
and nuclear envelope formation.
[0031] By "mitotic reprogramming media" is meant a media (e.g., a
mitotic cell extract) that induces chromatin condensation and
nuclear envelope breakdown.
[0032] By "reprogrammed cell" is meant a cell that has been exposed
to a reprogramming media. Preferably, at least 1, 5, 10, 15, 20,
25, 50, 75, 100, 150, 200, 300, or more mRNA or protein molecules
are expressed in the reprogrammed cell that are not expressed in
the donor or permeabilized cell. In another preferred embodiment,
the number of mRNA or protein molecules that are expressed in the
reprogrammed cell, but not expressed in the donor or permeabilized
cell, is between 1 and 5, 5 and 10, 10 and 25, 25 and 50, 50 and
75, 75 and 100, 100 and 150, 150 and 200, or 200 and 300,
inclusive. Preferably, at least 1, 5, 10, 15, 20, 25, 50, 75, 100,
150, 200, 300, or more mRNA or protein molecules are expressed in
the donor or permeabilized cell that are not expressed in the
reprogrammed cell. In yet another preferred embodiment, the number
of mRNA or protein molecules that are expressed in the donor or
permeabilized cell, but not expressed in the reprogrammed cell, is
between 1 and 5, 5 and 10, 10 and 25, 25 and 50, 50 and 75, 75 and
100, 100 and 150, 150 and 200, or 200 and 300, inclusive. In still
another preferred embodiment, these mRNA or protein molecules are
expressed in both the donor cell (i.e., the donor or permeabilized
starting cell) and the reprogrammed cell, but the expression levels
in these cells differ by at least 2, 5, 10, or 20-fold, as measured
using standard assays (see, for example, Ausubel et al., Current
Protocols in Molecular Biology, John Wiley & Sons, New York,
2000).
[0033] By "addition of a factor" is meant the binding of a factor
to chromatin, a chromosome, or a component of the nuclear envelope,
such as the nuclear membrane or nuclear matrix. Alternatively, the
factor is imported into the nucleus so that it is bounded or
encapsulated by the nuclear envelope. Preferably, the amount of
factor that is bound to a chromosome or located in the nucleus
increases by at least 25, 50, 75, 100, 200, or 500%.
[0034] By "removal of a factor" is meant the dissociation of a
factor from chromatin, a chromosome, or a component of the nuclear
envelope, such as the nuclear membrane or nuclear matrix.
Alternatively, the factor is exported out of the nucleus so that it
is no longer bounded or encapsulated by the nuclear envelope.
Preferably, the amount of factor that is bound to a chromosome or
located in the nucleus decreases by at least 25, 50, 75, 100, 200,
or 500%.
[0035] By "enrichment or depletion of a factor" is meant the
addition or removal of a naturally-occurring or recombinant factor
by at least 20, 40, 60, 80, or 100% of the amount of the factor
originally present in an reprogramming media (e.g., a cell
extract). Alternatively, a naturally-occurring or recombinant
factor that is not naturally present in the reprogramming media may
be added. Preferred factors include proteins such as DNA
methyltransferases, histone deacetylases, histones, protamines,
nuclear lamins, transcription factors, activators, and repressors;
membrane vesicles, and organelles. In one preferred embodiment, the
factor is purified prior to being added to the reprogramming media,
as described below. Alternatively, one of the purification methods
described below may be used to remove an undesired factor from the
reprogramming media.
[0036] By "purified" is meant separated from other components that
naturally accompany it. Typically, a factor is substantially pure
when it is at least 50%, by weight, free from proteins, antibodies,
and naturally-occurring organic molecules with which it is
naturally associated. Preferably, the factor is at least 75%, more
preferably, at least 90%, and most preferably, at least 99%, by
weight, pure. A substantially pure factor may be obtained by
chemical synthesis, separation of the factor from natural sources,
or production of the factor in a recombinant host cell that does
not naturally produce the factor. Proteins, vesicles, and
organelles may be purified by one skilled in the art using standard
techniques such as those described by Ausubel et al. (Current
Protocols in Molecular Biology, John Wiley & Sons, New York,
2000). The factor is preferably at least 2, 5, or 10 times as pure
as the starting material, as measured using polyacrylamide gel
electrophoresis, column chromatography, optical density, HPLC
analysis, or western analysis (Ausubel et al., supra). Preferred
methods of purification include immunoprecipitation, column
chromatography such as immunoaffinity chromatography, magnetic bead
immunoaffinity purification, and panning with a plate-bound
antibody.
[0037] By "recloned" is meant used in a second round of cloning. In
particular, a cell from an embryo, fetus, or adult generated from
the methods of the invention may be incubated in a mitotic
reprogramming media (e.g., a mitotic cell extract) to form a
chromatin mass for insertion into an enucleated oocyte, as
described above. Alternatively, the cell may be permeabilized,
incubated in a reprogramming media, and inserted into an enucleated
oocyte, as described above. Performing two or more rounds of
cloning may result in additional reprogramming of the donor
chromatin mass or donor cell, thereby increasing the chance of
generating a viable offspring after the last round of cloning.
[0038] By "viable offspring" is meant a mammal that survives ex
utero. Preferably, the mammal is alive for at least one second, one
minute, one hour, one day, one week, one month, six months, or one
year from the time it exits the maternal host. The mammal does not
require the circulatory system of an in utero environment for
survival.
[0039] By "nuclear transfer oocyte" or "nuclear transplant oocyte"
is meant an oocyte in which a donor cell, nucleus, or chromatin
mass is inserted or fused. An embryo formed from the oocyte is
referred to as a "nuclear transfer" or "nuclear transplant"
embryo.
[0040] By "embryo" or "embryonic" is meant a developing cell mass
that has not implanted into the uterine membrane of a maternal
host. Hence, the term "embryo" may refer to a fertilized oocyte; an
oocyte containing a donor chromatin mass, nucleus, or reprogrammed
cell; a pre-blastocyst stage developing cell mass; or any other
developing cell mass that is at a stage of development prior to
implantation into the uterine membrane of a maternal host and prior
to formation of a genital ridge. An embryo may represent multiple
stages of cell development. For example, a one cell embryo can be
referred to as a zygote; a solid spherical mass of cells resulting
from a cleaved embryo can be referred to as a morula, and an embryo
having a blastocoel can be referred to as a blastocyst. An
"embryonic cell" is a cell isolated from or contained in an
embryo.
[0041] By "cells derived from an embryo" is meant cells that result
from the cell division of cells in the embryo.
[0042] By "chimeric embryo" is meant an embryo formed from cells
from two or more embryos. The resulting fetus or offspring can have
cells that are derived from only one of the initial embryos or
cells derived from more than one of the initial embryos. If
desired, the percentage of cells from each embryo are incorporated
into the placental tissue and into the fetal tissue can be
determined using standard FISH analysis or analysis of a membrane
dye added to one embryo.
[0043] By "precompaction embryo" is meant an embryo prior to
compaction. A precompaction embryo expresses essentially no
E-cadherin on the surface of its blastomereres. Preferred
precompaction embryos express at least 3, 5, 10, 20, 30, or 40-fold
less E-cadherin than a fully compacted embryo of the same species,
or express no E-adherin.
[0044] By "compaction embryo" is meant an embryo undergoing
compaction or following compaction. The blastomeres of a compaction
embryo express E-cadherin on their surface. This E-cadherin
expression can be measuring using standard methods with an anti-
E-cadherin antibody. E-cadherin increases the adherence between
blastomeres. Preferred compaction embryos include embryos in which
the compaction process is completed. Other preferred compaction
embryos express at least 3, 5, 10, 20, 30, or 40-fold more
E-cadherin than a precompaction embryo of the same species.
[0045] By "fetus" is meant a developing cell mass that has
implanted into the uterine membrane of a maternal host. A fetus may
have defining features such as a genital ridge which is easily
identified by a person of ordinary skill in the art. A "fetal cell"
is any cell isolated from or contained in a fetus.
[0046] By "parthenogenesis" or "parthenogenetic activation" is
meant development of an oocyte or ovum without fusion of its
nucleus with a male pronucleus to form a zygote. For example, an
oocyte can be induced to divide without fertilization.
[0047] By "zona pellucida" is meant a translucent, elastic,
noncellular layer surrounding the oocyte or ovum of many
mammals.
[0048] By "trophectoderm" is meant the outermost layer of cells
surrounding the blastocoel during the blastocyst stage of mammalian
embryonic development. Trophectoderm gives rise to most or all of
the placental tissue upon further development.
[0049] By "inner cell mass" is meant the cells surrounded by the
trophectoderm. The inner cell mass cells give rise to most of the
fetal tissues upon further development.
[0050] By "mRNA or protein specific for one cell type" is meant an
mRNA or protein that is expressed in one cell type at a level that
is at least 10, 20, 50, 75, or 100 fold greater than the expression
level in all other cell types. Preferably, the mRNA or protein is
only expressed in one cell type.
[0051] By "mutation" is meant an alteration in a
naturally-occurring or reference nucleic acid sequence, such as an
insertion, deletion, frameshift mutation, silent mutation, nonsense
mutation, or missense mutation. Preferably, the amino acid sequence
encoded by the nucleic acid sequence has at least one amino acid
alteration from a naturally-occurring sequence. Examples of
recombinant DNA techniques for altering the genomic sequence of a
cell, embryo, fetus, or mammal include inserting a DNA sequence
from another organism (e.g., a human) into the genome, deleting one
or more DNA sequences, and introducing one or more base mutations
(e.g., site-directed or random mutations) into a target DNA
sequence. Examples of methods for producing these modifications
include retroviral insertion, artificial chromosome techniques,
gene insertion, random insertion with tissue specific promoters,
homologous recombination, gene targeting, transposable elements,
and any other method for introducing foreign DNA. All of these
techniques are well known to those skilled in the art of molecular
biology (see, for example, Ausubel et al., supra). Chromatin
masses, chromosomes, and nuclei from transgenic cells containing
modified DNA or donor transgenic cells may be used in the methods
of the invention.
[0052] By "immortalized" is meant capable of undergoing at least
25, 50, 75, 90, or 95% more cell divisions than a
naturally-occurring control cell of the same cell type, genus, and
species as the immortalized cell or than the donor cell from which
the immortalized cell was derived. Preferably, an immortalized cell
is capable of undergoing at least 2, 5, 10, or 20-fold more cell
divisions than the control cell. More preferably, the immortalized
cell is capable of undergoing an unlimited number of cell
divisions. Examples of immortalized cells include cells that
naturally acquire a mutation in vivo or in vitro that alters their
normal growth-regulating process. Still other preferred
immortalized cells include cells that have been genetically
modified to express an oncogene, such as ras, myc, abl, bcl2, or
neu, or that have been infected with a transforming DNA or RNA
virus, such as Epstein Barr virus or SV40 virus (Kumar et al.,
Immunol. Lett. 65:153-159, 1999; Knight et al., Proc. Nat. Acad.
Sci. USA 85:3130-3134, 1988; Shammah et al., J. Immunol. Methods
160-19-25, 1993; Gustafsson and Hinkula, Hum. Antibodies Hybridomas
5:98-104, 1994; Kataoka et al., Differentiation 62:201-211, 1997;
Chatelut et al., Scand. J. Immunol. 48:659-666, 1998). Cells can
also be genetically modified to express the telomerase gene (Roques
et al., Cancer Res. 61:8405-8507, 2001).
[0053] By "non-immortalized" is meant not immortalized as described
above.
[0054] By "fusigenic compound" is meant a compound that increases
the probability that a chromatin mass or nucleus is inserted into a
recipient cell when located adjacent to the cell. For example, the
fusigenic compound may increase the affinity of a chromatin mass or
a nucleus for the plasma membrane of a cell. The fusigenic compound
may also promote the joining of the nuclear membrane of a nucleus
with the plasma membrane of a cell.
[0055] By "substantially identical" is meant having a sequence that
is at least 60, 70, 80, 90, or 100% identical to that of another
sequence. Sequence identity is typically measured using sequence
analysis software with the default parameters specified therein
(e.g., Sequence Analysis Software Package of the Genetics Computer
Group, University of Wisconsin Biotechnology Center, 1710
University Avenue, Madison, Wis. 53705). This software program
matches similar sequences by assigning degrees of homology to
various substitutions, deletions, and other modifications.
[0056] The present invention provides a number of advantages
related to the cloning of mammals and the transfer of genomic
material into recipient cells. For example, the methods may result
in a higher percentage of viable offspring, increasing the number
of mammals that may be used for agricultural or medical
applications. Compared to microinjection, the method described
herein for the transfer of chromosomes, chromatin masses, or nuclei
into cells, called lipofusion, is a gentler and simpler means of
introducing genetic material into cells since it does not require
physical disruption of cellular structures or the technical skill
needed to pick up a nucleus or chromatin mass using a micropipette
and inject it into a cell. The present method may also be safer
than fusion methods involving viruses or viral components. Further,
lipofusion is believed to elicit minimal, if any, physiological
damage to the recipient cell and is therefore beneficial over
electrofusion which elicits signaling events inside the fused cells
that may impair cell cycle progression or development of the cloned
embryo.
[0057] Other features and advantages of the invention will be
apparent from the following detailed description and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0058] The application file contains drawings executed in color
(FIGS. 3, 6A-6C, 8A, and 8B). Copies of this patent or patent
application with color drawings will be provided by the Office upon
request and payment of the necessary fee.
[0059] FIGS. 1A and 1B illustrate the immunodetection of nuclear
envelope and nuclear matrix proteins in bovine preimplantation
embryos. FIG. 1A is a picture of in vitro-fertilized bovine embryos
at the pronuclear and 8-cell stage examined using the same
antibodies. Arrows in FIG. 1A to anti-NuMA and anti-AKAP95 labeling
in the female pronucleus of pronuclear stage embryos. Insets in
FIG. 1A are pictures of DNA labeled with 0.1 .mu.g/ml Hoechst 33342
(bars, 20 .mu.m). FIG. 1B is the immunoblotting analysis of bovine
fibroblasts (upper rows) and pronuclear stage embryos (lower rows).
Molecular weight markers are shown in kDa on the right of FIG.
1B.
[0060] FIG. 2 illustrates the dynamics of the nuclear envelope,
NuMA, and AKAP95 during premature chromatin condensation and
pronuclear assembly in nuclear transplant embryos. FIG. 2 is a
picture of bovine donor fibroblasts (Donor cell), nuclear
transplant embryos at the premature chromatin condensation stage
(three hours post-fusion), nuclear transplant embryos at the
pronuclear stage (19 hours post-fusion), and parthenogenetic
pronuclear stage embryos activated as described herein. Disassembly
of the donor nucleus and assembly of the new pronuclei were
monitored at the premature chromatin condensation stage three hours
post injection "hpi" ("PCC") and seven hours post injection ("NT
PN"), using anti-lamin B, lamins A/C, NuMA, and AKAP95 antibodies.
Female pronuclei formed after parthenogenetic activation of MII
oocytes with 10 mM SrCl.sub.2 were also analyzed five hours after
start of activation treatment ("Parth. PN"). Lamins A/C were
assembled in pronuclei of bovine pronuclear stage nuclear
transplant embryos. DNA was counterstained with 0.1 .mu.g/ml
Hoechst 33342. TRITC refers to labeling with TRITC-conjugated
secondary antibodies (bars, 20 .mu.m).
[0061] FIG. 3 is a graph demonstrating that AKAP95 is more strongly
anchored in pronuclei of nuclear transplant embryos compared to
parthenogenetic embryos. This graph shows the relative percent of
unextracted lamin B, AKAP95, and DNA labeling in pronuclei of
parthenotes, nuclear transplant embryos, and somatic donor nuclei
after in situ extraction with 0.1% Triton X-100 and 1 mg/ml DNAse I
together with 100 or 300 mM NaCl for 30 minutes at room temperature
prior to fixation with 3% paraformaldehyde. Localization of B-type
lamins (red) and AKAP95 (green) was examined by double
immunofluorescence. Fluorescence labeling intensity in each
channel--red, (lamin B), blue (DNA), and green (AKAP95)--was
quantified. The reference value (100% unextracted) represents
relative amounts of B-type lamins, DNA, and AKAP95 staining in
embryos or cells permeabilized with 0.1% Triton X-100 only prior to
fixation. Approximately 30 embryos were examined in each group.
[0062] FIG. 4 demonstrates that lamins A/C are transcribed de novo
upon pronuclear reconstitution in nuclear transplant embryos. FIG.
4 is a picture of bovine pronuclear nuclear transplant embryos
produced by fibroblast fusion and oocyte activation with either 5
.mu.M ionomycin for four minutes followed by 10 .mu.g/ml
cycloheximide/2.5 .mu.g/ml cytochalasin D for four hours (b', -),
ionomycin/cycloheximide/cytochalasin D as in (b') followed by an
additional nine hours of culture with 10 .mu.g/ml cycloheximide
(b'', CHX) or incubation as in (b') together with 1 .mu.g/ml
actinomycin D during the entire activation treatment (b''').
Anti-lamin B (rabbit polyclonal) and anti-lamins A/C (mAb)
antibodies were used on the same preparations. Insets are pictures
of DNA labeling with 0.1 .mu.g/ml Hoechst 33342 (bars, 20
.mu.m).
[0063] FIG. 5 is a graph of chromosome condensation and nuclear
envelope breakdown in mitotic cytoplasmic extract (M-S15), mitotic
cytosolic extract (M-S200), and oocyte extract (MII-S15) (n=300-400
nuclei examined in 3-5 replicates).
[0064] FIGS. 6A-6C are sets of pictures of immunofluorescence
analysis of purified input bovine fibroblast nuclei (FIG. 6A) and
condensed chromatin produced in mitotic cytosolic extract (FIG. 6B)
and oocyte extract (FIG. 6C). The indicated nuclear markers were
examined. DNA was counterstained with propidium iodide (red) (bars,
10 .mu.m).
[0065] FIG. 7 is a set of pictures of immunofluorescence analysis
of condensed chromatin obtained in oocytes following conventional
nuclear transplant (NT) or nuclear injection (NI) methods and
following injection of chromatin masses into oocytes (CT) using the
methods of the present invention. Both detectable lamins B and A/C
appear to be solubilized (bar, 10 .mu.m).
[0066] FIGS. 8A and 8B are sets of pictures of immunofluorescence
analysis of pronuclei resulting from chromatin transfer, nuclear
transplant, or nuclear injection. Embryos were fixed at 19 hours
post nuclear transplant, nuclear injection, or chromatin transfer
and labeled. Control parthenogenetic pronuclei (Part.) were also
examined. FIG. 8A shows the analysis of lamins A/C and B. FIG. 8B
shows the analysis of AKAP95 and NuMA. Lamins A/C (green label)
only appear in nuclear transplant and nuclear injection pronuclei
(bars, 30 .mu.m).
DETAILED DESCRIPTION
[0067] We have developed a novel method of cloning mammals that
involves remodeling of the donor genetic material before it is
inserted into the recipient oocyte. Remodeling refers to any
morphological change that improves development of the resulting
nuclear transplant oocyte over that derived from either
transferring whole cells or intact nuclei into a recipient oocyte.
Reprogramming is achieved by incubating a donor nucleus in a
reprogramming media (e.g., a mitotic extract, detergent and/or salt
solution, or protein kinase solution) resulting in nuclear envelope
dissolution and possibly chromatin condensation. This nuclear
envelope breakdown and chromatin condensation allows the release of
transcription regulatory proteins that were attached to the
chromosomes and that would otherwise promote the transcription of
genes undesirable for oocyte, embryo, or fetus development.
Additional regulatory proteins may be removed by purifying the
chromatin mass prior to transferring it into a recipient oocyte.
Alternatively, specific regulatory proteins that are released from
the chromosomes may be immunodepleted or otherwise removed from the
reprogrammed media (e.g., a cell extract) to prevent them from
re-binding the chromosomes. After nuclear transfer, new proteins
from the oocyte cytoplasm may be bound to the chromosomes during
decondensation of the chromatin and nuclear envelope formation in
the oocyte. These proteins promote the transcription of genes that
allow the oocyte to develop into a viable offspring.
[0068] This chromatin transfer cloning method produced embryos with
protein expression patterns that more closely resembled in vitro
fertilized embryos than cloned embryos produced using traditional
cloning methods. As illustrated in Examples 1 and 4, chromatin
transfer embryos expressed much less lamin A/C protein than
traditional nuclear transfer embryos. Lamins A/C are
somatic-specific components of the nuclear lamina that are
naturally expressed in differentiated cells, but not expressed in
embryos. Because of the reported interaction of lamins with
transcription factors, chromatin proteins, and DNA, it is likely
that the expression of lamins A/C in traditional nuclear transfer
embryos promotes the expression of proteins specific for somatic
cells that are undesirable for embryo development. Thus, the
chromatin transfer embryos of the present invention may express
fewer undesirable somatic-specific proteins than traditional
nuclear transfer embryos. Additionally, the chromatin transfer
embryos had expression patterns for NuMA, a main component of the
nuclear matrix that is implicated in transcriptional regulation,
that more closely resembled in vitro fertilized embryos than
traditional nuclear transplant embryos. This result also indicates
that chromatin transfer embryos are more efficiently reprogrammed
than traditional nuclear transplant embryos.
[0069] Another cloning method was developed that involves
reprogramming a permeabilized cell by incubating it in a
reprogramming media (e.g., a cell extract) to allow the addition or
removal of factors from the cell. The plasma membrane of the
permeabilized cell is preferably resealed to enclose the desired
factors and restore the membrane integrity of the cell. The
reprogrammed cell is then transferred into a recipient ooctye for
the production of a cloned mammal. This cloning method has been
used to produce fetuses that have survived past day 60. Preliminary
results indicate that fetal survival between day 40 and day 60 is
higher for fetuses formed using this method ( 7/10; 70%) than for
conventional nuclear transfer fetuses ( 8/16; 50%).
[0070] The invention also provides methods for generating chimeric
embryos in which the majority of the placental tissue is from one
genetic source and the majority of the fetal tissue is from another
genetic source. These chimeric embryos may have fewer placental
abnormalities and thus may have an increased survival rate. In one
such method, cells from an in vitro fertilized or
naturally-occurring embryo are contacted with cells from an embryo
produced using traditional nuclear transfer methods or any of the
novel cloning methods described herein. For example, cells from an
in vitro fertilized embryo can be injected into the periphery of a
nuclear transfer embryo (e.g., between the zona pellucida and the
embryo itself). This method was used to produce chimeric embryos
that had a 67% survival rate at day 40 compared to a 25% survival
rate for control nuclear transfer embryos. In an alternative
method, cells from a precompaction, in vitro fertilized or
naturally-occurring embryo are incubated with cells from a
precompaction nuclear transfer embryo under conditions that allow
cells from each embryo to reorganize to produce a single chimeric
embryo (Wells and Powell, Cloning 2:9-22, 2000). In both methods,
the cells from the in vitro fertilized or naturally-occurring
embryo are preferentially incorporated into the placenta, and the
cells from the nuclear transfer method are preferentially
incorporated into the fetal tissue.
[0071] The invention also features a novel method, denoted
lipofusion, for inserting a nucleus or chromosomes into cells. This
method involves incubating the nucleus or chromosomes and the
recipient cell with a fusigenic compound that allows the nucleus or
chromosomes to be transferred into the cytoplasm of the cell. This
method may generally be applied to nuclei and chromosomes from all
cell types and to recipient cells of all cell types.
[0072] These methods are described further below. It is noted that
any of the methods described below can also be performed with
reprogramming media other than cell extracts. For example, a
reprogramming media can be formed by adding one or more
naturally-occurring or recombinant factors (e.g., nucleic acids or
proteins such as DNA methyltransferases, histone deacetylases,
histones, protamines, nuclear lamins, transcription factors,
activators, repressors, chromatin remodeling proteins, growth
factors, interleukins, cytokines, or other hormones) to a solution,
such as a buffer. Preferably, one or more of the factors are
specific for oocytes or stem cells, such as embryonic stem
cells.
EXAMPLE 1
Evidence For Nuclear Reprogramming Deficiencies in Traditional
Bovine Nuclear Transplant Embryos
Distribution of Nuclear Envelope, Nuclear Matrix, and
Chromatin-Matrix Interface Components During Bovine Preimplantation
Development
[0073] To determine the distribution of nuclear envelope (B-type
and A/C-type lamins), nuclear matrix (NuMA), and chromatin-matrix
interface (AKAP95) components in preimplantation embryos, bovine
embryos were produced by in vitro fertilization (IVF) and examined
by immunofluorescence analysis. Bovine in vitro fertilization was
performed as described previously (Collas et al., Mol. Reprod.
Devel. 34:212-223, 1993). Briefly, frozen-thawed bovine sperm from
a single bull was layered on top of a 45-90% Percoll gradient and
centrifuged for 30 minutes at 700.times.g. The concentration of
sperm in the pellet was determined, and the sperm was diluted such
that the final concentration at fertilization was 10.sup.6
sperm/ml. At 22 hours post maturation, oocytes were washed three
times in TL HEPES and placed in 480 .mu.l fertilization medium.
Twenty .mu.l sperm suspension were added at 10.sup.6 sperm/ml for
50 oocytes. Embryos were placed in culture in four-well tissue
culture plates containing a monolayer of mouse fetal fibroblasts in
0.5 ml of embryo culture medium covered with 0.3 ml of embryo
tested mineral oil (Sigma). Between 25 and 50 embryos were placed
in each well and incubated at 38.5.degree. C. in a 5% CO.sub.2 air
atmosphere. Fertilization rates were over 90% as determined by
pronuclear development.
[0074] For the immunofluorescence analysis of these in vitro
fertilized bovine embryos, anti-human lamin B antibodies were
obtained from Dr. Jean-Claude Courvalin, CNRS, Paris, France.
Anti-lamins A/C monoclonal antibodies were purchased from
Santa-Cruz Biotechnology, and anti-NuMA antibodies were obtained
from Transduction Laboratories. Anti-rat AKAP95 affinity-purified
rabbit polyclonal antibodies were obtained from Upstate
Biotechnologies. The in vitro fertilized bovine embryos were
settled onto poly-L-lysine-coated glass coverslips, fixed with 3%
paraformaldehyde for 15 minutes, and permeabilized with 0.1% Triton
X-100 for 15 minutes (Collas et al., J. Cell Biol. 135:1715-1725,
1996). The proteins were blocked with 2% BSA in PBS/0.01% Tween 20
(PBST) for 15 minutes. Primary antibodies (anti-AKAP95, anti-lamin
B, anti-LBR, anti-NuMA, and anti-lamins A/C) and secondary
antibodies were incubated each for 30 minutes and used at a 1:100
dilution in PBST-BSA. DNA was counterstained with 0.1 .mu.g/ml
Hoechst 33342 incorporated in the antifade mounting medium. Samples
were mounted onto slides and coverslips sealed with nail polish.
Immunofluorescence observations were made on an Olympus BX60
epifluorescence microscope and photographs were taken with a JVC
CCD camera and AnalySIS software. Images were processed using the
Aldus Photostyler software. Relative quantification of fluorescence
signals was performed using the AnalySIS quantification program.
Data were expressed as mean relative fluorescence intensities.
[0075] Immunofluorescence analysis of bovine embryos showed that
B-type lamins were detected at the nuclear periphery (FIG. 1A).
Lamins A/C, however, were not detected at the pronuclear or 8-cell
stage. This failure to detect lamins A/C at these early cell stages
is expected for a marker of differentiated cells (Guilli et al.,
EMBO J. 6:3795-3799, 1987). The nuclear matrix structural protein,
NuMA, was detected in all the stages that were examined (FIG. 1A).
However, in bovine pronuclear stage embryos, NuMA labeling was
restricted to the female pronucleus (FPN), the smallest of both
pronuclei (FIG. 1A arrows). AKAP95, which was recently
characterized in early mouse embryos (Bomar et al., 2002 manuscript
submitted) and detected using affinity-purified anti-rat AKAP95
antibodies, was also restricted to the female pronucleus (FIG. 1A).
Nevertheless, intranuclear distribution of AKAP95 was observed in
nuclei of all blastomeres in subsequent developmental stages (FIG.
1A).
[0076] Specificity of immunofluorescence labeling was verified by
Western blot analysis of bovine primary fetal fibroblasts and
pronuclear stage in vitro fertilized embryos (FIG. 1B). For this
analysis, proteins were resolved by 10% SDS-PAGE at 40 mA per gel.
Proteins were electrophoretically transferred onto a nitrocellulose
membrane in transfer buffer (25 mM TrisHC1, pH 8.3, 192 mM glycine,
20% methanol, and 0.1% SDS) at 100 V for one hour. Membranes were
washed for 10 minutes with Tris-buffered saline (TBS; i.e., 140 mM
NaCl1, 2.7 mM KC1, and 25 mM Tris-HC1 at pH 8.0), blocked for one
hour with TBST (TBS with 0.05% Tween-20) containing 5% milk, and
incubated for 1.5 hours with the following primary antibodies:
anti-AKAP95 (1:250 dilution), anti-lamin B (1:1000), anti-LBR
(1:500), anti-NuMA (1:500), and anti-lamins A/C (1:500). Blots were
washed twice for 10 minutes in TBST and incubated for one hour with
horse radish peroxidase (HRP)-conjugated secondary antibodies.
Blots were washed twice for 10 minutes in TBS and developed using
enhanced chemiluminescence (ECL, Amersham).
[0077] All proteins were detected at their expected apparent
M.sub.r: 68 kDa (B-type lamins), 70 and 60 kDa (lamins A and C,
respectively), .about.180 kDa (NuMA), and 95 kDa (AKAP95).
Altogether, these results indicate that preimplantation bovine
embryos express nuclear structural proteins that can be detected
with cross-reacting antibodies. Notably, lamins A/C are not
immunologically detected in bovine preimplantation embryos. Because
lamins A/C are expressed in somatic cells (FIG. 1B), they
potentially constitute molecular markers for nuclear reprogramming
in nuclear transplant embryos.
Dynamics of Nuclear Envelope, NuMA and AKAP95 in Nuclear Transplant
Bovine Embryos
[0078] The dynamics of nuclear envelope and nuclear matrix
structures was examined during traditional nuclear transplantation
procedure in bovine. These structures were investigated using
antibodies to lamins A/C and B, NuMA, and AKAP95, respectively. To
determine the dynamics of these markers during nuclear remodeling,
bovine nuclear transplant embryos were produced using primary fetal
fibroblasts, which were isolated as described previously, as the
donor cells (Kasinathan et al., Biol. Reprod. 64:1487-1493, 2001).
Briefly, cells were harvested from bovine fetuses by trypsinization
using 0.08% trypsin and 0.02% EDTA in PBS (trypsin-EDTA). Cells
were seeded in a T75 culture flask (Corning) in .alpha.-MEM (Gibco)
supplemented with 10% fetal bovine serum (FBS; Hyclone), 0.15 g/ml
glutamine (Sigma), 0.003% .beta.-mercaptoethanol (Gibco), and an
antibiotic-antimycotic (Gibco). On day three after seeding, cells
were harvested with trypsin-EDTA and frozen in .alpha.-MEM/DMSO. G1
cells were isolated as described previously (Kasinathan et al.,
Biol. Reprod. 64:1487-1493, 2001). Briefly, 24 hours before
isolation, 5.0.times.10.sup.5 cells were plated in a T75 flask
containing 10 ml of MEM/FBS. The following day, the plates were
washed with PBS, the culture medium was replaced for 1-2 hours, and
the plates were shaken for 30-60 seconds on a Vortex at medium
speed. The medium was removed, centrifuged at 500.times.g for five
minutes, and the pellet was resuspended in 250 .mu.l of MEM/FBS.
Cell doublets attached by a cytoplasmic bridge were selected using
a micropipette and used for nuclear transfer.
[0079] Bovine nuclear transfer was carried out as described earlier
(Kasinathan et al., Biol. Reprod. 64:1487-1493, 2001). In
vitro-matured oocytes were enucleated 18-20 hours post-maturation.
After transferring G1 donor cells into the perivitelline space,
they were fused using a single electrical pulse of 2.4 kV/cm for 20
microseconds (Electrocell Manipulator 200, Genetronics). At 28-30
hours post maturation (i.e., 28-30 hours after oocytes were placed
in maturation medium after collection from ovaries and at least two
hours after fusion with donor cells) reconstructed oocytes and
parthenogenetic controls were activated with calcium ionophore (5
.mu.M) for four minutes (Cal Biochem) followed by 10 .mu.g
cycloheximide and 2.5 .mu.g cytochalasin D (Sigma) in ACM medium
(100 mM NaCl, 3 mM KCl, 0.27 mM CaCl.sub.2, 25 mM NaHCO.sub.3, 1 mM
sodium lactate, 0.4 mM pyruvate, 1 mM L-glutamine, 3 mg/ml BSA, 1%
BME amino acids, and 1% MEM nonessential amino acids, for five
hours (Liu et al., Mol. Reprod. Dev. 49:298-307, 1998). After
activation, nuclear transplant embryos or oocytes eggs were washed
five times and co-cultured with mouse fetal fibroblasts at
38.5.degree. C. in a 5% CO.sub.2 atmosphere.
[0080] Reconstituted embryos were activated using standard methods,
and three hours post-fusion, embryos at the premature chromatin
condensation (PCC) stage were fixed with paraformaldehyde and
analyzed by immunofluorescence using antibodies to lamins A/C,
lamin B, NuMA, and AKAP95 (FIG. 2, PCC). Furthermore, groups of
nuclear transplant embryos that were allowed to progress to the
pronuclear (PN) stage (i.e., 15 hour post-fusion bovine embryos)
were analyzed similarly (FIG. 2, nuclear transplant-PN). As
controls, parthenogenetic oocytes activated as described herein
were also examined at the pronuclear stage (FIG. 2, Parth. PN).
[0081] As expected, somatic donor cells (bovine fetal fibroblasts,
FIG. 2) expressed all markers with a distribution anticipated from
the literature. At the premature chromatin condensation stage,
distinct condensed chromosome masses were evidenced by DNA staining
with Hoechst 33342. Lamins A/C and B were not detected on or near
the condensed chromosomes (FIG. 2, PCC), presumably as a result of
their dispersal in the egg cytoplasm. Some labeled NuMA was
detected; this NuMA was presumably associated with the spindle
poles maintaining the condensed chromosomes. AKAP95, in contrast,
was associated with the condensed (PCC) chromosomes. This result is
reminiscent of AKAP95 labeling in mitotic human cells (Collas et
al., J. Cell Biol. 147:1167-1180, 1999; Steen et al., J. Cell Biol.
150:1251-1262, 2000). At the pronuclear stage, all markers were
detected. Lamins A/C were present at the pronuclear envelope (FIG.
2, nuclear transplant-PN). This contrasted with their absence from
the envelope of control parthenote pronuclei (FIG. 2) and from the
envelope of fertilized pronuclei (FIG. 1A). Lamin B was detected in
nuclear transplant pronuclei, as in control pronuclei. Likewise,
NuMA and AKAP95 decorated the nuclear interior except for the
nucleoli. NuMA labeling was consistently brighter in nuclear
transplant pronuclei than in control parthenogenetic pronuclei
(compare nuclear transplant PN and Parth. PN, FIG. 2).
Collectively, these observations indicate that pronuclei of nuclear
transplant embryos reassemble the somatic nuclear markers lamins A
and C and display strong NuMA staining.
Differential Anchoring of AKAP95 in Pronuclei of Parthenogenetic
Embryos and Nuclear Transplant Embryos
[0082] The A-kinase anchoring protein AKAP95 is a nuclear protein
implicated in mitotic chromosome condensation. For use as another
molecular marker affecting reprogramming of somatic nuclei after
nuclear transplant, the intranuclear anchoring properties of AKAP95
were characterized in bovine nuclear transplant pronuclear stage
embryos formed from fetal fibroblasts. Anchoring of AKAP95 in
pronuclei from parthenogenetic embryos and nuclei of somatic donor
cells was also examined.
[0083] Intranuclear anchoring of AKAP95 in pronuclear embryos was
examined in situ by extraction of embryos with 0.1% Triton X-100, 1
mg/ml DNAse I, and either 100 or 300 mM NaCl for 30 minutes at room
temperature. As noted above, male pronuclei did not harbor any
AKAP95. In contrast, a significant amount of AKAP95 and DNA was
resistant to DNAse I and 300 mM NaCl in pronuclei of nuclear
transplant embryos, and in donor nuclei in bovine (FIG. 3). B-type
lamins were not extracted by DNAse I and 300 mM NaCl in parthenote
or nuclear transplant pronuclei (FIG. 3), suggesting that
alterations in AKAP95 and DNA distributions did not result from
gross changes in nuclear architecture. These data indicate that, as
in somatic nuclei, AKAP95 is more tightly anchored to intranuclear
structures in nuclear transplant pronuclei than in parthenogenetic
pronuclei in the bovine. Whether this association imposes
constraints on DNA organization or results from altered genome
organization in nuclear transplant embryos remains to be
determined. As DNAse I-resistant DNA is transcriptionally silent,
incomplete remodeling of AKAP95 anchoring after nuclear
transplantation likely impairs expression of developmentally
important genes.
Transcriptional Misregulation of Lamins A/C in Nuclear Transplant
Bovine Embryos
[0084] A striking observation was that lamins A/C reassemble at the
periphery of pronuclei in bovine nuclear transplant embryos,
whereas this somatic-specific marker is absent from in vitro
fertilized, and parthenogenetic pronuclei. Thus, we investigated
whether reassembly of lamins A/C resulted from (i) re-targeting of
somatic lamins disassembled at the premature chromatin condensation
stage (FIG. 2), (ii) translation and assembly of lamins from a pool
of maternal lamin A/C mRNA, or (iii) de novo transcription of the
somatic lamin A (LMNA) gene in nuclear transplant pronuclei.
[0085] To distinguish between these possibilities, bovine nuclear
transplant embryos were produced by either the "traditional"
nuclear transplant procedure as described herein, nuclear
transplant followed by activation of reconstituted embryos with the
protein synthesis inhibitor cycloheximide (CHX), or by nuclear
transplant followed by activation in the presence of the RNA
polymerase II (PolII) inhibitor actinomycin D (ActD) to inhibit de
novo transcription. For culturing bovine nuclear transplant embryos
in cycloheximide, oocytes were activated after nuclear transfer as
described above except that oocytes were incubated for 14 hours in
cycloheximide (CHX). At 14 hours after activation, oocytes were
washed five times and placed in ACM culture medium containing 15
.mu.g/ml Hoechst 33342 (Sigma) for one hour. After incubation,
pronuclear development was observed by epifluorescence microscopy.
Pronuclear embryos were then fixed in 3% paraformaldehyde in PBS,
washed, and mounted on slides. For culturing bovine nuclear
transplant oocytes in actinomycin D, oocytes were activated after
nuclear transfer as described above except 5 .mu.g/ml actinomycin D
(ActD) was added to the cycloheximide incubation step. After five
hours, eggs were washed five times and placed in ACM culture medium
containing 5 .mu.g/ml actinomycin D. At 14 hours after activation,
eggs were washed five times and placed in ACM culture medium
containing 15 .mu.g/ml Hoechst 33342 (Sigma) for one hour. After
incubation, pronuclear development was observed by epifluorescence
microscopy. Pronuclear stage embryos were fixed in 3%
paraformaldehyde in PBS, washed, and mounted on slides.
[0086] Lamin B assembly around nuclear transplant pronuclei was not
affected by either protein or RNA synthesis inhibition. This result
indicates that lamin B was reassembled from either a previously
disassembled somatic pool and/or from a large pool of lamin B in
the oocyte cytoplasm. Lamins A/C, which were detected in nuclear
transplant pronuclei (FIG. 2), were absent from nuclei reformed
after activation with cycloheximide. This result indicates that
lamins A/C assembly requires de novo protein synthesis and that
these lamins are not re-targeted from a disassembled somatic pool
brought into the oocyte by donor nucleus injection or cell fusion.
Furthermore, lamins A/C are not reassembled when embryos are
activated in the presence of actinomycin D. This result indicates
that lamins A/C reassembly in nuclear transplant pronuclei results
from de novo transcription of the LMNA gene in the reconstituted
pronucleus. NuMA, which was detected in nuclear transplant
pronuclei, is not reassembled in pronuclei of nuclear transplant
embryos activated with cycloheximide, but is faintly detected in
pronuclei of actinomycin D-treated nuclear transplant embryos. This
finding strongly suggests that NuMA reassembly in nuclear
transplant pronuclei requires de novo translation that occurs, at
least in part, from a pool of maternal NuMA mRNA. The consistent
observation that anti-NuMA labeling is weaker in pronuclei of
actinomycin D-treated nuclear transplant embryos compared to
control untreated nuclear transplant embryos (compare b' and b'''
in FIG. 4) suggests that part of NuMA assembly in nuclear
transplant pronuclei results from de novo transcription of the NuMA
gene at the pronuclear stage.
[0087] Collectively, these results indicate that the LMNA gene is
not turned off upon nuclear remodeling after nuclear
transplantation. Similarly, the NuMA gene apparently remains active
in pronuclear nuclear transplant embryos. It is likely that
transient inactivation of these genes takes place during premature
chromatin condensation, as anticipated from the highly condensed
nature of the chromatin (FIG. 2). These results clearly illustrate
incomplete nuclear reprogramming in nuclear transplant embryos
produced under the conditions described herein. As discussed
earlier for AKAP95, we propose that the persistence of lamins A/C
in nuclear transplant pronuclei affects gene expression, such as
expression of developmentally important genes. The previously
reported interactions of lamins A and C with chromatin proteins and
DNA, and the association of these lamins with transcription factors
also support this hypothesis.
EXAMPLE 2
Use of Reprogrammed Donor Chromatin Masses to Clone Mammals
[0088] To overcome the problem of incomplete reprogramming in
traditional nuclear transfer embryos that was demonstrated in
Example 1, new methods were developed to more efficiently reprogram
donor chromatin prior to nuclear transfer. These methods involve
incubating a nucleus from a donor cell in a reprogramming media
(e.g., a cell extract) that results in nuclear envelope dissolution
and possibly chromatin condensation. This nuclear envelope
breakdown and chromatin condensation allows the release of
transcription regulatory proteins that were attached to the
chromosomes and that would otherwise promote the transcription of
genes undesirable for oocyte, embryo, or fetus development.
Additionally, regulatory proteins from the reprogramming media may
bind the chromatin mass and promote the transcription of genes
desirable for development.
Bulk Preparation of Donor Nuclei for Use in Cloning
[0089] As many as several million nuclei may be isolated from
synchronized or unsynchronized cell populations in culture. The
cell populations may be synchronized naturally or chemically.
Preferably, at least 40, 60, 80, 90, or 100% of the cells in a
population are arrested in G.sub.o or G.sub.1 phase. To accomplish
this, cells may be incubated, for example, in low serum, such as
5%, 2%, or 0% serum, for 1, 2, 3, or more days to increase the
percentage of cells in Go phase. To synchronize cells in G.sub.1,
the cells may be grown to confluence as attached cells and then
incubated in 0.5-1 .mu.g/ml nocodazole (Sigma Chemicals, St. Louis,
Mo.) for 17-20 hours, as described previously (see, for example,
Collas et al., J. Cell Biol. 147:1167-1180, 1999 and references
therein). The flasks containing the attached cells are shaken
vigorously by repeatedly tapping the flasks with one hand,
resulting in the detachment of mitotic cells and G.sub.1 phase
doublets. The G.sub.1 phase doublets are pairs of elongated cells
at the end of the division process that are still connected by a
thin bridge. Detached G.sub.1 phase doublets may be isolated from
the media based on this characteristic doublet structure. The
G.sub.1 phase doublets may remain attached or may divide into two
separate cells after isolation.
[0090] The synchronized or unsynchronized cells are harvested in
phosphate buffered saline (PBS) using standard procedures, and
several washing steps are performed to transfer the cells from
their original media into a hypotonic buffer (10 mM HEPES, pH 7.5,
2 mM MgCl.sub.2, 25 mM KCl, 1 mM DTT, 10 .mu.M aprotinin, 10 .mu.M
leupeptin, 10 .mu.M pepstatin A, 10 .mu.M soybean trypsin
inhibitor, and 100 .mu.M PMSF). For example, the cells may be
washed with 50 ml of PBS and pelleted by centrifugation at
500.times.g for 10 minutes at 4.degree. C. The PBS supernatant is
decanted, and the pelleted cells are resuspended in 50 ml of PBS
and centrifuged, as described above. After this centrifugation, the
pelleted cells are resuspended in 20-50 volumes of ice-cold
hypotonic buffer and centrifuged at 500.times.g for 10 min at
4.degree. C. The supernatant is again discarded and approximately
20 volumes of hypotonic buffer are added to the cell pellet. The
cells are carefully resuspended in this buffer and incubated on ice
for at least one hour, resulting in the gradual swelling of the
cells.
[0091] To allow isolation of the nuclei from the cells, the cells
are lysed using standard procedures. For example, 2-5 ml of the
cell suspension may be transferred to a glass homogenizer and
Dounce homogenized using an initial 10-20 strokes of a
tight-fitting pestle. Alternatively, the cell suspension is
homogenized using a motorized mixer (e.g., Ultraturrax). If
desired, cell lysis may be monitored using phase contrast
microscopy at 40-fold magnification. During this homogenization,
the nuclei should remain intact and most or preferably all of the
originally attached cytoplasmic components such as vesicles,
organelles, and proteins should be released from the nuclei. If
necessary, 1-20 .mu.g/ml of the cytoskeletal inhibitors,
cytochalasin B or cytochalasin D, may be added to the
aforementioned hypotonic buffer to facilitate this process.
Homogenization is continued as long as necessary to lyse the cells
and release cytoplasmic components from the nuclei. For some cell
types, as many as 100, 150, or more strokes may be required. The
lysate is then transferred into a 15 ml conical tube on ice, and
the cell lysis procedure is repeated with the remainder of the
suspension of swollen cells. Sucrose from a 2 M stock solution made
in hypotonic buffer is added to the cell lysate (e.g., 1/8 volume
of 2 M stock solution is added to the lysate), resulting in a final
concentration of 250 mM sucrose. This solution is mixed by
inversion, and the nuclei are pelleted by centrifugation at
400.times.g in a swing out rotor for 10 to 40 minutes at 4.degree.
C. The supernatant is then discarded, and the pelleted nuclei are
resuspended in 10-20 volumes of nuclear buffer (10 mM HEPES, pH
7.5, 2 mM MgCl.sub.2, 250 mM sucrose, 25 mM KCl, 1 mM DTT, 10 .mu.M
aprotinin, 10 .mu.M leupeptin, 10 .mu.M pepstatin A, 10 .mu.M
soybean trypsin inhibitor, and 100 .mu.M PMSF). The nuclei are
sedimented and resuspended in 1-2 volumes of nuclear buffer, as
described above. The freshly isolated nuclei may either be used
immediately for in vitro reprogramming and nuclear transfer as
described below or stored for later use. For storage, the nuclei
are diluted in nuclear buffer to a concentration of approximately
10.sup.6/ml. Glycerol (2.4 volumes of 100% glycerol) is added and
mixed well by gentle pipetting. The suspension is aliquoted into
100-500 .mu.l volumes in 1.5-ml tubes on ice, immediately frozen in
a methanol-dry ice bath, and stored at -80.degree. C. Prior to use,
aliquots of the nuclei are thawed on ice or at room temperature.
One volume of ice cold nuclear buffer is added, and the solution is
centrifuged at 1,000.times.g for 15 minutes in a swing out rotor.
The pelleted nuclei are resuspended in 100-500 .mu.l nuclear buffer
and centrifuged as described above. The pelleted nuclei are then
resuspended in a minimal volume of nuclear buffer and stored on ice
until use.
Preparation of Mitotic Extract or Media for use in Reprogramming
Donor Genetic Material
[0092] For the preparation of a mitotic extract, a somatic cell
line (e.g., fibroblasts) is synchronized in mitosis by incubation
in 0.5-1 .mu.g/ml nocodazole for 17-20 hours (e.g., Collas et al.,
J. Cell Biol. 147:1167-1180, 1999 and references therein) and the
mitotic cells are detached by vigorous shaking, as described above.
The detached G.sub.1 phase doublets may be discarded, or they may
be allowed to remain with the mitotic cells which constitute the
majority off the detached cells (typically at least 80%). The
harvested detached cells are centrifuged at 500.times.g for 10
minutes in a 10 ml conical tube at 4.degree. C. Several cell
pellets are pooled, resuspended in a total volume of 50 ml of cold
PBS, and centrifuged at 500.times.g for 10 minutes at 4.degree. C.
This PBS washing step is repeated. The cell pellet is resuspended
in approximately 20 volumes of ice-cold cell lysis buffer (20 mM
HEPES, pH 8.2, 5 mM MgCl.sub.2, 10 mM EDTA, 1 mM DTT, 10 .mu.M
aprotinin, 10 .mu.M leupeptin, 10 .mu.M pepstatin A, 10 .mu.M
soybean trypsin inhibitor, 100 .mu.M PMSF, and optionally 20
.mu.g/ml cytochalasin B), and the cells are sedimented by
centrifugation at 800.times.g for 10 minutes at 4.degree. C. The
supernatant is discarded, and the cell pellet is carefully
resuspended in no more than one volume of cell lysis buffer. The
cells are incubated on ice for one hour to allow swelling of the
cells. The cells are lysed by either sonication using a tip
sonicator or Dounce homogenization using a glass mortar and pestle.
Cell lysis is performed until at least 90% of the cells and nuclei
are lysed, which may be assessed using phase contrast microscopy.
The sonication time required to lyse at least 90% of the cells and
nuclei may vary depending on the type of cell used to prepare the
extract.
[0093] The cell lysate is placed in a 1.5-ml centrifuge tube and
centrifuged at 10,000 to 15,000.times.g for 15 minutes at 4.degree.
C. using a table top centrifuge. The tubes are removed from the
centrifuge and immediately placed on ice. The supernatant is
carefully collected using a 200 .mu.l pipette tip, and the
supernatant from several tubes is pooled and placed on ice. This
supernatant is the "mitotic cytoplasmic" or "MS15" extract. This
cell extract may be aliquoted into 50 .mu.l or 10 .mu.l volumes of
extract per tube on ice, depending on whether the regular or
micromethod for generation of chromatin masses will be used. The
extracts are immediately flash-frozen on liquid nitrogen and stored
at -80.degree. C. until use. Alternatively, the cell extract is
placed in an ultracentrifuge tube on ice (e.g., fitted for an SW55
Ti rotor; Beckman). If necessary, the tube is overlayed with
mineral oil to the top. The extract is centrifuged at
200,000.times.g for three hours at 4.degree. C. to sediment
membrane vesicles contained in the MS 15 extract. At the end of
centrifugation, the oil is discarded. The supernatant is carefully
collected, pooled if necessary, and placed in a cold 1.5 ml tube on
ice. This supernatant is referred to as "MS200" or "mitotic
cytosolic" extract. The extract is aliquoted and frozen as
described for the MS15 extract.
[0094] If desired, the extract can be enriched with additional
nuclear factors. For example, nuclei can be purified from cells of
the cell type from which the reprogramming extract is derived or
from cells of any other cell type and lysed by sonication as
described above. The nuclear factors are extracted by a 10-60
minute incubation in nuclear buffer containing NaCl or KCl at a
concentration of 0.15-800 mM under agitation. The lysate is
centrifuged to sediment unextractable components. The supernatant
containing the extracted factors of interest is dialyzed to
eliminate the NaCl or KCl. The dialyzed nuclear extract is
aliquoted and stored frozen. This nuclear extract is added at
various concentrations to the whole cell extract described above
prior to adding the nuclei for reprogramming.
[0095] Mitotic extracts can also be prepared from germ cells, such
as oocytes or male germ cells. For example, metaphase II oocytes
that are naturally arrested at this stage can be harvested, washed,
and lysed as described above for the generation of an oocyte
extract. To prepare a male germ cell extract, germ cells are
isolated from testes obtained from the abattoir by mincing the
organ and by differential centrifugation of the harvested cells on
a sucrose or percoll gradient. Germ cells are separated from
somatic (Leydig and Sertoli) cells, washed by suspension, and
sedimentation in PBS. The cells are then washed once in ice-sold
cell lysis buffer as described above and lysed by sonication. The
lysate is cleared by centrifugation at 15,000.times.g for 15
minutes at 4.degree. C., and the supernatant (i.e., the germ cell
extract) is aliquoted and snap-frozen in liquid nitrogen.
[0096] As an alternative to a cell extract, a reprogramming media
can also be formed by adding one or more naturally-occurring or
recombinant factors (e.g., nucleic acids or proteins such as DNA
methyltransferases, histone deacetylases, histones, protamines,
nuclear lamins, transcription factors, activators, repressors,
chromatin remodeling proteins, growth factors, interleukins,
cytokines, or other hormones) to a solution, such as a buffer.
Preferably, one or more of the factors are specific for oocytes or
stem cells.
Formation of Condensed Chromatin Masses by Exposure of Nuclei to a
Mitotic Extract or Media
[0097] An aliquot of MS15 or MS200 extract or the mitotic media is
thawed on ice. An ATP-generating system (0.6 .mu.l) is added to 20
.mu.l of extract or media and mixed by vortexing. For the
preparation of the ATP-generating system, equal proportions of 100
mM ATP stock, 1 M creatine phosphate, and 2.5 mg/ml creatine kinase
stock solutions (100.times.) made in H.sub.2O are mixed and stored
on ice until use. After addition of the ATP generating system to
the extract, the final concentrations are 1 mM ATP, 10 mM creatine
phosphate, and 25 .mu.g/ml creatine kinase.
[0098] The nuclei suspension is added to the extract or media at a
concentration of 1 .mu.l nuclei per 10 .mu.l of extract or media,
mixed well by pipetting, and incubated in a 30, 33, 35, 37, or
39.degree. C. water bath. The tube containing the mixture is tapped
gently at regular intervals to prevent chromosomes from clumping at
the bottom of the tube. Nuclear envelope breakdown and chromosome
condensation is monitored at regular intervals, such as every 15
minutes, under a microscope. When the nuclear envelope has broken
down and chromosomes have started to condense, the procedure for
recovery of chromatin masses from the extract or media is
started.
Formation of Decondensed Chromatin Masses by Exposure of Nuclei to
a Mitotic Extract or Media and Anti-NuMA Antibodies
[0099] Alternatively, chromatin masses that are not condensed or
only partially condensed may be formed by performing the above
procedure after pre-loading the isolated nuclei with an antibody to
the nuclear matrix protein NuMA (Steen et al., J. Cell Biol. 149,
531-536, 2000). This procedure allows the removal of nuclear
components from chromatin by the dissolution of the nuclear
membrane surrounding the donor nuclei; however, the condensation
step is inhibited by addition of the anti-NuMA antibody. Preventing
chromosome condensation may reduce the risk of chromosome breakage
or loss while the chromosomes are incubated in the mitotic
extract.
[0100] For this procedure, purified cell nuclei (2,000
nuclei/.mu.l) are permeabilized in 500 .mu.l nuclear buffer
containing 0.75 .mu.g/ml lysolecithin for 15 minutes at room
temperature. Excess lysolecithin is quenched by adding 1 ml of 3%
BSA made in nuclear buffer and incubating for 5 minutes on ice. The
nuclei are then sedimented and washed once in nuclear buffer. The
nuclei are resuspended at 2,000 nuclei/.mu.l in 100 .mu.l nuclear
buffer containing an anti-NuMA antibody (1:40 dilution;
Transduction Laboratories). After a one hour incubation on ice with
gentle agitation, the nuclei are sedimented at 500.times.g through
1 M sucrose for 20 minutes. The nuclei are then resuspended in
nuclear buffer and added to a mitotic extract or media containing
an ATP regenerating system, as described in the previous section.
Optionally, the anti-NuMA antibody may be added to the extract or
media to further prevent chromosome condensation.
Formation of Decondensed Chromatin Masses by Exposure of Nuclei to
a Detergent and/or Salt Solution or to a Protein Kinase
Solution
[0101] Chromatin masses that are not condensed or only partially
condensed may also be formed by exposure to a detergent or protein
kinase. Detergent may be used to solubilize nuclear components that
are either unbound or loosely bound to the chromosomes in the
nucleus, resulting in the removal of the nuclear envelope. For this
procedure, purified cell nuclei (2,000-10,000 nuclei/.mu.l) are
incubated in nuclear buffer supplemented with a detergent, such as
0.1% to 0.5% Triton X-100 or NP-40. To facilitate removal of the
nuclear envelope, additional salt, such as NaCl, may be added to
the buffer at a concentration of approximately 0.1, 0.15, 0.25,
0.5, 0.75, or 1 M. After a 30-60 minute incubation on ice with
gentle shaking, the nuclei are sedimented by centrifugation at
1,000.times.g in a swing-out rotor for 10-30 minutes, depending on
the total volume. The pelleted nuclei are resuspended in 0.5 to 1
ml nuclear buffer and sedimented as described above. This washing
procedure is repeated twice to ensure complete removal of the
detergent and extra salt.
[0102] Alternatively, the nuclear envelope may be removed using
recombinant or naturally-occurring protein kinases, alone or in
combination. Preferably, the protein kinases are purified using
standard procedures or obtained in purified form from commercial
sources. These kinases may phosphorylate components of the nuclear
membrane, nuclear matrix, or chromatin, resulting in removal of the
nuclear envelope (see, for example, Collas and Courvalin, Trends
Cell Biol. 10: 5-8, 2000). Preferred kinases include
cyclin-dependent kinase 1 (CDK1), protein kinase C (PKC), protein
kinase A (PKA), MAP kinase, calcium/calmodulin-dependent kinase
(CamKII), and CK1 casein kinase, or CK2 casein kinase. For this
method, approximately 20,000 purified nuclei are incubated in 20
.mu.l of phosphorylation buffer at room temperature in a 1.5 ml
centrifuge tube. A preferred phosphorylation buffer for CDK1
(Upstate Biotechnology) contains 200 mM NaCl, 50 mM Tris-HCl (pH
7.2-7.6), 10 mM MgSO.sub.4, 80 mM .beta.-glycerophosphate, 5 mM
EGTA, 100 .mu.M ATP, and 1 mM DTT. For PKC, a preferred buffer
contains 200 mM NaCl, 50 mM Tris-HCl (pH 7.2-7.6), 10 mM
MgSO.sub.4, 100 .mu.M CaCl.sub.2, 40 .mu.g/ml phosphatidylserine,
20 .mu.M diacylglycerol, 100 .mu.M ATP, and 1 mM DTT. If both PKC
and CDK1 are used simultaneously, the CDK1 phosphorylation buffer
supplemented with 40 .mu.g/ml phosphatidylserine and 20 .mu.M
diacylglycerol is used. A preferred phosphorylation buffer for PKA
includes 200 mM NaCl, 10 mM MgSO4, 10 mM Tris, pH 7.0, 1 mM EDTA,
and 100 .mu.M ATP. For MAP kinase, the PKA phosphorylation buffer
supplemented with 10 mM CaCl.sub.2, and 1 mM DTT may be used. For
CamKII, either PKA buffer supplemented with 1 mM DTT or a Cam
Kinase assay kit from Upstate Biotechnology (Venema et al. J. Biol.
Chem 272: 28187-90, 1997) is used.
[0103] The phosphorylation reaction is initiated by adding a
protein kinase to a final amount of 25-100 ng. The reaction is
incubated at room temperature for up to one hour. Nuclear envelope
breakdown may be monitored by microscopy during this incubation,
such as at 15 minute intervals. After nuclear envelope breakdown,
nuclei are washed three times, as described above for the removal
of the detergent solution.
Recovery of Chromatin Masses from the Media, Extract Detergent
and/or Salt Solution, or Protein Kinase Solution
[0104] The extract or solution containing the condensed, partially
condensed, or not condensed chromatin masses is placed under an
equal volume of 1 M sucrose solution made in nuclear buffer. The
chromatin masses are sedimented by centrifugation at 1,000.times.g
for 10-30 minutes depending on the sample volume in a swing out
rotor at 4.degree. C. The supernatant is discarded, and the
pelleted chromatin masses are carefully resuspended by pipetting in
0.1-1.0 ml nuclear buffer or lipofusion buffer (150 mM NaCl, 10
.mu.M aprotinin, 10 .mu.M leupeptin, 10 .mu.M pepstatin A, 10 .mu.M
soybean trypsin inhibitor, and 100 .mu.M PMSF in either 20 mM HEPES
around pH 7.0 or pH 7.5 or 20 mM MES around pH 6.2) and centrifuged
at 1,000.times.g for 10-30 minutes. The supernatant is discarded,
and the pelleted chromatin masses are resuspended in nuclear buffer
or lipofusion buffer and stored on ice until use. Each chromatin
mass is transferred to a 20 .mu.l drop of HEPES-buffered medium
under oil in a micromanipulation dish. One chromatin mass is
inserted into each enucleated oocyte, as described below.
Micromethod for Preparation of Chromatin Masses
[0105] A 10-20 .mu.l drop of MS15 or MS200 extract or mitotic media
containing an ATP generating system, a detergent and/or salt
solution, or a protein kinase solution as described above is placed
in a petri dish. A 50-.mu.l drop of isolated G.sub.1 phase cell
doublets or G.sub.0 phase cells in culture medium, a separate 50
.mu.l "lysis" drop of HEPES- or bicarbonate-buffered medium
containing 0.1% Triton X-100 or NP-40 for use in facilitating cell
lysis, and a 50-.mu.l drop of oocyte injection medium is then
added. Each of these drops is covered with CO.sub.2 equilibrated
mineral oil. A 50 .mu.l "wash drop" of culture medium is also added
to the petri dish for use in washing the lysed cells or nuclei.
[0106] Cells are transferred to the lysis drop using a
micropipette. The cell membranes are lysed in the pipette by gentle
repeated aspirations. When the cell is lysed, the lysate is gently
expelled into the wash drop, and the nucleus is immediately
reaspirated to remove detergent. Optionally, the nuclei may be
permeabilized and incubated with anti-NuMA antibodies prior to
being added to the mitotic extract or media. The nucleus is then
expelled into the drop of MS15, MS200, or media, detergent and/or
salt solution, or protein kinase solution. Nuclear breakdown and
chromosome condensation is monitored as described above. Once the
nuclear envelope has broken down and, if a mitotic extract without
anti-NuMA antibodies was used, the chromosomes have started to
condense, a single intact chromatin mass is isolated with a
micropipette and transferred to an enucleated recipient oocyte, as
described below.
Enucleation of Oocytes
[0107] Preferably, the recipient oocyte is a metaphase II stage
oocyte. At this stage, the oocyte may be activated or is already
sufficiently activated to treat the introduced chromatin mass as it
does a fertilizing sperm. For enucleatation of the oocyte, part or
preferably all of the DNA in the oocyte is removed or inactivated.
This destruction or removal of the DNA in the recipient oocyte
prevents the genetic material of the oocyte from contributing to
the growth and development of the cloned mammal. One method for
destroying the pronucleus of the oocyte is exposure to ultraviolet
light (Gurdon, in Methods in Cell Biology, Xenopus
Laevis:--Practical Uses in cell and Molecular Biology, Kay and
Peng, eds., Academic Press, California, volume 36:pages 299-309,
1991). Alternatively, the oocyte pronucleus may be surgically
removed by any standard technique (see, for example, McGrath and
Solter, Science 220:1300-1319, 1983). In one possible method, a
needle is placed into the oocyte, and the nucleus is aspirated into
the inner space of the needle. The needle may then be removed from
the oocyte without rupturing the plasma membrane (U.S. Pat. Nos.
4,994,384 and 5,057,420).
Lipofusion for Insertion of Chromatin Masses into Oocytes
[0108] Chromatin may be introduced into recipient oocytes by
lipofusion as described below or by standard microinjection or
electrofusion techniques (see, for example, U.S. Pat. Nos.
4,994,384 and 5,945,577). The following lipofusion method may also
be used in other applications to insert chromosomes into other
recipient cells.
[0109] Chromatin masses are isolated from the mitotic extract,
detergent and/or salt solution, or protein kinase solution by
centrifugation, and then washed with lipofusion buffer, as
described above. The chromatin masses may be in stored in ice-cold
lipofusion buffer until use. Alternatively, the chromatin masses
are aliquoted, frozen in liquid nitrogen or in a methanol-dry ice
bath, and stored frozen at -80.degree. C. The lipofusion solution
is prepared by mixing one or more fusigenic reagents with the
lipofusion buffer in respective proportions ranging from 5:1 to
1:10 approximately. The fusigenic reagents consist of, but are not
limited to, polyethylene glycol (PEG) and lipophilic compounds such
as Lipofectin.RTM., Lipofectamin.RTM., DOTAP.RTM., DOSPA.RTM.,
DOPE.RTM., and membrane vesicle fractions. For example, a cationic
lipid, such as DOTAP.RTM., may be used at a concentration of
approximately 0.1 to 30 .mu.g/ml in lipofusion buffer.
Alternatively, a liposome formulation consisting of a mixture of a
cationic lipid and a neutral lipid, such as DOPE.RTM., may be
used.
[0110] The chromatin masses, either freshly prepared or frozen and
thawed, are mixed with the lipofusion solution to allow coating of
the chromatin masses with the compound. Incubation takes place at a
temperature of 20-30.degree. C. for a period of approximately 10-30
minutes. Microdrops containing the chromatin masses in the
lipofusion solution are placed under CO.sub.2 equilibrated mineral
oil. A drop containing the enucleated recipient oocytes is also
prepared. The chromatin masses coated with the lipofusion reagent
are picked up in a micropipette and inserted in the perivitellin
space, between the oocyte cytoplasm and the zona pellucida. The
chromatin mass is placed next to the oocyte membrane to ensure
contact with the oocyte. The chromatin mass-oocyte complexes are
maintained at a temperature of 20-30.degree. C., and fusion is
monitored under the microscope. Once fusion has occurred,
reconstituted oocytes are activated as described below.
Activation Culturing, and Transplantation of Reconstituted
Oocytes
[0111] To prevent polar body extrusion and chromosome loss, the
oocyte may be activated in the presence of cytochalasin B, or
cytochalasin B may be added immediately after activation (Wakayama
et al., PNAS 96:14984-14989, 1999; Wakayama et al., Nature Genetics
24:108-109, 2000). Either electrical or non-electrical means may be
used for activating reconstituted oocytes. Electrical techniques
for activating cells are well known in the art (see, for example,
U.S. Pat. Nos. 4,994,384 and 5,057,420). Non-electrical means for
activating cells may include any method known in the art that
increases the probability of cell division. Examples of
non-electrical means for activating an oocyte include incubating
the oocyte in the presence of ethanol; inositol trisphosphate;
Ca.sup.++ ionophore and a protein kinase inhibitors; a protein
synthesis inhibitor; phorbol esters; thapsigargin, or any component
of sperm. Other non-electrical methods for activation include
subjecting the oocyte to cold shock or mechanical stress.
Alternatively, one to three hours after nuclear transfer, oocytes
may be incubated for approximately six hours in medium containing
Sr.sup.2+ to activate them and cytochalasin B to prevent
cytokinesis and polar body extrusion (Wakayama et al., PNAS
96:14984-14989, 1999; Wakayama et al., Nature Genetics 24:108-109,
2000). Depending on the type of mammal cloned, the preferred length
of activation may vary. For example, in domestic animals such as
cattle, the oocyte activation period generally ranges from about
16-52 hours or preferably about 28-42 hours.
[0112] After activation, the oocyte is placed in culture medium for
an appropriate amount of time to allow development of the resulting
embryo. At the two cell stage or a later stage, the embryo is
transferred into a foster recipient female for development to term.
For bovine species, the embryos are typically cultured to the
blastocyst stage (e.g., for approximately 6-8 days) before being
transferred to maternal hosts. For other cloned animals, an
appropriate length for in vitro culturing is known by one skilled
in the art or may be determined by routine experimentation.
[0113] Methods for implanting embryos into the uterus of a mammal
are also well known in the art. Preferably, the developmental stage
of the embryo is correlated with the estrus cycle of the host
mammal. Once the embryo is placed in the uterus of the mammal, the
embryo may develop to term. Alternatively, the embryo is allowed to
develop in the uterus until a chosen time, and then the embryo (or
fetus) is removed using standard surgical methods to determine its
health and viability. Embryos from one species may be placed into
the uterine environment of an animal from another species. For
example, bovine embryos can develop in the oviducts of sheep (Stice
and Keefer, Biology of Reproduction 48: 715-719, 1993). Any
cross-species relationship between embryo and uterus may be used in
the methods of the invention.
Lipofusion of Nuclei with Oocytes or Other Recipient Cells
[0114] The lipofusion solution is prepared by mixing one or more
fusigenic reagents with lipofusion buffer in respective proportions
ranging from approximately 5:1 to 1:10, as described above. Nuclei,
either freshly prepared or frozen and thawed as described above,
are mixed with the lipofusion solution to allow coating of the
nuclei with the compound. Incubation takes place at a temperature
of 20-30.degree. C. for a period of approximately 10-30 minutes.
Microdrops containing nuclei in the lipofusion solution are placed
under CO.sub.2 equilibrated mineral oil. A drop containing the
recipient cell, preferably an enucleated cell, is also prepared.
Enucleated recipient cells are prepared by physically removing the
chromosomes or the nucleus by micromanipulation or by damaging the
genetic material by exposure to UV light, as described above. For
insertion into oocytes, the nuclei coated with the lipofusion
reagent are picked up in a micropipette and inserted in the
perivitellin space, between the oocyte cytoplasm and the zona
pellucida. For insertion into other recipient cells, the coated
nuclei are preferably placed next to the cell membrane to ensure
contact with the cell. The nucleus-cell complexes are maintained at
a temperature of 20-30.degree. C., and fusion is monitored using a
microscope. Once fusion has occurred, reconstituted oocytes are
activated as described above.
EXAMPLE 3
Use of Reprogrammed Permeabilized Cells to Clone Mammals
[0115] Cells may also be reprogrammed without requiring the
isolation of nuclei or chromatin masses from the cells. In this
method, cells are permeabilized and then incubated in an interphase
or mitotic reprogramming media under conditions that allow the
exchange of factors between the media (e.g., a cell extract) and
the cells. If an interphase media is used, the nuclei in the cells
remain membrane-bounded; if a mitotic media is used, nuclear
envelope breakdown and chromatin condensation may occur. After the
nuclei are reprogrammed by incubation in this media, the plasma
membrane is preferably resealed, forming an intact reprogrammed
cell that contains desired factors from the media. If desired, the
media can be enriched with additional nuclear factors as described
in Example 2. The reprogrammed cells are then fused with recipient
oocytes, and embryos formed from the reconstituted oocytes are
inserted into maternal recipient mammals for the generation of
cloned mammals.
Permeabilization of Cells
[0116] Cells that may be reprogrammed using this procedure include
unsynchronized cells and cells synchronized in G.sub.o, G.sub.1, S,
G.sub.2, or M phase or a combination of these phases. The cells are
permeabilized using any standard procedure, such as
permeabilization with digitonin or Streptolysin O. Briefly, cells
are harvested using standard procedures and washed with PBS. For
digitonin permeabilization, cells are resuspended in culture medium
containing digitonin at a concentration of approximately 0.001-0.1%
and incubated on ice for 10 minutes. For permeabilization with
Streptolysin O, cells are incubated in Streptolysin O solution
(see, for example, Maghazachi et al., FASEB J. 11:765-74, 1997, and
references therein;) for .about.15, 30, or 60 minutes at room
temperature. After either incubation, the cells are washed by
centrifugation at 400.times.g for 10 minutes. This washing step is
repeated twice by resuspension and sedimentation in PBS. Cells are
kept in PBS at room temperature until use. Preferably, the
permeabilized cells are immediately added to the interphase or
mitotic media for reprogramming, as described below.
Preparation of the Reprogramming Media
[0117] To prepare an interphase reprogramming extract, interphase
cultured cells are harvested using standard methods and washed by
centrifugation at 500.times.g for 10 minutes in a 10 ml conical
tube at 4.degree. C. The supernatant is discarded, and the cell
pellet is resuspended in a total volume of 50 ml of cold PBS. The
cells are centrifuged at 500.times.g for 10 minutes at 4.degree. C.
This washing step is repeated, and the cell pellet is resuspended
in approximately 20 volumes of ice-cold interphase cell lysis
buffer (20 mM HEPES, pH 8.2, 5 mM MgCl.sub.2, 1 mM DTT, 10 .mu.M
aprotinin, 10 .mu.M leupeptin, 10 .mu.M pepstatin A, 10 .mu.M
soybean trypsin inhibitor, 100 .mu.M PMSF, and optionally 20
.mu.g/ml cytochalasin B). The cells are sedimented by
centrifugation at 800.times.g for 10 minutes at 4.degree. C. The
supernatant is discarded, and the cell pellet is carefully
resuspended in no more than one volume of interphase cell lysis
buffer. The cells are incubated on ice for one hour to allow
swelling of the cells. The cells are lysed by either sonication
using a tip sonicator or Dounce homogenization using a glass mortar
and pestle. Cell lysis is performed until at least 90% of the cells
and nuclei are lysed, which may be assessed using phase contrast
microscopy. The sonication time required to lyse at least 90% of
the cells and nuclei may vary depending on the type of cell used to
prepare the extract.
[0118] The cell lysate is placed in a 1.5-ml centrifuge tube and
centrifuged at 10,000 to 15,000.times.g for 15 minutes at 4.degree.
C. using a table top centrifuge. The tubes are removed from the
centrifuge and immediately placed on ice. The supernatant is
carefully collected using a 200 .mu.l pipette tip, and the
supernatant from several tubes is pooled and placed on ice. This
supernatant is the "interphase cytoplasmic" or "IS15" extract. This
cell extract may be aliquoted into 20 .mu.l volumes of extract per
tube on ice and immediately flash-frozen on liquid nitrogen and
stored at -80.degree. C. until use. Alternatively, the cell extract
is placed in an ultracentrifuge tube on ice (e.g., fitted for an
SW55 Ti rotor; Beckman). If necessary, the tube is overlayed with
mineral oil to the top. The extract is centrifuged at
200,000.times.g for three hours at 4.degree. C. to sediment
membrane vesicles contained in the IS15 extract. At the end of
centrifugation, the oil is discarded. The supernatant is carefully
collected, pooled if necessary, and placed in a cold 1.5 ml tube on
ice. This supernatant is referred to as "IS200" or "interphase
cytosolic" extract. The extract is aliquoted and frozen as
described for the IS15 extract.
[0119] If desired, the extract can be enriched with additional
nuclear factors. For example, nuclei can be purified from cells of
the cell type from which the reprogramming extract is derived or
from cells of any other cell type and lysed by sonication as
described above. The nuclear factors are extracted by a 10-60
minute incubation in nuclear buffer containing NaCl or KCl at a
concentration of 0.15-800 mM under agitation. The lysate is
centrifuged to sediment unextractable components. The supernatant
containing the extracted factors of interest is dialyzed to
eliminate the NaCl or KCl. The dialyzed nuclear extract is
aliquoted and stored frozen. This nuclear extract is added at
various concentrations to the whole cell extract described above
prior to adding the cells for reprogramming.
[0120] Interphase extracts can also be prepared from germ cells,
such as oocytes or male germ cells. For example, oocytes are
activated as described above and cultured for five hours to allow
entry into interphase. Oocytes are then treated as described in
Example 2 for metaphase II oocyte extracts except that EDTA is
omitted from the lysis buffer. Male germ cell extracts can be
prepared as described in Example 2.
[0121] As an alternative to a cell extract, a reprogramming media
can also be formed by adding one or more naturally-occurring or
recombinant factors (e.g., nucleic acids or proteins such as DNA
methyltransferases, histone deacetylases, histones, protamines,
nuclear lamins, transcription factors, activators, repressors,
chromatin remodeling proteins, growth factors, interleukins,
cytokines, or other hormones) to a solution, such as a buffer.
Preferably, one or more of the factors are specific for oocytes or
stem cells.
Reprogramming of Cells in a Media
[0122] The permeabilized cells are suspended in an interphase
reprogramming media described above or one of the mitotic
reprogramming medias described in Example 2 at a concentration of
approximately 100-1,000 cells/.mu.l. The ATP generating system and
GTP are added to the extract as described above, and the reaction
is incubated at 30-37.degree. C. for up to two hours to promote
translocation of factors from the extract into the cell and active
nuclear uptake or chromosome-binding of factors. The reprogrammed
cells are centrifuged at 800.times.g, washed by resuspension, and
centrifuged at 400.times.g in PBS. The cells are resuspended in
culture medium containing 20-30% fetal calf serum (FCS), RPMI1640
containing 2 mM CaCl.sub.2 (added from a 1 M stock in H.sub.2O), or
in .alpha.-MEM medium containing 2 mM CaCl.sub.2 and incubated for
1-3 hours at 37.degree. C. in a regular cell culture incubator to
allow resealing of the cell membrane. The cells are then washed in
regular warm culture medium (10% FCS) and cultured further using
standard culturing conditions.
Alternative Method of Reprogramming Permeabilized Cells on
Coverslips Instead of in Solution
[0123] Alternatively, the cells can be permeabilized while placed
on coverslips to minimize the handling of the cells and to
eliminate the centrifugation of the cells, thereby maximizing the
viability of the cells. Cells (e.g., fibroblasts) are grown on
16-mm poly-L-lysine-coated coverslips in RPMI1640 to 50,000-100,000
cells/coverslip in 12-well plates. Cells are permeabilized in 200
ng/ml Streptolysin O in Ca.sup.2+-free Hanks Balanced Salt Solution
(Gibco-BRL) for 50 minutes at 37.degree. C. in regular atmosphere.
If desired, the percent of cells that are permeabilized under these
conditions can be measured based on propidium iodide uptake.
Streptolysin O is aspirated; coverslips are overlaid with 80-100
.mu.l of reprogramming media; and the cells are incubated for
thirty minutes to one hour at 37.degree. C. in CO.sub.2 atmosphere.
The reprogramming media preferably contains the ATP generating
system and 1 mM each of ATP, CTP, GTP and UTP. To reseal plasma
membranes, .alpha.-MEM medium containing 2 mM CaCl.sub.2, medium
containing 20-30% fetal calf serum, or RPMI1640 containing 2 mM
CaCl.sub.2 is added to the wells, and the cells are incubated for
two hours at 37.degree. C.
Effect of Various Streptolysin O Treatments on the Percentage of
Permeabilized and Resealed Cells
[0124] To assess the percent of permeabilized and resealed cells,
dose and time titrations of Streptolysin O incubation were
performed (Table 1). Permeabilization of cells was assessed by
uptake of 0.1 .mu.g/ml of the DNA stain propidium iodide at the end
of Streptolysin O treatment. Resealing was assessed similarly at
the end of the resealing treatment in a separate group of cells.
TABLE-US-00001 TABLE 1 Permeabilization and resealing of
Streptolysin O (SLO)-treated bovine fibroblasts Permeabilization
Resealing ng/ml SLO N % pemeabilized +/- sd N % Resealed +/- sd 0
563 1 +/- 2.8 560 89.9 +/- 4.9 100 404 48.6 +/- 4.2 810 86.1 +/-
8.3 200 548 79.2 +/- 1.4 478 84.9 +/- 1.5 500 495 88.7 +/- 1.6 526
87.6 +/- 0.5 1000 425 84.9 +/- 0.7 544 86.4 +/- 1.4 2000 315 96.6
+/- 2.2 425 10.7 +/- 1 4000 200 99 +/- 1.4 200 11.2 +/- 5.3
Assessment of Viability of Bovine Fibroblasts Permeabilized with
Streptolysin O Treatment and Exposed to Mitotic Extract
[0125] TUNEL analysis was performed to evaluate apoptosis in cells
permeabilized with 0 or 500 ng/ml Streptolysin O and resealed, or
in cells permeabilized with Streptolysin O, exposed to mitotic
extract for 30 or 60 minutes, and resealed. TUNEL-positive cells
are cells undergoing apoptosis (i.e., cell death). The data show
that Streptolysin O itself does not induce apoptosis (Table 2).
Exposure of Streptolysin O-treated cells to the mitotic extract for
60 minutes, but not 30 minutes, induces a 10% increase in apoptotic
rate, based on TUNEL analysis (Table 2). Based on these data, a
30-minute incubation of donor cells in the extract is more
preferable than a 60 minute incubation. Thirty minute incubations
were shown by immunofluorescence analysis of cells to induce
nuclear envelope breakdown in the majority of nuclei examined
(.about.90%, n>100).
[0126] Additionally, purified nuclei incubated in extract and
washed in either buffer N or TL-HEPES and sucrose as described in
Example 4 for the chromatin transfer method do not undergo
apoptosis (2/34 and 3/47 TUNEL positive, respectively).
TABLE-US-00002 TABLE 2 TUNEL analysis of Streptolysin O and
Streptolysin O plus extract-treated bovine fibroblasts ng/ml SLO N
% TUNEL pos. +/- sd 0-Input cells 400 7.7 +/- 1.7 0 800 6.5 +/-
0.17 500 892 7.3 +/- 3.41 0 + extract 30' 400 5.5 +/- 1.12 500 +
extract 30' 400 8.2 +/- 1.1 0 + extract 60' 784 6.5 +/- 4.0 500 +
extract 60' 691 16.9 +/- 1.9
The permeabilization method chosen for these cloning methods was
500 ng/ml SLO for 30 minutes at 38.degree. C. The resealing method
chosen for forming an intact membrane surrounding the reprogrammed
cells was a two hour incubation in .alpha.-MEM medium containing 2
mM CaCl.sub.2. Formation Activation Culturing and Transplantation
of Reconstituted Oocytes
[0127] The reprogrammed cells are inserted into, or fused with,
recipient oocytes using standard microinjection or electrofusion
techniques (see, for example, U.S. Pat. Nos. 4,994,384 and
5,945,577). For example, the cells can be placed next to the
oocytes in standard cell medium in the presence or absence of
sucrose (e.g., 2.5% sucrose), and the cells can be drawn into an
injection pippette. The pipette is then aspirated a few times to
lyse the cells and remove cytoplasmic components from the nucleus
which is then injected into the oocyte. The reconstituted oocytes
are then activated, cultured, and transplanted into maternal
recipient mammals using standard methods such as those described in
Example 2 to produce cloned mammals.
EXAMPLE 4
Evidence for More Complete Nuclear Reprogramming using Two Novel
Cloning Procedures: Chromatin Transfer (CT) and Streptolysin
O-Transfer (SLOT)
[0128] As illustrated Example 1, incomplete nuclear remodeling and
reprogramming occurs in traditional nuclear transplant pronuclear
stage embryos. This finding was demonstrated by the assembly of
lamins A/C in the nuclear envelope of pronuclear nuclear transplant
embryos and excess NuMA immunofluorescence labeling. More complete
nuclear reprogramming was achieved using the chromatin mass
transfer method described in Example 2 and the cell
permeabilization and reprogramming method (also referred to as
SLOT) described in Example 3.
Assessment of in Vitro Nuclear Breakdown of Bovine Fibroblast
Nuclei Incubated in a Mitotic Extract and Characterization of the
Resulting Chromatin Masses
[0129] Extracts prepared from mitotic bovine fibroblasts
consistently supported breakdown of .about.80% of input purified
fibroblast nuclei (FIG. 5). An extract from metaphase II oocytes
(i.e., an extract from oocytes naturally arrested in metaphase II
prior to fertilization) also successfully supported nuclear
breakdown (75% of nuclei within 30 minutes).
[0130] Input interphase nuclei (FIG. 6A), chromatin masses obtained
from nuclei incubated in a MS15 mitotic extract (FIG. 6B), and
chromatin masses obtained from nuclei incubated in an oocyte
extract (FIG. 6C) were examined for the expression of the following
markers: lamin B receptor (LBR), an integral protein of the inner
nuclear membrane (membrane marker); lamin B, a ubiquitous component
of the nuclear lamina; lamins A/C, a somatic-specific component of
the nuclear lamina present only in differentiated cells and absent
in embryos; NuMA, a main component of the nuclear matrix; AKAP95, a
PKA-anchoring protein of the nucleus; and DNA. Both somatic
cytosolic MS15 and oocyte MS15 extracts induced solubilization of
lamin B, lamins A/C, LBR, and NuMA in .about.100% of chromatin
units examined (FIGS. 6B and 6C). As expected, AKAP95 remained
associated with chromosomes, as observed previously in mitotic
human cells (Collas et al., J. Cell Biol. 147:1167-1180, 1999).
This result was also described in Example 1 for bovine nuclear
transplant embryos at the premature chromatin condensation stage.
Both the mitotic extract and the oocyte extract appeared to be as
efficient as intact oocytes in promoting nuclear envelope
solubilization, regardless of the method used, i.e., traditional
nuclear transplant, nuclear injection (NI), or chromatin transfer
(FIG. 7).
Comparison of Pronuclear Embryos Produced by Chromatin Transfer and
Pronuclei from Nuclear Transplant and Nuclear Injection Embryos
[0131] To generate chromatin transfer embryos, in vitro-matured
oocytes were enucleated about 18-20 hours post maturation. Nuclei
from interphase bovine fetal fibroblasts were incubated in a MS15
mitotic extract that was prepared from bovine fetal cells as
described herein. Chromatin masses were isolated from the extract
when after nuclear envelope breakdown had occurred and before
chromatin condensation was completed. In particular, the chromatin
masses were isolated when the chromatin was approximately 50-60%
condensed, compared to the level of condensation of chromosomes in
interphase (designated 0% condensed) and the maximum level of
condensation of chromosomes in mitotsis (designated 100%
condensed.) At this stage, individual chromosomes in the chromatin
mass could not be distinguished and the edges of the chromatin mass
had an irregular shape. Chromatin masses that had been isolated
from the mitotic extract were placed in a microdrop of TL HEPES
with 2.5% sucrose along with enucleated oocytes. The sucrose was
added to the buffer to minimize damage to the ooctyes from the
subsequent injection procedure. Chromatin masses were injected into
the oocytes using a beveled microinjection pipette using a Burleigh
Piezo Drill (Fishers, N.Y.) (frequency 2 Hz for 75 microseconds at
an amplitude of 70 V). Typically multiple pulses, such as 2, 3, 4,
or 5 pulses, were performed so that the needle sufficiently
penetrated the oocyte for injection. After injection, oocytes were
washed in serial dilutions of TL HEPES in sucrose to minimize
osmotic shock. At 28-30 hours post maturation (i.e., 28-30 hours
after oocytes were placed in maturation medium after collection
from ovaries, which is also at least two hours after injection of
chromatin masses), reconstructed oocytes and controls for
parthenogenetic development were activated with calcium ionophore
(5 .mu.M) for four minutes (Cal Biochem, San Diego, Calif.) and 10
.mu.g/ml cycloheximide and 2.5 .mu.g/ml cytochalasin D (Sigma) in
ACM culture medium [100 mM NaCl, 3 mM KCl, 0.27 mM CaCl.sub.2, 25
mM NaHCO.sub.3, 1 mM sodium lactate, 0.4 mM pyruvate, 1 mM
L-glutamine, 3 mg/ml BSA (fatty acid free), 1% BME amino acids, and
1% MEM nonessential amino acids (Sigma)], for five hours as
described earlier (Liu et al., Mol. Reprod. Dev. 49:298-307, 1998).
After activation, eggs were washed five times and placed in culture
in four-well tissue culture plates containing mouse fetal
fibroblasts and 0.5 ml of embryo culture medium covered with 0.3 ml
of embryo tested mineral oil (Sigma). Between 25 and 50 embryos
were placed in each well and incubated at 38.5.degree. C. in a 5%
CO.sub.2 air atmosphere. If desired, calcium (e.g., .about.0.5,
1.0, 1.5 , 2.0, 2.5, 3, 3.5, 5 mM, or more CaCl.sub.2) can be added
to the culture medium for .about.0.5, 1.0, 1.5, 2.0, 2.5, 3.0, or
more hours to promote resealing of the oocyte after injection. The
resealed oocytes are likely to have increased survival rates due to
the intact layer surrounding the oocytes when they are implanted
into the recipient mammal using the standard methods described
herein.
[0132] Nuclear injection embryos were formed as described above for
chromatin transfer embryos, except that interphase bovine fetal
fibroblasts nuclei that had not been incubated in an extract were
injected into the ooctyes instead of chromatin masses. Nuclear
transplant embryos were generated using the conventional methods
described in Example 1.
[0133] Nuclear transplant, nuclear injection, and chromatin
transfer pronuclei reassemble lamin B (FIG. 8A, red label) and
AKAP95 (FIG. 8B, red label) as anticipated. Nuclear transplant and
nuclear injection pronuclei also reassemble lamins A/C, a
somatic-specific component (FIG. 8A, green label), consistent with
the results reported above for nuclear transplant embryos. However,
chromatin transfer pronuclei and control parthenote pronuclei do
not reassemble lamins A/C (FIG. 8A). Nuclear transplant pronuclei
also contain NuMA (green label), unlike most chromatin transfer or
parthenote pronuclei (FIG. 8B, green label). A proportion of
parthenote nuclei and chromatin transfer nuclei assemble a low
level of NuMA, as reported above.
[0134] In vitro disassembly of nuclei followed by chromatin
transfer results in pronuclei that are morphologically similar to
control parthenote pronuclei. In contrast, nuclear transplant and
nuclear injection pronuclei harbor somatic-specific components
(lamins A/C and extensive NuMA labeling). This result is indicative
of incomplete nuclear remodeling after traditional nuclear
transplant or nuclear injection procedures. As described above,
lamins A/C detected in nuclear transplant and nuclear injection
pronuclei originate from lamins transcribed de novo at the
pronuclear stage. Because nuclear lamins and possibly NuMA are
implicated in transcription regulation and disease in humans,
persistence of lamins A/C in conventional nuclear transplant
pronuclei might be indicative of improper functional reprogramming.
We conclude that in vitro nuclear disassembly and chromatin
transfer produces more normal pronuclei than traditional nuclear
transplant or nuclear injection.
Cloning Efficiency using Reprogrammed Chromatin Masses or
Permeabilized Cells as Donor Source
[0135] As described in Example 3, a novel cloning procedure denoted
"SLOT" was developed that involves Streptolysin O (SLO)-induced
permeabilization of primary fetal bovine fibroblasts, exposure of
permeabilized cells to a reprogramming media (e.g., a mitotic
extract) for 30 minutes, resealing of the fibroblasts with 2 mM
calcium in culture, and transfer of the chromatin into oocytes
using standard cell fusion methods.
[0136] For this cloning method, a vial of Streptolysin O (Sigma
S-5265; 25,000 units stored in store powder form at 4.degree. C.)
was dissolved in 400 .mu.l H.sub.2O and mixed well. All contents
were transferred to a 15-ml conical tube, and then 3.6 ml H.sub.2O
was added and mixed by vortexing. Aliquots of 10 .mu.l were frozen
at -20.degree. C. at a stock concentration of 0.062 U/.mu.l. Cells
(.about.100,000) were suspended in 100 .mu.l HBSS (Gibco BRL, cat.
No. 14170-120) at room temperature. These cells were confluent, and
thus .about.80-85% of the cells were in G1 phase, and the majority
of the other cells were in S phase. Streptolysin O stock solution
(5 .mu.l) (i.e., 500 ng/ml or 0.3 U/.mu.l final concentration) was
added, and the mixture was incubated at 38.degree. C. for 25
minutes in a water bath. The tube was gently tapped 2-3 times
during incubation to ensure that the cells remained in suspension.
Room temperature PBS (200 .mu.l) was added and mixed well by gentle
pipetting. The cells were centrifuged cells at 5,000 rpm for five
minutes at room temperature in a table top centrifuge. All the
supernatant was discarded. At this stage, the pellet is small and
may not be clearly visible. Mitotic extract containing the
ATP-generating system (40 .mu.l, "MS15") was added and mixed well.
The extract was prepared during the centrifugation of the cells by
thawing one vial of 40 .mu.l extract and adding 1.2 .mu.l of
ATP-generating system, mixing well, and incubating at room
temperature. This mitotic extract was the same extract used for the
generation of chromatin masses in the section above. The mixture
was incubated at 38.degree. C. in water bath for 30 minutes, and
the tube was occasionally gently tapped. Room temperature resealing
medium (RM, 500 .mu.L) (complete .alpha.-MEM [Bio-Whittaker] medium
supplemented with CaCl.sub.2 to 2 mM from a 1 M stock) was added.
The tube was left open and incubated in a CO.sub.2 incubator for
two hours with occasional tapping of the tube to ensure that the
cells remained in suspension. The cells were centrifuged at 5,000
rpm for five minutes at room temperature in a table top centrifuge.
The cell pellet was resuspended in 100 .mu.l of room temperature TL
HEPES (Bio-Whittaker, cat. No. 04-616F), and another 900 .mu.l TL
HEPES was added. The nuclear transfer was performed using standard
procedures. Oocytes were activated and transferred to recipient
mammals as described in the previous section for chromatin
transfer.
[0137] The development of embryos formed using this SLOT method and
the chromatin transfer method of the present invention is
summarized in Table 3. Development to the blastocyst stage was
slightly lower for SLOT embryos compared to conventional nuclear
transfer embryos. The differences between SLOT and nuclear transfer
development at the blastocyst stage could be due to the effect of
using a greater precentage of cells in the G1 phase of the cell
cycle for nuclear transfer than for SLOT. The survival rate was
lower for chromatin transfer embryos, which is expected for an
invasive procedure.
[0138] Pregnancy rates were comparable for nuclear transfer and
SLOT embryos at 40 days of gestation (Table 3). Survival from 40
days of pregnancy to 60 days tended to be higher for SLOT embryos
than for nuclear transfer embryos produced using conventional
methods. TABLE-US-00003 TABLE 3 Development of chromatin transfer
(CT), nuclear transplant, and SLOT- produced bovine embryo clones
No. No. No. No. Survived No. Survived PN stage No. Cleaved
Blastocysts No. 40 day 40-60 days/total transferred (%) (%) (%) (%)
Preg. (%) (%) CT 1503 736 (49) 355 (23.5) 81 (5.3) 3 0 ND SLOT 1884
1802 (97) ND 575 (30.5) 156 (8.3) 24/65 (37) 7/10 (70) nuclear 1821
1682 (92) ND 764 (41.9) 235 (12.9) 39/103 (36) 8/16 (50)
transplant
[0139] As noted above, the survival rate for chromatin transfer
embryos may be increased by incubating the reconstituted oocytes in
calcium for a few hours to allow the oocytes to reseal prior to be
inserted into recipient mammals. Survival rates for SLOT embryos
may also be increased by reducing the amount of time between when
the cells are taken out of culture and when they are fused with
oocytes. For example, the length of time for the incubation in
Streptolysin O, the incubation in the reprogramming medium, and/or
the incubation in the resealing medium may be decreased. In
particular, the incubation in the resealing medium may be decreased
to approximately one hour or less. This shortened resealing
treatment may be performed in the presence of 2 mM calcium as
described above or in the presence of a higher concentration of
calcium (e.g., .about.2.5, 3.0, 3.5, 4.0, 4.5, 5.0, or 6.0 mM
calcium) to increase the rate of resealing. By reducing the amount
of time the cells are treated prior to being fused with oocytes,
the cells are less likely to enter S phase and begin DNA
replication which reduces the survival rate of the reconstituted
oocyte.
EXAMPLE 5
Methods for the Generation of Chimeric Mammals
[0140] Many spontaneous abortions that occur using traditional
methods to clone mammals are thought to result from placental
abnormalities rather than from problems with the fetus. Thus,
methods have been developed to produce chimeric embryos with
placental tissue primarily from one origin (e.g., an in vitro
fertilized, naturally-occurring, or parthenogenetically activated
embryo) and fetal tissue primarily from another origin (e.g., a
nuclear transfer embryo). Chimeric embryos with placental tissue
derived primarily from cells from in vitro fertilized,
naturally-occurring, or parthenogenetically activated embryos may
better resemble naturally-occurring placental tissue and result in
increased production of viable offspring. Preferably, the majority
of the cells of the offspring are derived from cells from the
nuclear transfer embryo and thus have a genome that is
substantially identical to that of the donor cell used to generate
the nuclear transfer embryo.
[0141] In one such method, cells from an in vitro fertilized embryo
are injected into the periphery of a compaction embryo (e.g.,
between the zona pellucida and the embryo itself) that was produced
using traditional nuclear transfer methods or any of the novel
cloning methods described herein. In an alternative method, cells
from a precompaction, in vitro fertilized embryo are incubated with
cells from a precompaction embryo produced using one of the cloning
methods of the present invention (e.g., using a reprogrammed
chromatin mass or a permeabilized cell as the donor source) under
conditions that allow cells from each embryo to reorganize to
produce a single chimeric embryo (Wells and Powell, Cloning 2:9-22,
2000). In both methods, the cells from the in vitro fertilized
embryo are preferentially incorporated into the placenta, and the
cells from the nuclear transfer method are preferentially
incorporated into the fetal tissue. These methods are described
further below.
Isolation of G1 Fibroblasts
[0142] For the isolation of G1 fibroblasts as donor cells to
produce nuclear transfer embryos, the previously described "shake
off" method was used (Kasinathan et al., Nature biotech
19:1176-1178, 2001). Briefly, 24 hours prior to isolation,
5.0.times.10.sup.5 cells were plated onto 100 mm tissue culture
plates containing 10 ml of .alpha.-MEM plus FCS. The following day,
plates were washed with PBS, and the culture medium was replaced
for one to two hours before isolation. The plates were then shaken
for 30-60 seconds on a Vortex-Genie 2 (Fisher Scientific, Houston,
Tex., medium speed). The medium was removed, spun at 500.times.g
for five minutes, and the pellet was re-suspended in 250 .mu.l of
MEM plus FCS. This cell suspension consisted of newly divided cell
doublets attached by a cytoplasmic bridge, some single cells, and
metaphase or anaphase cells. The cell doublets attached by a
cytoplasmic bridge were used as donor cells for nuclear
transfer.
Nuclear Transplantation, Activation, and Embryo Culture
[0143] The nuclear transfer procedure using the isolated G1
fibroblasts was performed essentially as previously described
(Cibelli et al., Nature Biotech. 16(7):642-646, 1998; Kasinathan et
al., Biol. Reprod. 64(5):1487-1493, 2000). In vitro matured oocytes
were enucleated about 18-20 hours post maturation, and chromosome
removal was confirmed by bisBenzimide (Hoechst 33342, Sigma)
labeling under UV light. These cytoplast-donor cell couplets were
fused using a single electrical pulse of 2.4 kV/cm for 20
mircoseconds (Electrocell manipulator 200, Genetronics, San Diego,
Calif.). At 30 hours past maturation, reconstructed oocytes and
controls were activated with calcium ionophore (5 .mu.) for four
minutes (Cal Biochem, San Diego, Calif.) and 10 .mu.g cycloheximide
and 2.5 .mu.g cytochalasin D (Sigma) in ACM culture medium (100 mM
NaCl, 3 mM KCl, 0.27 Mm CaCl.sub.2, 25 mM NaHCO.sub.3, 1 mM sodium
lactate, 0.4 mM Pyruvate, 1 mM L-glutamine, 3 mg/ml BSA (fatty acid
free), 1% BME amino acids, and 1% MEM nonessential amino acids; all
from Sigma) for six hours as described previously (Liu et al., Mol.
Reprod. Dev. 49:298-307, 1998; Presicce et al., Mol. Reprod. Dev.
38:380-385, 1994). After activation, eggs were washed in HEPES
buffered hamster embryo culture medium (HECM-HEPES, 114 mM NaCl,
3.2 mM KCl, 2 mM CaCl.sub.2, 10 mM Sodium Lactate, 0.1 mM sodium
pyruvate, 2 mM NaHCO.sub.3, 10 mM HEPES, and 1% BME amino acids;
Sigma) five times and placed in culture in 4-well tissue culture
plates containing mouse fetal fibroblasts and 0.5 ml of embryo
culture medium covered with 0.2 ml of embryo tested mineral oil
(Sigma). Twenty five to 50 embryos were placed in each well and
incubated at 38.5.degree. C. in a 5% CO.sub.2 in air atmosphere. On
day four, 10% FCS was added to the culture medium. On days seven
and eight, development to the blastocyst stage was recorded.
Bovine In Vitro Fertilization
[0144] In vitro fertilization was performed as described earlier to
produce bovine in vitro fertilized embryos (Collas et al., Mol.
Reprod. Dev. 34:224-231, 1993). A 45% and 90% isotonic Percoll
gradient was prepared with sperm TL stock (Parrish et al,
Theriogenology 24:537-549, 1985). Frozen-thawed bovine sperm from a
single bull was layered on top of the gradient and centrifuged for
30 minutes at 700.times.g (2000 rpm using a 6.37 inch tip radius).
The concentration of sperm in the pellet was determined, and the
sperm was diluted in sperm TL (sperm TL stock, 1 mM pyruvate, 6
mg/ml BSA, and 1% PS) such that the final concentration at
fertilization was 10.sup.6 sperm/ml. At 22 hours post maturation,
oocytes were wash three times in TL HEPES and placed in 480 ul of
fertilization TL (Bavister et al., Biol. Reprod. 28:235-247, 1983)
in Nunc wells containing 6 mg/ml BSA, 0.2 mM pyruvate, 20 uM
penicillamine, 10 uM hypotaurine, 1 mM epinepherine (Leibfried et
al., J. Reprod. Fertil. 66:87-93, 1982), and 0.004 ug/ml heparin.
Twenty microliters of sperm were added to generate a final
concentration of 10.sup.6 sperm/ml to 50 oocytes. Culture
conditions were the same as those described above for nuclear
transfer. Fertilization rates were over 90% based on pronuclear
development.
Chimeric Nuclear Transfer Embryos
[0145] In vitro fertilized embryos at 8-cell stage (6-12
blastomeres) were harvested at approximately 96 hours post
fertilization, prior to compaction. The zona pellucida was removed
with protease (3 mg/ml in TL-HEPES). The zona dissolution was
carefully monitored using a dissecting microscope. When the zona
first appeared to dissolve (.about.two minutes), the embryos were
removed and washed in TL-HEPES and transferred to 30 mm petri
dishes containing Hank's balanced salt solution and incubated at
37.5.degree. C. for 30 minutes. The blastomeres from these
precompaction embryos were transferred into microdrops (50 .mu.l)
of TL-HEPES under mineral oil in 100 mm petridish. Nuclear transfer
embryos on day four at the 8-16 cell stage were selected and
transferred into the same microdrops containing the blastomeres.
These nuclear transfer embryos included both precompaction embryos
(e.g., 8 cell stage embryos) and compaction embryos (e.g., 16 stage
embryos). Then 4-6 blatomeres were transferred into the nuclear
transfer embryos with the beveled micro pipette (35 .mu.m diameter)
using standard micromanipulation techniques. After transferring the
blastomeres, the embryos were cultured as described for nuclear
transfer embryos.
[0146] On days seven and eight, the development to blastocyst of
the chimeric embryos was evaluated. The blastocysts were also
analyzed for the presence of the membrane dye DiI that was added to
the cells from the in vitro fertilized embryo before they were
injected into the nuclear transfer embryo. The cells were labeled
on day four and observed on day seven. This dye is maintained for a
few cell divisions in the progeny of the originally dyed cells,
allowing the chimeric embryo to be analyzed after a few cell
divisions. Based on this analysis, cells from the in vitro
fertilized embryo were incorporated into the chimeric embryo. If
desired, fluorescence in situ hybridization (FISH) with a probe
specific for a nucleic acid in either the in vitro fertilized
embryo or the nuclear transfer embryo can be performed using
standard methods (see, for example, Ausubel et al., Current
Protocols in Molecular Biology, John Wiley & Sons, New York,
pp. 14.7.1-14.7.12, 1995). This FISH analysis can be used to
determine the distribution of cells derived from each embryo in the
chimeric embryo (e.g., to determine what percent of the cells are
incorporated into the inner cell mass and what percent are
incorporated into the trophectoderm) while it is cultured in vitro
and in the fetus or the offspring generated from the embryo.
Alternatively, a reporter gene such as green fluorescent protein
can be added to cells from one of the embryos and used to monitor
the incorporation of the cells into the placenta and various fetal
tissues of the chimeric embryo.
Embryo Transfer
[0147] Days seven and eight, nuclear transfer blastocysts of grade
1 and 2, derived from nuclear transfer embryos and chimeric nuclear
transfer embryos were transferred into day six and seven
synchronized recipient heifers. Recipients were synchronized using
a single injection of Lutalyse (Parmacia & Upjohn, Kalamazoo,
Mich.) followed by estrus detection. The recipients were examined
on days 30 and 60 after embryo transfer by ultrasonography for the
presence of conceptus and thereafter every 30 days by rectal
palpation until 240 days. The pregnancy results at day 40 for the
chimeric embryos and for control embryos produced by fusing a
transgenic bovine fibroblast with an oocyte are compared in Table
4. These results indicate that a greater number of chimeric embryos
survived until day 40. TABLE-US-00004 TABLE 4 Embryo transfers and
pregnancies Control Nuclear transfers Chimeric Nuclear Transfers 40
day 40 day Implant No of recipients Pregnancy No of recipients
Pregnancy First 2 1 2 1 Second 6 1 4 3 Total 8 2 (25%) 6 4
(67%)
Alternative Methods for Production of Chimeric Embryos
[0148] Standard methods can be used to modify the above method for
producing chimeric embryos. For example, a naturally-occurring
embryo can be surgically isolated from a mammal (e.g., a bovine) or
an oocyte can be parthenogenetically activated using standard
techniques and used instead of the in vitro fertilized embryo. If
desired, fewer cells from the in vitro fertilized,
naturally-occurring, or parthenogenetically activated embryos
(e.g., 1, 2, 3, 4, or 5 cells) can be injected into the nuclear
transfer embryo to reduce the percent of the injected cells and
their progeny that become incorporated into fetal tissue.
Alternatively, more cells (e.g., 6, 7, 8, 9, 10, 11 or more cells)
can be injected to increase the percent of the injected cells and
their progeny that are incorporated into placental tissue.
Moreover, cells from embryos in other cell stages can be used. For
example, in vitro fertilized, naturally-occurring, or
parthenogenetically activated embryos at the 4, 8, 16, 32, 64, 128,
256, 512, or later cell stage can be injected into nuclear transfer
embryos at the 4, 8, 16, 32, 64, 128, 256, 512, or later cell
stage. The injected cells and the nuclear transfer embryo can be at
the same cell stage or at different cell stages. In one embodiment,
the in vitro fertilized, naturally-occurring, or
parthenogenetically activated embryo has increased ploidy (e.g., a
DNA content of 4n) relative to the nuclear transfer embryo, which
further biases the injected cells to the trophectoderm (i.e., the
outermost layer of cells of the embryo that primarily forms the
placental tissue). If desired, all or part of the zona pellucida
can be kept surrounding the injected cells, rather than removed
prior to injection.
[0149] In other alternative methods, cells from a precompaction or
compaction in vitro fertilized, naturally-occurring, or parthenote
embryo are incubated with cells from a precompaction nuclear
transfer embryo under conditions that allow cells from each embryo
to reorganize to produce a single chimeric embryo (Wells and
Powell, Cloning 2:9-22, 2000). Cells from in vitro fertilized,
naturally-occurring, or parthenote embryo are expected to
contribute primarily to the trophectoderm and eventually to the
placental tissue, and cells from the nuclear transfer embryo are
expected to contribute primarily to the inner cell mass and
eventually to the fetal tissue. Cells from both embryos can be at
the same cell stage or at different cell stages, and the same or
different numbers of cells from each embryo can be combined to form
the aggregation embryo.
Other Embodiments
[0150] From the foregoing description, it will be apparent that
variations and modifications may be made to the invention described
herein to adopt it to various usages and conditions. Such
embodiments are also within the scope of the following claims.
[0151] All publications mentioned in this specification are herein
incorporated by reference to the same extent as if each independent
publication or patent application was specifically and individually
indicated to be incorporated by reference.
* * * * *