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ScienceWeek
SCIENCEWEEK
ScienceWeek
March 14, 2003
Vol. 7 Number 11
An Online Digest of Research in the Sciences
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Between the fifth and tenth days the lump of stem cells
differentiates into the overall building plan of the mouse embryo
and its organs. It is a bit like a lump of iron turning into the
space shuttle. In fact it is the profoundest wonder we can still
imagine and accept, and at the same time so usual that we have to
force ourselves to wonder about the wondrousness of this wonder.
-- Miroslav Holub
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Section 1
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Thematic Issue: Cloning and Somatic Cell Nuclear Transfer
1. Introduction
2. Somatic Cell Nuclear Transfer
3. Differentiation and Nuclear Transfer
4. Epigenetic Aspects
5. Abnormal Gene Expression and Nuclear Transfer
6. An Application in Neurobiology
Notices and Subscription Information
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Section 2
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1. INTRODUCTION
Broadly defined, cloning of a complete organism is asexual
reproduction that results in progeny genetically identical to the
parent. To many people, cloning was invented with the birth of
the sheep Dolly. In fact, cloning has been practiced for
millennia with plants, and for decades with mammals, and Dolly's
birth followed an orderly progression of experiments that started
with cloning mammalian embryos in the 1970s. The first successful
mammalian cloning by nuclear transfer, in which cells from
cleavage-stage sheep embryos were fused with unfertilized sheep
eggs, was reported in 1986. Successful cloning from older embryos
(and ultimately from an adult cell, in the case of Dolly)
challenged conclusions from previous work that the nuclei of
differentiated cells are unable to support normal development.
In general, the term "stem" cells refers to undifferentiated
cells that upon differentiation can give rise to various
specialized cell lines such as blood cells, skin cells, nerve
cells, etc. Adult bone marrow, for example, contains stem cells
that are the precursors of the various specialized types of blood
cells. "Embryonic" stem cells are specifically stem cells derived
from the embryo only, and such cells are "totipotent", i.e., they
have the ability to differentiate into any type of cell and thus
form a new organism or regenerate any part of an organism.
ON ANIMAL CLONING
"The crowning -- provisionally? -- achievement [of genetic
engineering] has been the cloning of animals, that is, the
production of animals almost identical genetically to a chosen
individual. In this technique, the nucleus of an egg cell is
removed and replaced by the nucleus of a somatic cell -- from the
intestine, for instance, or the mammary gland -- of the organism
to be cloned. The egg cell produced in this way contains two sets
of chromosomes, just like a normally fertilized egg cell. But,
contrary to the normal case, the chromosomes do not originate
from two parental germ cells, of which each has provided one of
the two sets, but from the differentiated cell whose nucleus has
been used. Thus, the individual born from the development of the
renucleated egg cell in a surrogate mother is a genetic copy of
the donor of the nucleus, except for mitochondrial genes and
other forms of cytoplasmic heredity, which are provided by the
recipient egg cell. This technique was first successfully applied
to amphibians, in the 1970s, by the British biologist John
Gurdon. Then a Swiss investigator claimed to have cloned mice,
but later had to retreat in some confusion, perhaps unjustly in
view of what is now known of the hazards of the technique, when
his results proved not to be reproducible. Cloning made a
spectacular comeback in 1997, with the announcement, by a
Scottish laboratory directed by lan Wilmut, of the birth of the
now world-famous Dolly, a ewe six years younger than its almost
identical twin, the donor of the nucleus used to replace the
egg's own nucleus. Since then, mammalian cloning has produced
mice, calves, goats, and pigs. Application of the technique to
primates has proved more difficult but will presumably be
successfully accomplished some time in the future. Human cloning
is already being contemplated, in spite of strong ethical
objections.
"Note that the technology still remains highly problematic. Even
under the best conditions, many attempts at cloning are
unsuccessful or yield severely abnormal offspring. Dolly, for
example, was the single product of 277 attempts. In general, the
present success rate varies between 0.1 and 1.0 percent. These
difficulties are understandable. In reality, what is puzzling is
the successes, rather than the failures... Body [somatic] cells
originate from embryonic cells by progressive differentiation, a
process in which only certain genes -- such as those that specify
a brain, liver, or skin cell, depending on the cells' location in
the developing embryo -- come to be expressed, while all others
are silenced. Thus, when the nucleus of such a cell is used for
transfer, it must be deprogrammed so as to recover all the
potentialities needed for the formation of the cells of the
cloned organism. This reversal is beset by many difficulties. The
fact that cloning can be done shows that complete deprogramming
of a body cell nucleus is possible, presumably thanks to the
special environment provided by the egg-cell cytoplasm. However,
the stage at which it can occur may be highly critical, thus
accounting for the many failures."
Christian de Duve: Live Evolving: Molecules, Mind, and Meaning.
Oxford University Press 2002, p.253.
ON CYTOPLASMIC CONTROL OF GENE EXPRESSION
"A considerable body of evidence supports the notion that the
spectrum of genes expressed in eukaryotic cells is influenced by
the cytoplasmic environment in which the nucleus resides. The
technique of nuclear transplantation has been especially useful
in demonstrating this point. In one type of transplantation
study, transcriptionally inactive nuclei have been taken out of
cells that are synthesizing neither DNA nor RNA and transplanted
into egg cells whose nuclei were previously removed. When placed
in this new cytoplasmic environment, a nucleus that had
previously been inactive will begin to synthesize RNA and, in
some cases, DNA. In the most dramatic experiments of this type,
carried out on the African clawed toad Xenopus laevis, John
Gurdon removed the nuclei from cells lining the intestine of
swimming tadpoles and transplanted them into egg cells whose
nuclei had already been removed. Although the transplanted nuclei
were derived from intestinal cells, they were able to direct the
development of the egg cell into a complete new toad. Clearly the
egg cytoplasm must exert profound effects on nuclear activity if
it can induce the nucleus of an intestinal cell to behave in this
way."
L.J. Kleinsmith and V.M. Kish: Principles of Cell and Molecular
Biology. HarperCollins 1995, p.445.
ON THE PLURIPOTENCY OF SOMATIC CELLS
"Is it possible that some differentiated cell nuclei differ from
others in their ability to direct development? John Gurdon and
his colleagues, using... methods of nuclear transplantation on
the frog Xenopus, obtained results suggesting that the nuclei of
some differentiated cells can remain totipotent. [Like other
researchers], Gurdon... found a progressive loss of potency with
increasing developmental age, although Xenopus cells retained
their potencies for a longer period than did the cells of Rana.
The exceptions to this rule, however, proved very interesting.
Gurdon transferred nuclei from the intestinal endoderm of feeding
Xenopus tadpoles into activated enucleated eggs. These donor
nuclei contained a genetic marker (one nucleolus per cell instead
of the usual two) that distinguished them from host nuclei. Out
of 726 nuclei transferred, only 10 (1.4%) promoted development to
the feeding tadpole stage. Serial transplantation (transplanting
an intestinal nucleus into an egg and, when the egg had become a
blastula, transferring the nuclei of the blastula cells into
several more eggs) increased the yield to 7% (Gurdon 1962). In
some instances, nuclei from tadpole intestinal cells were capable
of generating all the cell lineages -- neurons, blood cells,
nerves, and so forth -- of a living tadpole. Moreover, seven of
these tadpoles (from two original nuclei) metamorphosed into
fertile adult frogs (Gurdon and Uehlinger 1966); these two nuclei
were totipotent.
"King and his colleagues criticized these experiments, pointing
out that (1) not enough care was taken to make certain that
primordial germ cells, which can migrate through to the gut, were
not used as sources of nuclei, and (2) the intestinal epithelial
cells of such a young tadpole may not qualify as a truly
differentiated cell type because such cells of feeding tadpoles
still contain yolk platelets (Di Berardino and King 1967;
McKinnell 1978; Briggs 1979). To answer these criticisms, Gurdon
and his colleagues cultured epithelial cells from adult frog foot
webbing. These cells were shown to be differentiated; each of
them contained a specific keratin, the characteristic protein of
adult skin cells. When nuclei from these cells were transferred
into activated, enucleated Xenopus oocytes, none of the first-
generation transfers progressed further than the formation of the
neural tube shortly after gastrulation. By serial
transplantation, however, numerous tadpoles were generated
(Gurdon et al. 1975). Although these tadpoles all died prior to
feeding, they showed that a single differentiated cell nucleus
still retained incredible potencies.
"In 1997, lan Wilmut announced that a sheep had been cloned from
a somatic cell nucleus from an adult female sheep. This was the
first time that an adult vertebrate had been successfully cloned
from another adult. To do this, Wilmut and his colleagues (1997)
took cells from the mammary gland of an adult (6-year-old)
pregnant ewe and put them into culture. The culture medium was
formulated to keep the nuclei in these cells at the resting stage
of the cell cycle (Gg). They then obtained oocytes (the maturing
egg cell) from a different strain of sheep and removed their
nuclei. The fusion of the donor cell and the enucleated oocyte
was accomplished by bringing the two cells together and sending
electrical pulses through them. The electric pulses destabilized
the cell membranes, allowing the cells to fuse together.
Moreover, the same pulses that fused the cells activated the egg
to begin development. The resulting embryos were eventually
transferred into the uteri of pregnant sheep. Of the 434 sheep
oocytes originally used in this experiment, only one survived:
Dolly. DNA analysis confirmed that the nuclei of Dolly's cells
were derived from the strain of sheep from which the donor
nucleus was taken (Ashworth et al. 1998; Signer et al. 1998).
Thus, it appears that the nuclei of adult somatic cells can be
totipotent. No genes necessary for development have been lost or
mutated in a way that would make them nonfunctional. This result
has been confirmed in cows (Kato et al. 1998) and mice (Wakayama
et al. 1998). In mice, somatic cell nuclei from the cumulus cells
of the ovary were injected directly into enucleated oocytes.
These re-nucleated oocytes were able to develop into mice at a
frequency of 2.5%. Interestingly, nuclei from other somatic cells
(such as neurons or Sertoli cells) that are similarly blocked at
the Gg stage did not generate any live mice. Cumulus cell nuclei
from cows have also directed the complete development of oocytes
into mature cows (Kato et al. 1998)."
Scott F. Gilbert: Developmental Biology, 6th Edition. Sinauer
Associates 2000, p.85.
ON TERMINOLOGY IN CLONING
B. Vogelstein et al (Johns Hopkins University, US) discuss
current terminology concerning cloning, the authors making the
following points:
1) Scientists rely on a dialect of specialized terminology to
communicate precise descriptions of scientific phenomena to each
other. In general, that practice has served the scientific
community well, and novel terms are generally created when needed
to document new findings, behaviors, structures, or principles.
However, when the scientific shorthand makes it way to the
nonscientific public, there is a potential for precise meaning to
be lost or misunderstood and for the terminology to become
associated with research or applications for which it is
inappropriate.
2) In scientific parlance, "cloning" is a broadly used shorthand
term that refers to producing a copy of some biological entity --
a gene, an organism, a cell -- an objective that in many cases
can be achieved by means other than the technique known as
"somatic cell nuclear transfer". Bacteria clone themselves by
repeated fission. Plants reproduce clonally via sexual means and
by vegetative regeneration.
3) Much confusion has arisen in the public, in that cloning seems
to have become almost synonymous with somatic cell nuclear
transfer, a procedure that can be used for many different
purposes. However, only one of these purposes involves an
intention to create a clone of the entire organism (for example,
a human being). Unfortunately, legislation currently under
consideration by the US Congress does not adequately distinguish
cloning in general from human cloning, and this legislation would
prohibit a wide range of experimental procedures that in the near
future might become both medically useful and morally acceptable.
4) The authors offer the following tabulation of crucial
differences between nuclear transplantation in general and human
reproductive cloning in particular:
Scientific Usage of the Term "Cloning":
End product: In nuclear transplantation (NT), cells growing in a
petri dish; in human reproductive cloning (HRC), a human being.
Purpose: In NT, to treat a specific disease involving tissue
degeneration; in HRC, to replace or duplicate a human.
Time frame: In NT, a few weeks, (growth in culture); in HRC, 9
months.
Surrogate mother needed?: In NT, no; in HRC, yes.
Sentient human created?: In NT, no; in HRC, yes.
Ethical implications: In NT, similar to all embryonic cell
research; in HRC, highly complex issues.
Medical implications: in NT, similar to any cell-based therapy;
in HRC, safety and long-term efficacy concerns.
Science 2002 295:1237
ON MISUNDERSTANDINGS OF CLONING
Lee M. Silver (Princeton University, US) discusses current
confusions concerning cloning. Herbert J. Webber coined the word
"clone" in 1903 to describe a colony of organisms derived
asexually from a single progenitor, and the term was quickly
adopted by biologists. A clone of animal siblings can form
naturally, on occasion, as a result of asexual reproduction from
a single progenitor embryo. However, in contrast to plants, whole
animals cannot be grown directly from cells that have begun to
differentiate into a specialized form. The popular understanding
of cloning has its roots in Alvin Toffler's 1970 book _Future
Shock_, in which Toffler took a clear scientific concept and
muddled it into the fantastical prediction that "man will be able
to make biological carbon copies of himself". Unfortunately,
this fictitious version of cloning was presented in a highly-
influential non-fiction book, and in one fell swoop, clones
morphed from the simple progeny of asexual reproduction into
sophisticated products of biological engineering created by
scientists bent on controlling nature. Through the popular media,
this version of a clone was rapidly integrated into every major
language. The concept of a clone was extended to inanimate
objects such as computers ("PC clone"), as well as becoming a
figure of speech to describe people. No technology, however,
exists for making copies of people: real cloning technology can
lead only to the birth of a unique and unpredictable child who
has the same DNA sequence as someone else, but nothing more. The
scientific community has lost control over the term: cloning has
a popular connotation that is impossible to dislodge, and we must
accept that democratic debate on cloning is bereft of any
meaning. Science and scientists would be better served by
choosing other words to explain advances in developmental
biotechnology to the public.
Nature 2001 412:21
ON THE FUTURE OF CLONING
J.B. Gurdon and A. Coleman (2 installations, UK) review the
current state of the science and technology of cloning, the
authors making the following points:
1) The authors point out that cloning techniques have been in use
for centuries. The practice of taking cuttings is universal among
gardeners, and large companies now propagate desirable plant
strains in large quantities. Lower invertebrates can be easily
cloned: for example, if one cuts an earthworm or flatworm in
half, the halves will regenerate to create two genetically
identical individuals. Although this method does not work in
vertebrates, identical twins are naturally occurring genetic
clones, and the method of nuclear transplantation, first used 40
years ago in frogs, has been successfully used to make clones of
various mammals, and could probably be applied to humans.
2) Of importance in nuclear transfer techniques is the general
scheme of natural fertilization: In vertebrates, fertilization
begins with the union of the sperm cell and the egg cell. Prior
to fertilization, the egg cell has been stopped at a certain
stage of the *cell-division cycle. The sperm provides an
activation stimulus that triggers the resumption and completion
of cell division. The egg and sperm "*pronuclei" then swell,
their chromosomes unravel from the tightly packed condensed state
in which they are stored, and DNA replication can proceed. The
chromosomes then recondense, the nuclear membrane dissolves, and
the fertilized egg cell divides into two identical daughter
cells.
3) The technique of nuclear transfer subverts fertilization by
replacing the female genetic material of an unfertilized egg cell
with the nucleus from a different cell. The general procedure of
nuclear transfer in mammals is as follows:
... ... a) The genetic material is removed from the recipient
cell (an unfertilized egg cell).
... ... b) The genetic material of this egg cell is replaced by a
nucleus from a donor cell, the donor nucleus containing donor DNA
(the donor genome).
... ... c) The egg cell, now containing the donor nucleus, begins
the series of divisions that normally follow fertilization.
... ... d) At a very early embryonic stage (*blastocyst), the
embryo is transferred to a surrogate mother.
... ... e) In the surrogate mother, the embryo (fetus) develops
to term, is delivered as a neonate, and the neonate is
genetically identical to the donor of the original donor nucleus.
4) The authors suggest that for successful cloning, it is
probably essential for donor nuclei to contain a full complement
of genes. For nuclear transfer to work, an adult cell that has
already been programmed (differentiated) into a specific cell
type needs to be somehow reprogrammed so that it regains the
genetic totipotency of sperm cells and egg cells (germline cells)
-- the ability to guide the formation of all the different cell
types that make up an animal. An important conclusion to come
from nuclear transfer experiments is that the processes of cell
differentiation and ageing do not lead to permanent genetic
changes in non-germline cells (somatic cells).
5) The pattern of gene expression in adult cells is very
different from that in embryonic cells. In amphibians, for
example, a number of genes expressed in embryos 5 hours after
fertilization are not expressed in differentiated adult cells.
Conversely, some genes are expressed in adult cells but not in
early embryos. When embryos are analyzed a few hours after the
transfer of adult cell nuclei, gene expression cannot be
distinguished from that in embryos grown from normal fertilized
eggs. This indicates that the exchange of cytoplasm around a
nucleus, from the cytoplasm of an adult cell to that of an egg
cell, causes a dramatic switch in gene expression in only a few
hours. A nucleus that was once part of an intestine, skin, or
muscle cell is therefore transformed into that of an embryonic
cell. Key molecules found in egg cells that may bring about
reprogramming of the genome include *nucleoplasmin and certain
embryo-specific proteins around which the DNA is wrapped (embryo-
specific *histones).
6) The authors suggest that one of the major uses for cloning in
the future may be "therapeutic cloning" -- the use of cloning to
generate tissue to replace tissue that has been damaged or
diseased. The essential idea here is to use as a donor nucleus in
nuclear transfer the nucleus of a somatic cell of the individual
requiring therapeutic tissue replacement. The major advantage of
the technique is that transplantation of the cloned tissue into
the original donor should occur without the tissue rejection that
now compromises the success of transplantation procedures: the
cloned tissue would be genetically identical to the patient's
tissue. The authors suggest that therapeutic cloning is
"ethically less contentious because a new person is not
produced." However, since an unfertilized human egg cell must be
used in the procedure, "as for abortion, the issue of the
deliberate destruction of a potential person is raised."
Nature 1999 402:743
Notes:
... ... *cell-division cycle: The term "cell division cycle"
(cell cycle) refers to the ordered sequence of phases through
which a cell passes from one mitotic cell division to the next.
... ... *pronuclei: The term "pronucleus" refers to the nucleus
of either the egg cell (ovum) or the sperm cell following
fertilization. Once the ovum is fertilized, there are two
pronuclei, one originating from the ovum, the other from the
sperm cell that produced fertilization. The two nuclei do not
fuse until immediately before the first cleavage, when each
pronucleus loses its membrane to release its contents.
... ... *blastocyst: A mammalian egg in the later stages of
*cleavage but before implantation in the uterus. The blastocyst
consists of a hollow fluid-filled ball of cells and an inner cell
mass (embryonic stem cells) from which the embryo develops.
... ... *cleavage: The early and rapid division stage that
divides the fertilized egg into smaller and smaller cells
(blastomeres) while retaining the same overall size of the
embryo.
... ... *nucleoplasmin: A heat-stable acidic protein present in
the nucleus of many cell types. It forms complexes with
*histones.
... ... *histones: In *eukaryotic chromosomes, about every 200
nucleotides, the DNA double helix is coiled around a complex of 8
histone proteins, the entire assembly having the appearance of
beads on a string. The beads (nucleosomes) are in turn
supercoiled into a solenoid structure, and the entire complex of
the eukaryotic chromosome is called "chromatin". The small
histone proteins are basic (as opposed to acidic) proteins, and
they are essential in forming nucleosomes. Chemically, histones
are single polypeptide chains, molecular mass 11 to 21
kilodaltons, 25 percent lysine and arginine amino acids.
... ... *eukaryotic: Eukaryotic cells are cells having internal
membrane-bound organelles such as a nucleus.
ON THE MEDICAL APPLICATIONS OF CLONING
Ian Wilmut, who led the research team that cloned the sheep
Dolly, presents an essay describing the general techniques of
cloning and the possible medical applications. The author makes
the following points:
1) The author says the announcement of the sheep Dolly's birth in
February 1997 attracted enormous press interest, perhaps because
Dolly drew attention to the theoretical possibility of cloning
humans. The author says this is an outcome he hopes never comes
to pass. But the ability to make clones from cultured cells
derived from easily obtained tissue should bring numerous
practical benefits in animal husbandry and medical science, as
well as answer critical biological questions.
2) The ability to produce offspring from cultured cells opens up
relatively easy ways to make genetically modified (transgenic)
animals. Such animals are important for research and can produce
medically valuable human proteins.
3) Cloning offers many other possibilities. One is the generation
of genetically modified animal organs that are suitable for
transplantation into humans.
4) Another promising area is the rapid production of large
animals carrying genetic defects that mimic human diseases such
as *cystic fibrosis.
5) The power to make animals with precisely engineered genetic
constitution could also be employed more directly in cell-based
therapies for important diseases, including Parkinson's disease,
diabetes, and muscular dystrophy.
6) Cloning could also be a means of producing herds of cattle
that lack the prion protein gene, which makes cattle susceptible
to infection with prions, the agents that cause "*mad cow
disease".
7) The cloning technique might curtail the transmission of
genetic disease by treating an embryo with advanced forms of gene
therapy to modify the nuclei of embryonic cells so that the
subsequent fetus and child was free of a specific genetic disease
and unable to pass the disease to the next generation.
8) The author states that none of the suggested uses of cloning
for making copies of existing people is ethically acceptable to
his way of thinking, "because they are not in the interests of
the resulting child. It should go without saying that I strongly
oppose allowing cloned human embryos to develop so that they can
be tissue donors." The author concludes: "It nonetheless seams
clear that cloning from cultured cells will offer important
medical opportunities. Predictions about new technologies are
often wrong; societal attitudes change; unexpected developments
occur. Time will tell. But biomedical researchers probing the
potential of cloning now have a full agenda."
Scientific American December 1998
Related Background:
DEVELOPMENTAL POTENTIAL OF CLONED MAMMALIAN EMBRYOS
James C. Cross (University of Calgary, CA) discusses the
developmental potential of cloned mammalian embryos, the author
making the following points:
1) Few recent scientific advances have captured the imagination
of biologists and the general public like the prospect of animal
cloning. The procedure is elegantly simple: a nucleus from a
mature cell is transferred into the cytoplasm of an enucleated
egg and the nucleus becomes "reprogrammed" to reexecute
embryogenesis. That cloning has been successful at all seems
biologically remarkable and has forced biologists to assess what
cell differentiation is all about.
2) However, although possible, the process has many
complications. Fetal and placental weight are often dramatically
increased. Animals also frequently suffer from congenital
anomalies and die within hours of birth. Embryonic and fetal
losses are also extremely high, such that far less than 1 percent
of manipulated embryos give rise to live-born animals. These grim
facts, collectively termed the "cloned offspring syndrome", have
raised considerable concern about the cloning process.
3) The reasons for these complications have remained a mystery,
but recent work has revealed surprising conclusions, and results
indicate that different aspects of cloned offspring syndrome are
attributable to distinct methodological problems. In particular,
poor embryonic and postnatal survival is not specifically
associated with cloning, and the underlying mechanistic defects
can now better be addressed by focusing on the effects of cell
culture conditions, embryo transfer, and so on.
Proc. Nat. Acad. Sci. 2001 98:5949
ScienceWeek http://www.scienceweek.com
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2. SOMATIC CELL NUCLEAR TRANSFER
I. Wilmut et al (Roslin Institute, UK) discuss somatic cell
nuclear transfer, the authors making the following points:
1) Cloning by present methods is very inefficient owing to the
extraordinary demands placed on the oocyte cytoplasm in
reprogramming a somatic nucleus rather than a sperm nucleus. The
cumulative loss observed throughout development is assumed to
reflect inappropriate expression of many genes whose harmful
effect is exerted at different stages of development. These
fundamental limitations to cloning are being addressed by
analyses of the underlying cellular mechanisms. In time, this
information may be used in the development of treatments to cause
cells of one phenotype to "transdifferentiate" to another.
2) Several laboratories have used a variety of somatic cell types
to create cloned sheep, cattle, mice, pigs, goats, rabbits and
cats (1). However, those laboratories have failed so far to
obtain offspring in other species, including the rat, rhesus
monkey and dog. Consistent among all published research is that
only a small proportion of embryos reconstructed using adult or
fetal somatic cells developed to become live young, typically
between 0 and 4%. The low overall success rate is the cumulative
result of inefficiencies at each stage of the process.
3) In addition to embryonic loss, somatic cell nuclear transfer
is also associated with very high rates of fetal, perinatal and
neonatal loss, and production of abnormal offspring. Not all of
these effects are due solely to nuclear transfer, since similar
problems are reported after embryo culture(2). Typically, at
least one-third of the cattle and sheep confirmed pregnant with
cloned embryos lose their fetuses during gestation(1). Abnormal
development of the placenta, including vascular reduction, is a
principal contributor to loss particularly during early pregnancy
in sheep and cattle(3). It may also contribute to some of the
defects reported in neonates(4). In cattle the rate of loss is
also increased in the second and third trimesters of pregnancy
after nuclear transfer (compared with in vitro fertilization
(IVF)), with greater losses when adult rather than fetal or
embryonic nuclei are used(5). The overaccumulation of placental
fluid in hydroallantois occurs rarely in natural cattle
pregnancies, but can affect up to 2% and 40% of pregnancies
established with IVF and cloned embryos, respectively. In cloned
mice, the placentae are often 2 3-fold heavier than from natural
mating8, although a lack of vascularization has not been
reported.
4) As the fate of cloned embryos is determined by molecular
events within hours of nuclear transfer, it is disappointing that
so little is known about these events during the early
development of cloned embryos. During somatic cell nuclear
transfer a great deal is asked of the molecular mechanisms that
have evolved to regulate fertilization and pregnancy. Viewed in
this light, it is still surprising that somatic cell cloning ever
produces viable offspring. Although some improvements in
efficiency are to be expected from optimization of present
procedures, greater benefits might be expected from intervention
to assist reprogramming of the transferred nucleus. At present
the means to enhance the success of nuclear transfer are not
known, but may involve the use of remodeling complexes and
factors that remove somatic epigenetic modifications before
transfer. In addition to application of this information in
nuclear transfer, new understanding of mechanisms that regulate
developmental plasticity will lead to methods to change cells of
one phenotype to another as a means of providing histocompatible
cells for treatment of degenerative diseases. A new era of
developmental biology and regenerative medicine awaits.
5) In summary: Despite present experimental difficulties, cloning
by nuclear transfer from adult somatic cells is a remarkable
demonstration of developmental plasticity. When a nucleus is
placed in oocyte cytoplasm, the changes in chromatin structure
that govern differentiation can be reversed, and the nucleus can
be made to control development to term.
References (abridged):
1. Wilmut, I. & Peterson, L. A. Somatic cell nuclear transfer
(cloning) efficiency [online]
http://www.roslin.ac.uk/public/webtablesGR.pdf (2002).
2. Young, L. E. & Fairburn, H. R. Improving the safety of embryo
technologies: possible role of genomic imprinting. Theriogenology
53, 627-648 (2000)
3. Hill, J. R. et al. Evidence for placental abnormality as the
major cause of mortality in first-trimester somatic cell cloned
bovine fetuses. Biol. Reprod. 63, 1787-1794 (2000)
4. Hill, J. R. et al. Clinical and pathologic features of cloned
transgenic calves and fetuses (13 case studies). Theriogenology
51, 1451-1465 (1999)
5. Heyman, Y. et al. Frequency and occurrence of late-gestation
losses from cattle cloned embryos. Biol. Reprod. 66, 6-13 (2002)
Nature 2002 419:583
Related Background Brief:
IMPROVING THE SAFETY OF EMBRYO TECHNOLOGIES: POSSIBLE ROLE OF
GENOMIC IMPRINTING. Although developments in mammalian in vitro
embryo technologies have allowed many new clinical and
agricultural achievements, their application has been hindered by
limitations in the developmental potential of resulting embryos.
Low efficiencies of development to the pre-implantation
blastocyst stage have been consistently observed in most species,
including humans, rabbits, pigs and ruminants. Furthermore, in
cattle and sheep a wide range of congenital abnormalities
currently termed "Large Offspring syndrome" (LOS) are commonly
observed as a result of several embryo culture and manipulation
procedures. The authors review the hypothesis that at least some
of the problems associated with embryo technologies may result
from disruptions in imprinted genes. Several imprinted genes
(i.e. genes which express only the maternal or paternal allele)
are known to have significant effects on fetal size and survival
in other species and are possible candidates for involvement in
livestock LOS. Major changes in putative imprinting mechanisms
such as DNA methylation of imprinted genes occur in the mouse
embryo during pre-implantation development. Alterations in DNA
methylation are transmitted with stability through repeated cell
cycles such that changes in the embryo may still act at the fetal
stages. Thus any disruption in establishment and/or maintenance
of imprinting during the vulnerable periods of embryo culture or
manipulation is a plausible candidate mechanism for inducing
fetal loss and Large Offspring Syndrome. Identification of these
disruptions may provide crucial means to improve the success of
current procedures. L.E. Young and H.R. Fairburn: Theriogenology
2000 53:627.
Related Background:
CLONED CALVES PRODUCED FROM ADULT CELLS AFTER LONG-TERM CULTURE
C. Kubota et al (7 authors at 2 installations, JP US) report the
birth of 6 clones of an aged (17-year-old) Japanese Black Beef
bull using ear-skin *fibroblast cells as nuclear donor cells
after up to 3 months of in vitro culture (10 to 15 *passages).
The authors report they observed higher developmental rates for
embryos derived from later passages (10 to 15) as compared with
those embryos from an early passage (passage #5). Four surviving
clones are now 10 to 12 months of age and appear normal, similar
to their naturally reproduced peers. The authors suggest these
data indicate that fibroblasts of aged animals remain competent
for cloning, and that prolonged culture does not affect the
cloning competence of adult somatic cells. The authors conclude:
"In this study, we demonstrated that adult somatic cells remained
*totipotent for cloning after long-term culture. This suggests
the feasibility of targeted genetic manipulations such as *gene
knockout using cultured somatic cells before cloning to produce
knockouts or other types of genetically engineered cloned
animals. Cloning using site-specific genetically manipulated
cells would be a valuable tool with applications in agriculture,
medicine, and basic biological research."
Proc. Nat. Acad. Sci. 2000 97:990
Notes:
... ... *passages: In this context, the term "passage" refers to
a replication of cells in culture, with each passage referring to
a single replication.
... ... *fibroblast cells: (fibroblasts) Fibroblasts are a type
of connective tissue cell, secreting structural proteins such as
collagen, the proteins forming a matrix in which the fibroblasts
become embedded. These cells can be easily obtained from skin,
and they can be easily cultured outside the body.
... ... *totipotent: "Totipotent" cells have the ability to
differentiate into any type of cell and thus form a new organism
or regenerate any part of an organism.
... ... *gene knockout: In this context, the term "knockout"
refers to an organism in which function of a specific gene has
been deleted by genetic engineering techniques.
Related Background Brief:
FULL-TERM DEVELOPMENT OF GOLDEN HAMSTER OOCYTES FOLLOWING
INTRACYTOPLASMIC SPERM HEAD INJECTION. The golden hamster is the
mammalian species in which intracytoplasmic sperm injection
(ICSI) was first tried to produce fertilized oocytes. Thus far,
however, there are no reports of full-term development of hamster
oocytes fertilized by ICSI. The authors report the birth of
hamster offspring following ICSI. Keys to success were 1)
performing ICSI in a dark room with a small incandescent lamp and
manipulating both oocytes and fertilized eggs under a microscope
with a red light source and 2) injecting sperm heads without
acrosomes. All oocytes injected with acrosome-intact sperm heads
died within 3 h after injection, while those oocytes injected
with acrosomeless sperm heads survived injection. Under
illumination with red light in a dark room, the majority of the
oocytes injected with acrosomeless sperm heads were fertilized
normally (77%), cleaved (91%), and developed into morulae (49%).
Of the 47 morulae transferred to five recipient females, nine
(19%) developed to live offspring. Y. Yamauchi et al: Biology of
Reproduction 2002 67:534
Related Background Brief:
FERTILIZATION OF EGGS OF ZEBRAFISH, DANIO RERIO, BY
INTRACYTOPLASMIC SPERM INJECTION. To evaluate the potential for
fertilization by sperm injection into fish eggs, sperm from
zebrafish, Danio rerio, were microinjected directly into egg
cytoplasm of two different zebrafish lines. To evaluate
physiological changes of gametes on the possible performance of
intracytoplasmic sperm injection (ICSI), four different
combinations of injection conditions were conducted using
activated or nonactivated gametes. From a total of 188 zebrafish
eggs injected with sperm in all treatments, 31 (16%) developed to
blastula, 28 (15%) developed to gastrula, 10 (5%) developed
abnormally to larval stages, and another 3 (2%) developed
normally and hatched. The highest fertilization rate (blastodisc
formation) was achieved by injection of activated spermatozoa
into nonactivated eggs (35%). Injections were most effective when
performed within the first hour after egg collection. Flow
cytometric analysis of the DNA content of the developing ICSI
embryos revealed diploidy, and the use of a dominant pigment
marker confirmed paternal inheritance. The authors suggest their
study indicates that injection of a single sperm cell into the
cytoplasm of zebrafish eggs allows fertilization and subsequent
development of normal larvae to hatching and beyond. G.A. Poleo
et al: Biology of Reproduction 2001 65:961
SERIAL PRONUCLEAR TRANSFER INCREASES THE DEVELOPMENTAL POTENTIAL
OF IN VITRO-MATURED OOCYTES IN MOUSE CLONING. In vitro-matured
germinal vesicle oocytes are an interesting source of cytoplast
recipients in both animal and human nuclear transfer (NT)
experiments. The authors investigated two technical aspects that
might improve the developmental potential of nuclear transfer
mouse embryos constructed from in vitro-matured germinal vesicle
oocytes. In a first step, the effect of two maturation media on
the embryonic development of NT embryos originating from in
vitro-matured oocytes was compared. Supplementation of the oocyte
maturation medium with serum and gonadotrophins improved the
developmental rate of NT embryos constructed from in vitro-
matured oocytes, but it was still inferior to that obtained with
in vivo-matured metaphase II (MII) oocytes. Second, the authors
investigated the effect of serial pronuclear transfer from NT
zygotes originating from both in vitro- and in vivo-matured
oocytes to in vivo-fertilized zygotic cytoplasts. Blastocyst
quality was evaluated by counting nuclei from trophectoderm and
inner cell mass cells using a differential staining. Sequential
pronuclear transfer significantly improved the blastocyst
formation rate of NT embryos originating from in vitro-matured
oocytes up to the rate obtained with in vivo-matured MII oocytes.
The authors conclude that the developmental potential of NT
embryos constructed from in vitro-matured oocytes can be
optimized by serial pronuclear transfer to in vivo-produced
zygotic cytoplasts. B. Heindryckx et al: Biology of Reproduction
2002 67:1790
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3. DIFFERENTIATION AND NUCLEAR TRANSFER
DIFFERENTIATION OF EMBRYONIC STEM CELL LINES GENERATED FROM ADULT
SOMATIC CELLS BY NUCLEAR TRANSFER
T. Wakayama et al (Rockefeller University, US) discuss
differentiation and nuclear transfer, the authors making the
following points:
1) Stem cells are able to differentiate into multiple cell types,
representatives of which might be harnessed for tissue repair in
degenerative disorders such as diabetes and Parkinson's disease
(1). One obstacle to therapeutic applications is obtaining stem
cells for a given patient. A solution would be to derive stem
cells from embryos generated by cloning from the nuclei of the
individual's somatic cells. The authors have previously cloned
mice by microinjection using a variety of cell types as nucleus
donors, including embryonic stem (ES) cells (2-4). The authors
sought to perform the converse experiment by deriving ES cell
lines in vitro from the inner cell mass (ICM) of blastocysts
clonally produced by nuclear transfer.
2) To this end, nuclei from adult-derived somatic donor cells of
five strains, including inbred (e.g., 129/Sv and C57BL/6nu/nu,
nude) and F1 hybrid (e.g., C57BL/6 ˛ DBA/2) representatives were
transferred by microinjection to produce cloned blastocysts. When
plated on fibroblast feeder layers in culture medium, cloned
blastocysts from all five strains tested yielded at least one
nuclear transfer ES (ntES) cell line. Cultures were established
from XX embryos derived via cumulus cell nuclear transfer (14.2%
of blastocysts) and both XX and XY embryos derived from tail-tip
cells (6.5%). In total, 35 successfully cryopreserved stable ntES
cell lines were produced.
3) In summary: Embryonic stem (ES) cells are fully pluripotent in
that they can differentiate into all cell types, including
gametes. The authors have derived 35 ES cell lines via nuclear
transfer (ntES cell lines) from adult mouse somatic cells of
inbred, hybrid, and mutant strains. ntES cells contributed to an
extensive variety of cell types, including dopaminergic and
serotonergic neurons in vitro and germ cells in vivo. Cloning by
transfer of ntES cell nuclei could result in normal development
of fertile adults. The authors suggest these studies demonstrate
the full pluripotency of ntES cells.(5)
References (abridged):
1. R. McKay, Nature 406, 361 (2000).
2. T. Wakayama, A. C. F. Perry, M. Zuccotti, K. R. Johnson, R.
Yanagimachi, Nature 394, 369 (1998).
3. T. Wakayama and R. Yanagimachi, Nature Genet. 22, 127 (1999).
4. T. Wakayama, I. Rodriguez, A. C. F. Perry, R. Yanagimachi, P.
Mombaerts, Proc. Natl. Acad. Sci. U.S.A. 96, 14984 (1999).
5. M. P. Matise, W. Auerbach, A. L. Joyner, Gene Targeting: A
Practical Approach, A. L. Joyner, Ed. (Oxford Univ. Press, New
York, 1993), pp. 129-131.
Science 2001 292:740
Related Background Brief:
FULL-TERM DEVELOPMENT OF MICE FROM ENUCLEATED OOCYTES INJECTED
WITH CUMULUS CELL NUCLEI. Until recently, fertilization was the
only way to produce viable mammalian offspring, a process
implicitly involving male and female gametes. However, techniques
involving fusion of embryonic or fetal somatic cells with
enucleated oocytes have become steadily more successful in
generating cloned young. Dolly the sheep was produced by
electrofusion of sheep mammary-derived cells with enucleated
sheep oocytes. The authors report an investigation of the factors
governing embryonic development, the study involving the
introduction of nuclei from somatic cells (Sertoli, neuronal and
cumulus cells) taken from adult mice into enucleated mouse
oocytes. The authors found that some enucleated oocytes receiving
Sertoli or neuronal nuclei developed in vitro and implanted
following transfer, but none developed beyond 8.5 days post
coitum; however, a high percentage of enucleated oocytes
receiving cumulus nuclei developed in vitro. Once transferred,
many of these embryos implanted and, although most were
subsequently resorbed, a significant proportion (2 to 2.8%)
developed to term. The authors suggest these experiments
demonstrate that for mammals, nuclei from terminally
differentiated adult somatic cells of known phenotype introduced
into enucleated oocytes are capable of supporting full
development. T. Wakayama et al: Nature. 1998 394:303.
Related Background Brief:
MECHANISMS AND CONTROL OF EMBRYONIC GENOME ACTIVATION IN
MAMMALIAN EMBRYOS. Activation of transcription within the
embryonic genome (EGA) after fertilization is a complex process
requiring a carefully coordinated series of nuclear and
cytoplasmic events, which collectively ensure that the two
parental genomes can be faithfully reprogrammed and restructured
before transcription occurs. Available data indicate that
inappropriate transcription of some genes during the period of
nuclear reprogramming can have long-term detrimental effects on
the embryo. Therefore, precise control over the time of EGA is
essential for normal embryogenesis. In most mammals, genome
activation occurs in a stepwise manner. In the mouse, for
example, some transcription occurs during the second half of the
one-cell stage, and then a much greater phase of genome
activation occurs in two waves during the two-cell stage, with
the second wave producing the largest onset of de novo gene
expression. Changes in nuclear structure, chromatin structure,
and cytoplasmic macromolecular content appear to regulate these
periods of transcriptional activation. A model is presented in
which a combination of cell cycle-dependent events and both
translational and posttranslational regulatory mechanisms within
the cytoplasm play key roles in mediating and regulating EGA.
K.E. Latham: Int Rev Cytol 1999 193:71
Related Background Brief:
COMPLETION OF MOUSE EMBRYOGENESIS REQUIRES BOTH THE MATERNAL AND
PATERNAL GENOMES. Transplantation of pronuclei between one-cell-
stage embryos was used to construct diploid mouse embryos with
two female pronuclei (biparental gynogenones) or two male
pronuclei (biparental androgenones). The ability of these embryos
to develop to term was compared with control nuclear-transplant
embryos in which the male or the female pronucleus was replaced
with an isoparental pronucleus from another embryo. The results
show that diploid biparental gynogenetic and androgenetic embryos
do not complete normal embryogenesis, whereas control nuclear
transplant embryos do. The authors conclude that the maternal and
paternal contributions to the embryonic genome in mammals are not
equivalent and that a diploid genome derived from only one of the
two parental sexes is incapable of supporting complete
embryogenesis. J. McGrath and D. Solter: Cell 1984 37:179.
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4. EPIGENETIC ASPECTS
ON DNA METHYLATION AND EPIGENETICS
In general, the term "epigenetics" refers to influences on gene
expression other than those produced by direct changes in the
nucleotides of the genome. In recent years, it has become
increasingly apparent that in living cells complex epigenetic
processes that control gene expression are as important as the
genome code itself in determining cellular biochemistry and
physiology, cell differentiation, and the development of tissues,
organs, and the organism.
The term "DNA methylation" refers to the occurrence in vertebrate
DNA of varying amounts of 5-methylcytosine, which arises from
methylation of certain cytosine bases. The methylation status of
DNA correlates with its functional activity: in general, inactive
genes are more heavily methylated.
W. Reik et al (Babraham Institute Cambridge, UK) discuss DNA
methylation and epigenetics, the authors making the following
points:
1) DNA methylation is one of the best-studied epigenetic
modifications of DNA in all unicellular and multicellular
organisms. In mammals and other vertebrates, methylation occurs
predominantly at the symmetrical cytosine-guanine dinucleotide
(CpG). Symmetrical methylation and the recent discovery of a DNA
methyltransferase that prefers a hemi-methylated substrate, have
suggested a mechanism by which specific patterns of methylation
in the genome could be maintained. Thus, patterns imposed on the
genome at defined developmental time points in precursor cells
could be maintained by the enzyme and would lead to predetermined
programs of gene expression during development in descendants of
the precursor cells. This provided a means to explain how
patterns of differentiation could be maintained by populations of
cells.
2) In addition, specific demethylation events in differentiated
tissues could then lead to further changes in gene expression as
needed. Neat and convincing as this model is, it is still largely
unsubstantiated. While effects of methylation on expression of
specific genes, particularly imprinted genes and some
retrotransposons, have been demonstrated in vivo, it is still
unclear whether or not methylation is involved in the control of
gene expression during normal development. Although enzymes have
been identified that can methylate DNA de novo, it is unknown how
specific patterns of methylation are established in the genome.
Science 2001 293:1089
Related Background:
EPIGENETIC REPROGRAMMING AND DNA METHYLATION
H.R. Fairburn et al (Roslin Institute, UK) discuss epigenetic
reprogramming, the authors making the following points:
1) Despite the high interest in the feasibility of cloning
animals from adult cells, cloning remains an extremely
inefficient process [1]. Recent studies have found evidence that
somatic donor genomes can be incompletely reprogrammed by oocyte
cytoplasm after nuclear transfer, providing a potential
explanation for the low success rate seen in nuclear cloning. Of
the potential kinds of epigenetic modifications likely to be
involved in reprogramming, DNA methylation is by far the best
studied. Patterns of DNA methylation in preimplantation murine
embryos are known to be dynamic [2], and are decidedly different
from those seen in somatic nuclei. Three groups have now
investigated DNA methylation patterns in preimplantation cloned
bovine embryos, all identifying aberrant DNA methylation patterns
compared to those produced by in vitro fertilization (IVF). These
studies provide a potential clue to the molecular reasons behind
the low efficiency of cloning in mammalian systems.
2) Shortly after fertilization, and before the first cell
division, the murine paternal genome is abruptly and rapidly
demethylated in what is believed to be an active process [3,4],
the molecular nature of which is not clear. Despite being present
in the same egg cytoplasm as the demethylating paternal genome,
the maternal genome is resistant to this process and remains
methylated. Methylation of the maternal genome is gradually and
passively lost during subsequent cell divisions by the failure to
maintain methylation patterns after DNA replication [5]. Thus
both parental genomes are largely demethylated by the time the
embryo reaches the blastocyst stage. This is followed by de novo
methylation around the time of implantation.
3) The evolutionary conservation of DNA methylation reprogramming
in the early embryo of other species was not investigated until
recently. Studies in both fish (Danio rario) and frogs (Xenopus
leavis) failed to identify any evidence for active demethylation
immediately post-fertilization. The absence of global
demethylation in these two species is intriguing, although it is
possible that other forms of epigenetic reprogramming, such as
chromatin remodeling, may be more important in these animals. It
is worth noting, however, that cloning from frogs was achieved
decades before cloning from mammals, indicating that perhaps
amphibian somatic nuclei are easier to reprogram than are
mammalian nuclei, or perhaps that amphibian oocytes are better at
reprogramming than are mammalian oocytes.
4) In summary: DNA methylation patterns are dynamic in cleavage-
stage embryos of a number of mammalian species. A failure to
properly recapitulate preimplantation DNA methylation patterns in
embryos derived by nuclear transfer may contribute to the low
efficiency of nuclear transfer in producing live offspring.
References (abridged):
1. Solter D. (2000) Mammalian cloning: advances and limita tions.
Nat. Rev. Genet., 1:199-207.
2. Reik W., Dean W. and Walter J. (2001) Epigenetic reprogramming
in mammalian development. Science, 293:1089-1093.
3. Mayer W., Niveleau A., Walter J., Fundele R. and Haaf T.
(2000) Demethylation of the zygotic paternal genome. Nature,
403:501-502.
4. Oswald J., Engemann S., Lane N., Mayer W., Olek A., Fundele
R., Dean W., Reik W. and Walter J. (2000) Active demethylation of
the paternal genome in the mouse zygote. Curr. Biol., 10:475-478.
5. Rougier N., Bourc'his D., Molina Gomes D., Niveleau A.,
Plachot M., P…ldi A. and Viegas-P‚quignot E. (1998) Chromosome
methylation patterns during mammalian preimplantation
development. Genes Dev., 12:2108-2113.
Current Biology 2002 12:R68
Related Background:
EPIGENETIC REPROGRAMMING IN MAMMALIAN DEVELOPMENT
W. Reik et al (Babraham Institute Cambridge, UK) discuss
epigenetic reprogramming, the authors making the following
points:
1) DNA methylation is a major epigenetic modification of the
genome that regulates crucial aspects of its function. Genomic
methylation patterns in somatic differentiated cells are
generally stable and heritable. However, in mammals there are at
least two developmental periods -- in germ cells and in
preimplantation embryos -- in which methylation patterns are
reprogrammed genome wide, generating cells with a broad
developmental potential.
2) Epigenetic reprogramming in germ cells is critical for
imprinting; reprogramming in early embryos also affects
imprinting. Reprogramming is likely to have a crucial role in
establishing nuclear totipotency in normal development and in
cloned animals, and in the erasure of acquired epigenetic
information. A role of reprogramming in stem cell differentiation
is also envisaged.
3) DNA methylation is one of the best-studied epigenetic
modifications of DNA in all unicellular and multicellular
organisms. In mammals and other vertebrates, methylation occurs
predominantly at the symmetrical dinucleotide CpG. Symmetrical
methylation and the discovery of a DNA methyltransferase that
prefers a hemimethylated substrate, Dnmt1, suggested a mechanism
by which specific patterns of methylation in the genome could be
maintained. Patterns imposed on the genome at defined
developmental time points in precursor cells could be maintained
by Dnmt1, and would lead to predetermined programs of gene
expression during development in descendants of the precursor
cells. This provided a means to explain how patterns of
differentiation could be maintained by populations of cells. In
addition, specific demethylation events in differentiated tissues
could then lead to further changes in gene expression as needed.
4) Neat and convincing as this model is, it is still largely
unsubstantiated. While effects of methylation on expression of
specific genes, particularly imprinted ones and some
retrotransposons, have been demonstrated in vivo, it is still
unclear whether or not methylation is involved in the control of
gene expression during normal development. Although enzymes have
been identified that can methylate DNA de novo, it is unknown how
specific patterns of methylation are established in the genome.
Mechanisms for active demethylation have been suggested, but no
enzymes have been identified that carry out this function in vivo
(15-17). Genome-wide alterations in methylation -- brought about,
for example, by knockouts of the methylase genes -- result in
embryo lethality or developmental defects, but the basis for
abnormal development still remains to be discovered.
5) What is clear, however, is that in mammals there are
developmental periods of genome-wide reprogramming of methylation
patterns in vivo. Typically, a substantial part of the genome is
demethylated, and after some time remethylated, in a cell- or
tissue-specific pattern. The developmental dynamics of these
reprogramming events, as well as some of the enzymatic mechanisms
involved and the biological purposes, are beginning to be
understood.
Science 2001 293:1089
Related Background:
NUCLEAR CLONING AND EPIGENETIC REPROGRAMMING OF THE GENOME
W.M. Rideout et al (Whitehead Institute for Biomedical Research,
US) discuss epigenetic reprogramming and nuclear cloning, the
authors making the following points:
1) Epigenetic modification of the genome ensures proper gene
activation during development and involves (i) genomic
methylation changes, (ii) the assembly of histones and histone
variants into nucleosomes, and (iii) remodeling of other
chromatin-associated proteins such as linker histones, polycomb
group, nuclear scaffold proteins, and transcription factors (1).
2) The two parental genomes are formatted during gametogenesis to
respond to the oocyte environment and proceed through
development. The zygote biochemically remodels the paternal
genome shortly after fertilization and before embryonic genome
activation (EGA) occurs. To successfully recapitulate these
processes, the somatic nuclei transferred into an oocyte must be
quickly reprogrammed to express genes required for early
development.
3) The programming of the genome that occurs as primordial germ
cells (PGCs) differentiate into mature gametes establishes the
markedly different chromatin configurations of sperm and oocyte.
As demonstrated by normal preimplantation development of
uniparental embryos, both parental genomes share the ability to
independently direct cleavage (early development to the
blastocyst stage) despite profound differences in their
epigenetic organization (2,3). In spermatogenesis, chromatin is
sequentially remodeled, silenced, and ultimately compacted with
protamines (4), processes crucial for normal fertilization (5).
However, completion of these events is not strictly required for
development as normal pregnancies can result from
intracytoplasmic sperm injection with round spermatids or
secondary spermatocytes. In contrast, the genome of the oocyte is
organized in a structure more like that of a somatic cell, with
chromatin whose nucleosomes contain an oocyte-specific linker
histone. In comparison with the male pronucleus, the female
pronucleus is more transcriptionally repressive, contains
relatively deacetylated histone H4, and is deficient in
generalized transcription factors. This repressive chromatin
structure may protect the oocyte genome against the extensive
epigenetic modifications imposed on the paternal genome after
fertilization.
4) In summary: Cloning of mammals by nuclear transfer (NT)
results in gestational or neonatal failure with at most a few
percent of manipulated embryos resulting in live births. Many of
those that survive to term succumb to a variety of abnormalities
that are likely due to inappropriate epigenetic reprogramming.
Cloned embryos derived from donors, such as embryonic stem cells,
that may require little or no reprogramming of early
developmental genes develop substantially better beyond
implantation than NT clones derived from somatic cells. Although
recent experiments have demonstrated normal reprogramming of
telomere length and X chromosome inactivation, epigenetic
information established during gametogenesis, such as gametic
imprints, cannot be restored after nuclear transfer. Survival of
cloned animals to birth and beyond, despite substantial
transcriptional dysregulation, is consistent with mammalian
development being rather tolerant to epigenetic abnormalities,
with lethality resulting only beyond a threshold of faulty gene
reprogramming encompassing multiple loci.
References (abridged):
1. K. E. Latham, Int. Rev. Cytol. 193, 71 (1999).
2. M. H. Kaufman, S. C. Barton, M. A. Surani, Nature 265, 53
(1977).
3. J. McGrath and D. Solter, Cell 37, 179 (1984).
4. K. Steger, Anat. Embryol. 199, 471 (1999).
5. C. Cho, et al., Nature Genet. 28, 82 (2001).
Science 2001 293:1093
Related Background Brief:
EFFECT OF NUTRITION OF OOCYTE DONOR ON THE OUTCOMES OF SOMATIC
CELL NUCLEAR TRANSFER IN THE SHEEP. The purpose of this study was
to determine if the nutrition of the oocyte donor ewe influenced
the success of somatic cell cloning. Merino ewes were fed at
either a high- or a low-nutrition level for 3 5 mo before
superovulation treatments. Freshly ovulated oocytes were
enucleated and fused with serum-starved adult granulosa cells,
and resulting reconstructed embryos were cultured for 6 days in
modified synthetic oviduct fluid. Embryo cleavage and development
to blastocysts were recorded, and good-quality embryos were
transferred to synchronized recipient ewes either fresh or, on a
few occasions, after vitrification. Pregnancies were monitored by
ultrasonography from day 40 of pregnancy, and offspring were
delivered by either cesarean section or vaginal delivery. No
differences occurred in the numbers of follicles aspirated, of
oocytes recovered, or of oocytes utilizable for cloning between
the high and low groups. Neither were there treatment differences
in development to the blastocyst stage. However, transfer of
embryos from the high group led to significantly more pregnancies
and implanted fetuses. Also, more of the established pregnancies
from the high group were carried to term, although this
difference was not statistically significant. Lamb mortality was
high, with half the live-born perishing soon after birth and more
succumbing to various infections within days or weeks of birth,
but no clear association between the offspring fate and the
treatment group could be established. The authors suggest these
results indicate that more research into the effect of nutrition
on oocyte quality and its subsequent effect on cloning is
warranted. Biology of Reproduction 2003 68:45.
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5. ABNORMAL GENE EXPRESSION AND NUCLEAR TRANSFER
ABNORMAL GENE EXPRESSION IN CLONED MICE DERIVED FROM EMBRYONIC
STEM CELL AND CUMULUS CELL NUCLEI
D. Humpherys et al (Whitehead Institute for Biomedical Research,
US) discuss abnormalities in cloned animals, the authors making
the following points:
1) The majority of cloned mammals derived by nuclear transfer
(NT) die during gestation, display neonatal phenotypes resembling
large offspring syndrome (1,2), often with respiratory and
metabolic abnormalities, and have enlarged and dysfunctional
placentas (3-5). For a donor nucleus to support development in a
clone, it must be reprogrammed to a state compatible with
embryonic development. The transferred nucleus must properly
activate genes important for early embryonic development and also
adequately suppress differentiation-associated genes that had
been transcribed in the original donor cell. Because few clones
survive to birth, the question remains whether survivors are
normal or merely the least severely affected animals, making it
to adulthood despite harboring subtle abnormalities originating
from inadequate nuclear reprogramming.
2) Given the long generational time of most animal species
cloned, the long-term consequences of cloning on health have been
difficult to assess. Evidence that cloned animals retain
abnormalities capable of causing severe health consequences has
been obtained for mice cloned from Sertoli cells that, in
comparison to normally developing controls of the same sex and
background, had reduced lifespans and frequent pneumonia and
hepatic failure. Additionally, mice cloned from cumulus cell
donor nuclei were obese with increased body fat and size. Because
obesity was not passed on to the offspring of the clones it is
unlikely to reflect any genetic changes in the clones but instead
to reflect epigenetic abnormalities arising from inadequate
nuclear reprogramming. Examination of adult clones in other
species has been described only for younger animals and limited
to physical examinations and blood and urine chemistry.
3) Development of clones derived from embryonic stem (ES) cell
nuclei to the blastocyst stage is less efficient than that of
clones derived from somatic donor nuclei because the majority of
ES cells are in S phase, a stage of the cell cycle that is
incompatible with survival of clones. However, survival to birth
or adulthood of blastocysts derived from ES cell donor nuclei is
about 10-20 times more efficient than that of clones derived from
somatic donor nuclei. This striking increase in development rate
suggests that less reprogramming is needed for nuclei of
embryonically derived cells and that reprogramming is important
for post-implantation development. Despite this enhanced
developmental rate, it has been argued that epigenetic
instability described in ES cells during in vitro culturing makes
them a poor choice for NT donors. However, this argument is based
largely on the expression of imprinted genes known to be
particularly affected in ES cells. Nevertheless, common
phenotypes, including dramatically overgrown placentas, have been
described when using either ES cell or somatic cell donor nuclei
for NT.
4) In summary: To assess the extent of abnormal gene expression
in clones, the authors assessed global gene expression by
microarray analysis on RNA from the placentas and livers of
neonatal cloned mice derived by nuclear transfer (NT) from both
cultured embryonic stem cells and freshly isolated cumulus cells.
Direct comparison of gene expression profiles of more than 10,000
genes showed that for both donor cell types 4% of the expressed
genes in the NT placentas differed dramatically in expression
levels from those in controls and that the majority of abnormally
expressed genes were common to both types of clones. Importantly,
however, the expression of a smaller set of genes differed
between the embryonic stem cell- and cumulus cell-derived clones.
The livers of the cloned mice also showed abnormal gene
expression, although to a lesser extent, and with a different set
of affected genes, than seen in the placentas. The authors
suggest their results demonstrate frequent abnormal gene
expression in clones, in which most expression abnormalities
appear common to the NT procedure whereas others appear to
reflect the particular donor nucleus.
References (abridged):
1. Young, L. E. , Sinclair, K. D. & Wilmut, I. (1998) Rev.
Reprod. 3, 155-163.
2. Chavatte-Palmer, P. , Heyman, Y. & Renard, J. P. (2000)
Gynecol. Obstet. Fertil. 28, 633-642.
3. Wakayama, T. & Yanagimachi, R. (1999) Nat. Genet. 22, 127-128.
4. Hill, J. R. , Roussel, A. J. , Cibelli, J. B. , Edwards, J. F.
, Hooper, N. L. , Miller, M. W. , Thompson, J. A. , Looney, C. R.
, Westhusin, M. E. , Robl, J. M. & Stice, S. L. (1999)
Theriogenology 51, 1451-1465.
5. Hill, J. R. , Burghardt, R. C. , Jones, K. , Long, C. R. ,
Looney, C. R. , Shin, T. , Spencer, T. E. , Thompson, J. A. ,
Winger, Q. A. & Westhusin, M. E. (2000) Biol. Reprod. 63, 1787-
1794.
Proc. Nat. Acad. Sci. 2002 99:12889.
Related Background Brief:
LARGE OFFSPRING SYNDROME IN CATTLE AND SHEEP. Bovine and ovine
embryos exposed to a variety of unusual environments prior to the
blastocyst stage have resulted in the development of unusually
large offspring which can also exhibit a number of organ defects.
In these animals, the increased incidence of difficult
parturition and of fetal and neonatal losses has limited the
large-scale use of in vitro embryo production technologies
commonly used in humans and other species. Four different
situations have been identified that result in the syndrome: in
vitro embryo culture, asynchronous embryo transfer into an
advanced uterine environment, nuclear transfer and maternal
exposure to excessively high urea diets. However, programming of
the syndrome by all of these situations is unpredictable and not
all of the symptoms described have been observed universally.
Neither the environmental factors inducing the large offspring
syndrome nor the mechanisms of perturbation occurring in the
early embryo and manifesting themselves in the fetus have been
identified. L.E. Young et al: Rev Reprod 1998 3:155.
Related Background Brief:
CLONING AND ASSOCIATED PHYSIOPATHOLOGY OF GESTATION. Normal
fertile offspring can be produced with nuclear transfer (NT) of
somatic cells. This technique is associated with important
gestational losses in early pregnancy during the first two to
three months and in the late fetal and perinatal periods in cows.
In cows and sheep, recent studies suggest that early losses may
be associated with placental vascularization deficiencies. In
late gestation and at term, a syndrome, commonly called the large
offspring syndrome (LOS), causes important perinatal deaths. This
syndrome is associated with increased fetal and placental growth,
disturbed placental function and fetal abnormalities. Moreover,
prolonged gestations are common. Live offspring occasionally
exhibit a respiratory distress syndrome and several types of
abnormalities that may hinder their survival. The authors review
current knowledge of these pathologies and their incidence in
somatic and embryonic cloning. P. Chavatte-Palmer et al: Gynecol
Obstet Fertil 2000 28:633.
Related Background Brief:
CLINICAL AND PATHOLOGIC FEATURES OF CLONED TRANSGENIC CALVES AND
FETUSES (13 CASE STUDIES). The neonatal abnormalities, treatments
and outcomes in a group of 13 cloned transgenic calves and
fetuses that progressed into the third trimester of pregnancy are
described by the authors. From these 13 fetuses, 8 calves were
born live, 4 stillborn fetuses were recovered from 3 cows that
died 7 d to 2 mo before term, and 1 aborted fetus was recovered
at 8 mo gestation. All fetuses and calves were derived from the
same male fetal Holstein fibroblast cell line transfected with a
beta-galactosidase marker gene. Six calves were delivered by
Cesarian section and two by vaginal delivery between 278 and 288
d of gestation. Birth weights ranged from 44 to 58.6 kg. Five of
the 8 live born calves were judged to be normal within 4 h of
birth based on clinical signs and blood gas measurements. One of
these 5 calves died at 6 wk of age from a suspected dilated
cardiomyopathy. Three of the 8 calves were diagnosed with
neonatal respiratory distress immediately following birth, one of
which died (at 4 d of age) as a result of pulmonary surfactant
deficiency coupled with pulmonary hypertension and elevated
systemic venous pressures. Similar findings of chronic pulmonary
hypertension were also observed in 2 of 5 fetuses. Placental
edema was present in both calves that later died and in the 2
fetuses with cardiopulmonary abnormalities. Hydrallantois
occurred with or without placental edema in 6 cows, and only 1
calf from this group survived. The 6 cows without hydrallantois
or placental edema produced 5 live calves and 1 aborted fetus.
The cardiopulmonary abnormalities observed in the calves and
fetuses occurred in utero in conjunction with placental
abnormalities, and it is likely that the cloning technique and/or
in vitro embryo culture conditions contributed to these
abnormalities, although the mechanism remains to be determined.
J.R. Hill et al: Theriogenology 1999 51:1451.
Related Background:
CLONED CATTLE CAN BE HEALTHY AND NORMAL
R.P. Lanza et al (Advanced Cell Technology, US) discuss cloned
cattle, the authors making the following points:
1) The possibility of cloning humans has raised questions as to
whether nuclear transfer can be used to reproducibly generate
healthy adult animals. Reports in the popular and scientific
press on genetic, immunological, and other developmental problems
raise the question of whether there are "any normal clones in
existence".
2) The authors evaluated a series of 24 surviving sexually mature
cattle successfully cloned from nonquiescent somatic cells as
described in reports in 1998 and 2000. Results of physical
examination were normal for all animals, including objective
(temperature, pulse, respiratory rate) and subjective (general
appearance, eyes, lymph nodes, and cardiac and pulmonary
auscultation) findings. Results of abdominal palpation of
reproductive and gastrointestinal organs and kidneys were normal.
Social interaction and behavior of the cloned animals are normal.
They have normal conditioned responses such as reacting to farm
equipment used for feeding. They have developed a social
dominance hierarchy and the full spectrum of behavioral traits.
The cloned animals exhibited puberty at the expected age and body
weight. Conception rates after artificial insemination have been
excellent, and two of the cloned animals have give birth to
calves that appeared normal in every respect.
3) Consistent with published reports, several of the cloned
calves experienced pulmonary hypertension and respiratory
distress at birth and fever after vaccination at 4 months.
However, the authors report they did not observe genetic defects,
immune deficiencies, gross obesity, or other drastic
abnormalities cited by other researchers. The authors suggest it
remains to be determined whether such abnormalities occur in
other species and/or are due to differences in nuclear transfer
techniques.
Science 2001 294:1893
ScienceWeek http://www.scienceweek.com
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6. AN APPLICATION IN NEUROBIOLOGY
ASSESSMENT OF THE DEVELOPMENTAL TOTIPOTENCY OF NEURAL CELLS IN
THE CEREBRAL CORTEX OF MOUSE EMBRYO BY NUCLEAR TRANSFER
Y. Yamazaki et al (University of Hawaii, US) discuss
developmental totipotency of neural cells, the authors making the
following points:
1) Animal cloning has been achieved for many years by
transferring the nucleus of a somatic embryonic or fetal cell
into an enucleated oocyte (1). Successful cloning by using adult
somatic cells first was reported in the sheep (2), then in mice
(3), cattle (4-5), goats, and pigs. The cloning technique is a
very powerful tool to analyze the developmental potentials and
genomic status of various somatic cell nuclei.
2) Site-specific DNA rearrangement in developing lymphocytes of
the immune system is largely responsible for generating the
highly diverse array of immunoglobulins and T cell receptors.
Whereas neurons do not proliferate, there are superficial
similarities between the immune system and the central nervous
system. Extreme complexity, the capacity for memory, and
extensive apoptosis during development are examples. These
similarities have led to the hypothesis that the central nervous
system and the immune system use similar somatic DNA
rearrangement strategies during their development. The
rearrangement activating gene, RAG-1 in the immune system also
was detected in the central nervous system. Furthermore, knockout
mice lacking DNA-repair enzymes DNA ligase IV and XRCC4 failed to
repair DNA double-stranded breaks, causing defects in the immune
system, and had gross cell death along neural differentiation in
the embryonic cortex. It has been suggested recently that DNA
rearrangement may play a role in neural cell development.
3) In summary: When neural cells were collected from the entire
cerebral cortex of developing mouse fetuses (15.5-17.5 days post-
coitum) and their nuclei were transferred into enucleated
oocytes, 5.5% of the reconstructed oocytes developed into normal
offspring. This success rate was the highest among all previous
mouse cloning experiments that used somatic cells. Forty-four
percent of live embryos at 10.5 days post-coitum were
morphologically normal when premature and early-postmitotic
neural cells from the ventricular side of the cortex were used.
In contrast, the majority (95%) of embryos were morphologically
abnormal (including structural abnormalities in the neural tube)
when postmitotic-differentiated neurons from the pial side of the
cortex were used for cloning. Whereas 4.3% of embryos cloned with
ventricular-side cells developed into healthy offspring, only
0.5% of those cloned with differentiated neurons in the pial side
did so. The authors suggest these facts seem to indicate that the
nuclei of neural cells in advanced stages of differentiation had
lost their developmental totipotency. The underlying mechanism
for this developmental limitation could be somatic DNA
rearrangements in differentiating neural cells.
References (abridged):
1. DiBerardino, M. A. (1997) Genomic Potential of Differentiated
Cells (Columbia Univ. Press, New York), pp. 180-213.
2. Campbell, K. H. S. , McWhir, J. , Ritchie, W. A. & Wilmut, I.
(1996) Nature (London) 380, 64-66.
3. Wakayama, T. , Perry, A. C. F. , Zuccotti, M. , Johnson, K. R.
& Yanagimachi, R. (1998) Nature (London) 394, 369-374.
4. Kato, Y. , Tani, T. , Sotomaru, Y. , Kurokawa, K. , Kato, J. ,
Doguchi, H. , Yasue, H. & Tsunoda, Y. (1998) Science 282, 2095-
2098.
5. Cibelli, J. B. , Stice, S. L. , Golueke, P. , Kane, J. J. ,
Blackwell, C. , Ponce de L‚on, F. A. & Robl, J. M. (1998) Science
280, 1256-1258.
Proc. Nat. Acad. Sci. 2001 98:14022
ScienceWeek http://www.scienceweek.com
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