327
Using Xenopus to Understand Pluripotency
23.2.2. NUCLEAR REPROGRAMMING USING XENOPUS
LAEVIS OOCYTES, EGGS, AND EGG EXTRACTS
In amphibians, eggs develop from germ cells called oocytes,
which harbor a nucleus called a germinal vesicle (GV). The
GV is densely packed with components essential for development. Oocytes are arrested at prophase of meiosis I, and
hormones such as progesterone induce their progression past
meiosis I and into metaphase of meiosis II, at which they
are stalled again. These cells are then ready for fertilization—in mammals, this stage is termed the “MII oocyte”; in
Xenopus, it is called the egg. All early SCNT experiments
were performed at this second stage of arrest, as the closest means of mimicking natural fertilization, and hence this
method is also termed “egg-NT.” In an egg-NT experiment
(Figure 23.1), microinjection of the somatic nucleus causes
activation of the egg, which, like in fertilized embryos, is
then followed by several rapid, transcriptionally quiescent
cell divisions that are accompanied with DNA replication until the zygotic genome is ultimately activated. In an
optimal NT experiment, cell-type specifc gene expression
patterns are established and the cell types are correctly
formed, allowing the embryo to further develop successfully. Xenopus egg-NT as an experimental system provides
several advantages, as it represents a unique model to study
successful reprogramming but also to investigate why reprogramming so often fails due to unsuccessful erasure of the
previous somatic cell identity. For example, it can help to
identify the barriers present in somatic cells that confer
cellular memory of its differentiated state and that prevent
reprogramming. It can help to reveal the propagation mechanisms of cellular memory and the associated chromatin
factors from the differentiated donor nucleus to all cells of
the NT embryo throughout several cell divisions, independently of transcription. Furthermore, it offers the advantage
that reprogramming effciencies can be directly measured
by monitoring the establishment of cell-type-specif c transcriptional networks and the formation of functional cell
types in the developing NT embryo. However, the rapid early
cleavage cycles contrast with the typical slow division rate
of the somatic nucleus, and this transition can take a toll on
the nucleus, exposing the genome to chromosomal damage.
Thus, despite its many advantages, this can make it more
challenging to use this method for studying the alterations in
transcription throughout the reprogramming procedure and
the chromatin modifcations driving resistance.
The oocyte GV, when arrested in prophase I, does not
undergo cell division or DNA replication and can be a
highly transcriptionally active region. It acts as a reserve
for the generation and accumulation of large numbers of
maternal transcripts that will drive early development of the
embryo after fertilization. Due to this quality, it also serves
as a prime opportunity for the study of nuclear reprogramming. The transplantation of donor nuclei directly into the
GV of an amphibian oocyte—a method also called “oocyteNT” (Figure 23.1)—induces rapid de-differentiation of
the somatic nuclei along with reactivation and increased
expression of previously repressed genes and pluripotency
genes (Halley-Stott et al., 2010; Pasque et al., 2011). Several
hundred donor nuclei can be transplanted at a time into the
GV of a single oocyte. The high number of transcripts resulting from this technique facilitates both the analysis of gene
expression trends during reprogramming and the resistance
that the donor nuclei experience by means of epigenetic
modifcations. Although this technique generates oocytes
arrested in meiotic prophase I and cannot produce embryos
that support normal growth through specifcation of cell lineages, it is a useful system for studying reprogramming in
a purely transcriptional context. Xenopus oocytes also have
the handy ability to specifcally transcribe nuclei from different species, due to which it is possible to transfer mammalian or cultured donor cells and study transcription distinctly
induced by reprogramming without the noise of endogenous
maternal transcripts (De Robertis and Gurdon, 1977).
In addition to the use of oocytes as NT recipients to study
nuclear reprogramming in vitro, the use of Xenopus egg
extracts and oocyte extracts has also proved surprisingly
benefcial (Hansis et al., 2004). Initially used to study the
fertilization process more closely (Lokha and Masui, 1983),
the use of this cell-free system has since been adapted as
an excellent tool to mimic the reprogramming environment. Cultured cells display striking reprogramming activity when introduced to metaphasic Xenopus egg extracts,
including alterations in their replicative nature, epigenetic
signature, and chromatin organization (Figure 23.1; Ganier
et al., 2011). This occurs initially in the absence of transcription, which is mostly inhibited in eggs and embryos until
zygotic genome activation at mid-blastula transition. It has
been shown that transient exposure of donor nuclei to egg
extracts followed by typical NT to an enucleated egg primes
them for signifcantly more effcient reprogramming (Ganier
et al., 2011). Similar experiments have been carried out on
oocyte extracts, and this cell-free system has also been
developed for use in mammals (Miyamoto et al., 2009). The
ability to easily manipulate the composition of egg extracts
through biochemical methods and the ease with which the
resulting effects can be analyzed and transferred to an in
vivo setting make the use of egg extracts for reprogramming
invaluable.
23.3. NUCLEAR REPROGRAMMING TAKES A
LEAP FROM FROG TO MAMMALS AND
CLINICAL APPLICATION
23.3.1. MAMMALIAN NUCLEAR TRANSFER AND
GENERATION OF PLURIPOTENT STEM CELL LINES
It took a little over three decades for the early results in
Xenopus to be reproduced in mammals, but f nally, the
generation of the frst clones in a number of different
mammals succeeded: cloning of mouse embryos ( Tsunoda
et al., 1987 ; Wakayama et al., 1998 ), bovine embryos
( Prather et al., 1987 ), sheep ( Campbell et al., 1996 ;
Wilmut et al., 1997 ), and pigs ( Polejaeva et al., 2000 ). Not
Using Xenopus to Understand Pluripotency
23.2.2. NUCLEAR REPROGRAMMING USING XENOPUS
LAEVIS OOCYTES, EGGS, AND EGG EXTRACTS
In amphibians, eggs develop from germ cells called oocytes,
which harbor a nucleus called a germinal vesicle (GV). The
GV is densely packed with components essential for development. Oocytes are arrested at prophase of meiosis I, and
hormones such as progesterone induce their progression past
meiosis I and into metaphase of meiosis II, at which they
are stalled again. These cells are then ready for fertilization—in mammals, this stage is termed the “MII oocyte”; in
Xenopus, it is called the egg. All early SCNT experiments
were performed at this second stage of arrest, as the closest means of mimicking natural fertilization, and hence this
method is also termed “egg-NT.” In an egg-NT experiment
(Figure 23.1), microinjection of the somatic nucleus causes
activation of the egg, which, like in fertilized embryos, is
then followed by several rapid, transcriptionally quiescent
cell divisions that are accompanied with DNA replication until the zygotic genome is ultimately activated. In an
optimal NT experiment, cell-type specifc gene expression
patterns are established and the cell types are correctly
formed, allowing the embryo to further develop successfully. Xenopus egg-NT as an experimental system provides
several advantages, as it represents a unique model to study
successful reprogramming but also to investigate why reprogramming so often fails due to unsuccessful erasure of the
previous somatic cell identity. For example, it can help to
identify the barriers present in somatic cells that confer
cellular memory of its differentiated state and that prevent
reprogramming. It can help to reveal the propagation mechanisms of cellular memory and the associated chromatin
factors from the differentiated donor nucleus to all cells of
the NT embryo throughout several cell divisions, independently of transcription. Furthermore, it offers the advantage
that reprogramming effciencies can be directly measured
by monitoring the establishment of cell-type-specif c transcriptional networks and the formation of functional cell
types in the developing NT embryo. However, the rapid early
cleavage cycles contrast with the typical slow division rate
of the somatic nucleus, and this transition can take a toll on
the nucleus, exposing the genome to chromosomal damage.
Thus, despite its many advantages, this can make it more
challenging to use this method for studying the alterations in
transcription throughout the reprogramming procedure and
the chromatin modifcations driving resistance.
The oocyte GV, when arrested in prophase I, does not
undergo cell division or DNA replication and can be a
highly transcriptionally active region. It acts as a reserve
for the generation and accumulation of large numbers of
maternal transcripts that will drive early development of the
embryo after fertilization. Due to this quality, it also serves
as a prime opportunity for the study of nuclear reprogramming. The transplantation of donor nuclei directly into the
GV of an amphibian oocyte—a method also called “oocyteNT” (Figure 23.1)—induces rapid de-differentiation of
the somatic nuclei along with reactivation and increased
expression of previously repressed genes and pluripotency
genes (Halley-Stott et al., 2010; Pasque et al., 2011). Several
hundred donor nuclei can be transplanted at a time into the
GV of a single oocyte. The high number of transcripts resulting from this technique facilitates both the analysis of gene
expression trends during reprogramming and the resistance
that the donor nuclei experience by means of epigenetic
modifcations. Although this technique generates oocytes
arrested in meiotic prophase I and cannot produce embryos
that support normal growth through specifcation of cell lineages, it is a useful system for studying reprogramming in
a purely transcriptional context. Xenopus oocytes also have
the handy ability to specifcally transcribe nuclei from different species, due to which it is possible to transfer mammalian or cultured donor cells and study transcription distinctly
induced by reprogramming without the noise of endogenous
maternal transcripts (De Robertis and Gurdon, 1977).
In addition to the use of oocytes as NT recipients to study
nuclear reprogramming in vitro, the use of Xenopus egg
extracts and oocyte extracts has also proved surprisingly
benefcial (Hansis et al., 2004). Initially used to study the
fertilization process more closely (Lokha and Masui, 1983),
the use of this cell-free system has since been adapted as
an excellent tool to mimic the reprogramming environment. Cultured cells display striking reprogramming activity when introduced to metaphasic Xenopus egg extracts,
including alterations in their replicative nature, epigenetic
signature, and chromatin organization (Figure 23.1; Ganier
et al., 2011). This occurs initially in the absence of transcription, which is mostly inhibited in eggs and embryos until
zygotic genome activation at mid-blastula transition. It has
been shown that transient exposure of donor nuclei to egg
extracts followed by typical NT to an enucleated egg primes
them for signifcantly more effcient reprogramming (Ganier
et al., 2011). Similar experiments have been carried out on
oocyte extracts, and this cell-free system has also been
developed for use in mammals (Miyamoto et al., 2009). The
ability to easily manipulate the composition of egg extracts
through biochemical methods and the ease with which the
resulting effects can be analyzed and transferred to an in
vivo setting make the use of egg extracts for reprogramming
invaluable.
23.3. NUCLEAR REPROGRAMMING TAKES A
LEAP FROM FROG TO MAMMALS AND
CLINICAL APPLICATION
23.3.1. MAMMALIAN NUCLEAR TRANSFER AND
GENERATION OF PLURIPOTENT STEM CELL LINES
It took a little over three decades for the early results in
Xenopus to be reproduced in mammals, but f nally, the
generation of the frst clones in a number of different
mammals succeeded: cloning of mouse embryos ( Tsunoda
et al., 1987 ; Wakayama et al., 1998 ), bovine embryos
( Prather et al., 1987 ), sheep ( Campbell et al., 1996 ;
Wilmut et al., 1997 ), and pigs ( Polejaeva et al., 2000 ). Not