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First published online June 14, 2005
Stem Cells Vol. 23 No. 8 September 2005, pp. 1089 -1104
doi:10.1634/stemcells.2004-0352; www.StemCells.com
© 2005 AlphaMed Press

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Variable Reprogramming of the Pluripotent Stem Cell Marker Oct4 in Mouse Clones: Distinct Developmental Potentials in Different Culture Environments

Michele Boiania, Luca Gentileb, Vivian V. Gamblesa, Fatima Cavaleria, Carlo A. Redib, Hans R. Schölera

a Germline Development Group, Center for Animal Transgenesis and Germ Cell Research, New Bolton Center, University of Pennsylvania, Kennett Square, Pennsylvania, USA;
b Laboratory of Developmental Biology, Department of Animal Biology, University of Pavia, Pavia, Italy

Key Words. Embryo • Embryonic stem cell • Nuclear transfer • Oct4 • Reprogramming

Correspondence: Michele Boiani, Ph.D., Max Planck Institute for Molecular Biomedicine, Mendelstraße 7, D-48149 Münster, Germany. Telephone: 0049-0251-980-2864; Fax: 0049-0251-980-2992; e-mail: mboiani{at}mpi-muenster.mpg.de


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 
A prevailing view of cloning by somatic-cell nuclear transfer is that reprogramming of gene expression occurs during the first few hours after injection of the nucleus into an oocyte, that the process is stochastic, and that the type of reprogramming needed for cloning success is foreign and unlikely to be readily achieved in the ooplasm. Here, we present evidence that the release of reprogramming capacity is contingent on the culture environment of the clone while the contribution of aneuploidy to altered gene expression is marginal. In particular, the rate of blastocyst formation in clones and the regional distribution of mRNA for the pluripotent stem cell marker Oct4 in clonal blastocysts was highly dependent on the culture environment after cumulus cell nuclear transfer, unlike that in genetically equivalent zygotes. Epigenetic modifications of genetically identical somatic nuclei continue after the first cell division of the clones and are amenable to a degree of experimental control, and their development to the blastocyst stage and appropriate expression of Oct4 predict further outcome, such as derivation of embryonic stem (ES) cells, but not fetal development. This observation indicates that development to the blastocyst stage is not equivalent to full reprogramming and lends support to the novel concept that ES cells are not the equivalent of the inner cell mass, hence the discrepancy between ES cell derivability and fetal development of clones.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 
Nuclear transfer techniques are used to study fundamental biologic issues, such as reversibility of differentiated cellular states and interactive processes between the nucleus and the cytoplasm. These aspects are collectively dubbed reprogramming—a fashionable term in cloning research to indicate that the cellular state of a nucleus changes from that of a differentiated somatic cell into a pluripotent embryonic cell after the nucleus is transplanted into an oocyte. Cellular states are largely determined by the coordinated expression of thousands of genes. Epigenetic mechanisms (including DNA methylation and demethylation, post-translational histone modifications, and swapping of histone variants as well as other chromatin-associated proteins) are thought to underlie the somatic-to-embryonic transition of gene expression in the transplanted nucleus and the maternal-to-embryonic transition of gene expression after fertilization [1, 2]. Although these epigenetic changes always take place to some extent in clonal embryos, they are not always consequential in terms of gene expression [3]. Therefore, reprogramming at the DNA template level demands phenotypic assessment through studies of gene expression and cellular function in clones.

The initial experiments on cloning in amphibia by Briggs, King, and Gurdon demonstrated that the potential of the nucleus to direct normal embryonic development decreases progressively with the differentiation stage of the donor cell [4]. This phenomenon is notably evident in mouse cloning experiments using donor nuclei from somatic cells and pluripotent embryonic stem (ES) cells [5, 6]. After birth, Sertoli cell–cloned mice tend to die prematurely of hepatic failure or tumors [7] and cumulus cell–cloned mice tend to grow obese [8], whereas ES cell–cloned mice survive in larger numbers—provided they originate from F1 nucleus donors [5]. In these cases, the epigenetic make up of the donor nucleus exerts some priming and lasting effects on the clonal phenotype. Similarly, the proportion of fibroblast-derived and cumulus cell–derived bovine clones having zygote-like epigenotypes (as defined by patterns of methylation and acetylation of histone H3 lysine 9) correlates closely with the proportion developing to the blastocyst stage [3].

Although a nucleus-driven scenario for clones is logically appealing, environmental effects should be considered as well. In isogenic Avy and Axin(Fu) mice, the coat color and tail phenotypes show extremely variable expressivity linked to the activity of an inserted retrotransposon [9, 10], and diet can alter the methylation status of the retrotransposon and cause the phenotype change [11]. Differences between embryos of mouse strains that are traditionally considered blocking and nonblocking in vitro at the two-cell stage are contingent on the culture medium osmolarity [12]. In somatic cells, cytoplasmic compartments and pools of molecules are able to reallocate in response to changes in the culture medium composition [13]. In somatic cell–derived clonal embryos, certain idiosyncrasies of myoblast and cumulus cell clones referred to as somatic cell–like features are well documented but their causes remain elusive [14]. Environmental cues seem to merely modulate the developmental competence of already established zygotes [15], but the developmental competence of somatic cell clones is not yet established at nuclear transfer. In intact mouse oocytes, nuclear remodeling activities continue to operate after activation [16–18], albeit not to the extent observed when donor nuclei are exposed to metaphase II ooplasms [19, 20]. Alteration of pronuclear remodeling by culture conditions after oocyte fertilization would lead to a suboptimal, yet still zygotic, genetic program of embryonic development. In contrast, alteration of nuclear remodeling after somatic-cell nuclear transfer may either support or prevent the establishment of a zygotic genetic program, depending on the specific cues from the environment.

Is the reprogramming of the donor nucleus also contingent on the culture environment of the clone? The focus of this study was to investigate whether developmental pluripotency after cumulus-cell nuclear transfer ensues independently from, or is conditioned by, the culture environment as tested in a variety of forms.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Note Added in Proof
 References
 
Mice
Eight- to 10-week old B6C3F1 (C57Bl/6J X C3H/HeN) female mice (Taconic, Germantown, NY, http://www.taconic.com) were used to collect oocytes and cumulus cells after superovulation, to obtain zygotes after mating with F1 males, and to provide the female donors of Oct4-GFP cumulus cells after mating with OG2 [21] males. Superovulation and collection of oocytes and cumulus cells were performed as described [22]. Mice were maintained and used for experimentation in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Pennsylvania, Philadelphia.

Embryo and ES Cell Culture
Handling of oocytes, nuclear transfer from ovarian cumulus cells, and activation of reconstructed oocytes were performed essentially as described [23]. Six hours after the onset of activation, the constructs were washed carefully in {alpha} minimal essential medium ({alpha}-MEM) without cytochalasin B and randomly assigned to six culture groups (CZB, Dulbecco’s modified Eagle’s medium [DMEM], G1/G2, KSOM(aa), M16, and {alpha}-MEM; Table 1Go [24]). Zygotes were recovered after mating and cultured in parallel as controls. All embryos were cultured in 25-µl drops overlaid with washed mineral oil (catalog no. 151694; MP Biomedicals, Irvine, CA, http://www.mpbio.com) in 35-mm Petri dishes (catalog no. 430588; Corning, Corning, NY, http://www.corning.com) under 5% CO2 in air. All culture media were supplemented with 0.4% wt/vol bovine serum albumin (Pentex, code no. 81-068, lot no. 742 [Serologicals Corporation, Protein Fractionation Manufacturing, Kankakee, IL, http://www.serologicals.com/web/homepage.asp]) and 10 µg/ml gentamicin sulphate (catalog no. 194530; MP Biomedicals). At 48 hours of culture (four-cellstage), the CZB medium was supplemented with 5.56 mM D(+)glucose, because the basal CZB is devoid of glucose [25]; G1 medium was replaced with G2 [26] for the same reason (Table 1Go).


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  		Table 1. Culture media compositions (mg/L)
 
When required, clonal embryos that had developed to the four-cell stage were sampled for chromosomal analysis (see below; Fig. 1Go); clonal embryos that had developed up to blastocyst stage were freed of zonae pellucidae and plated onto feeder cells to derive ES cells following a standard method [27] with certain modifications (see below).



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  		Figure 1. Proof of genomic integrity is an essential prerequisite to perform gene expression studies in which altered levels of gene expression are attributed to epigenetic factors in reprogramming. Genomic integrity of a clonal four-cell-stage embryo derived from a cumulus cell. Each of the four blastomeres (A, B, C, D) has a normal complement of 20 chromosomes.

 
Isolation of RNA and Synthesis of cDNA from Single Blastocysts
Total RNA was extracted from single blastocysts using the RNeasy Micro Kit (Qiagen Inc., Valencia, CA, http://www1.qiagen.com/SelectCountry.aspx) according to the manufacturer’s instructions; all centrifugations were performed at 18,000g at 20°C. Blastocysts were washed three times in Na-citrate (3% wt/vol in Milli-Q water) supplemented with 0.1% wt/vol polyvinylpyrrolidone (40 kDa, catalog no. 529504; Calbiochem, San Diego, http://splash.emdbiosciences.com/default.asp?s=www.calbiochem.com&p=%2F&q=), transferred to 75-µl RLT buffer containing 1% vol/vol ß-mercaptoethanol, and disrupted by vortexing (30 seconds). Five microliters of a 4ng/µl solution of poly-(A) carrier RNA was added to the lysate, followed by 70% ethanol (1:1 volume). The mixture was loaded onto a MinElute Spin Column (provided in RNeasy Micro Kit) and centrifuged for 15 seconds, washed with buffer RW1, and centrifuged for another 15 seconds. To remove contaminating genomic DNA, the column bedding was treated with deoxyribonuclease (DNase) using (ribonuclease) RNase-free grade DNase-1 and then centrifuged for 15 seconds. After washes with buffer RW1, RPE, and 80% ethanol, RNA was eluted using 12.5 µl of Milli-Q water (>80% volume recovery).

cDNA synthesis was performed immediately after RNA extraction using the High Capacity cDNA Archive Kit (catalog no. 4322171; Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) following the manufacturer’s instructions. The flow-through of a MinElute Spin Column, as described above (10–11 µl), was transferred into a 0.2-ml thin-wall polymerase chain reaction (PCR) tube and mixed with 90 µl of reverse transcription (RT) reaction mixture as follows (final concentrations): 1 x RT buffer; 1 x random hexamers; 4 mM dNTP mix; 250 IU MultiScribe RT (provided in the High Capacity cDNA Archive Kit). Binding of random hexamers was allowed for 10 minutes at 25°C, and RT was carried out for 2 hours at 37°C, per the manufacturer’s instructions, on the heating block of a GenAmp 9700 thermalcycler (Applied Biosystems). Because mRNA RT did not involve an oligo-(dT) primer, but rather random hexamers, the possibility that poly(A) of different sizes may have altered the efficiency of RT and, therefore, the detection of such transcripts can be discounted.

Total mRNA was extracted from each blastocyst separately, reverse-transcribed, and split into 12 equal volumes of cDNA aliquots (five target genes, in duplicate, and two spare volumes). All transcripts, except Oct4 mRNA, were detected by quantitative RT-PCR (Q-RT-PCR) in pooled (n = 50) cumulus cells but at less than 1% of the oocyte level (five to seven amplification cycles later; data not shown). When spare cDNA volumes from the same embryo were processed, amplifications were reproduced within ± 0.4 SDs of the crossover threshold cycle (Ct), in line with previous reports [28, 29].

Light Cycler Q-RT-PCR: Quantification of Oct4, Bex1, Gapdh, Cpt2, and Hprt Gene Transcripts in Blastocysts
Total RNA was extracted from single blastocysts in a 75-µl volume using the RNeasy Micro Kit (Qiagen Inc.) according to the manufacturer’s instructions. cDNA synthesis was performed immediately after RNA extraction using the High Capacity cDNA Archive Kit following the manufacturer’s instructions. Transcript levels were determined using the Light Cycler Real-Time PCR system (Roche, Basel, Switzerland, http://www.roche.com) with SYBR green I as the fluorochrome [30, 31].

The individual embryo cDNAs pool was split into 12 reactions (one for each of the five genes, in duplicate, and two spare reactions), and the following four negative controls were run in parallel: H2O; H2O eluted on MinElute column; H2O reverse-transcribed; and sample RNA not reverse-transcribed. Five microliters of template was added to 15 µl of the following reaction mixture in a glass capillary tube (catalog no. 1909339; Roche): 1 x LightCycler FastStart DNA Master Plus SYBR green (catalog no. 03515885; Roche), 500 nM each primer (final concentrations). Primers for Gapdh (GenBank M32599 [GenBank] ) were 5'-TGGTTCCAGTATGACTCCACTCAC-3' and 5'-GAT-GACAAGCTTCCCATTCTCG-3' (107-bp amplicon); primers for Hprt (GenBank J00423 [GenBank] ) were 5'-GAAAGACTTGCTCGA-GATGTCATG-3' and 5'-ACAGAGGGCCACAATGTGATG-3' (60-bp amplicon); primers for Cpt2 (GenBank U01163 [GenBank] ) were 5'-CGGACCCGAAGTCTGAGTATAATG-3' and 5'-GAAACCG-CACTGCAGAAACAG-3' (72-bp amplicon); primers for Oct4 (GenBank M34381 [GenBank] ) were 5'-GTTGGAGAAGGTGGAAC-CAACTC-3' and 5'-CTTCAGCAGCTTGGCAAACTG-3' (88-bp amplicon); primers for Bex1 (GenBank AF051347 [GenBank] ) were 5'-TGACCACCATGATGAGTTTTGC-3' and 5'-GGTCCCCAT-GTCATCTTCAGAG-3' (69-bp amplicon).

The LightCycler profile was as follows: first denaturation and Taq polymerase activation at 95°C for 10 minutes; first 10 cycles of amplification at 95°C for 10 seconds, 65°C to 61°C for 10 seconds (touchdown of 0.4°C per cycle), 72°C for 10 seconds; followed by 35 cycles of amplification at 95°C for 10 seconds, 60°C for 10 seconds, and 72°C for 10 seconds. SYBR green I fluorescence was measured at the end of each extension step. Melting curve analysis was undertaken to determine the specificity of the PCR products, which were further confirmed by sequencing and Basic Local Alignment Search Tool (BLAST). Comparison of all amplicons with actual targets revealed greater than 99% homology for all amplicons. Sequencing and BLASTing of a product that was occasionally amplified by Oct4 primers in the cumulus cell cDNAs pool, but not in that of zygotes nor of clones, matched an X-linked sequence (GenBank LOC385358) similar to Oct4 that may have been previously encountered [32].

Transcript amounts were expressed relative to that of Hprt using the equation 2– (Ctclonal–Ctctrl), where Ct is the number of amplification cycles at which the fluorescent signal is first detected at a level that is 10 times above that of background for zygotes (Ct ctrl) or clones (Ct clonal).

When analysis of Xist (GenBank AJ421479 [GenBank] ) was required, the RLT lysate was split into two aliquots: 37.5 µl was used for RT and PCR of Xist RNA (with DNase treatment performed on column) with primers 5'-CATAGCCCCTTCCCAATAGGTC-3' and 5'-CTACTTGGGCGTTCACTTCAGAG-3' (143-bp amplicon); 37.5 µl was used for sexing with Y chromosome (Zfy locus)-specific primers and X chromosome (DXNds3 locus)-specific primers [33].

Chromosomal Analysis of Four-Cell Embryos and Analysis of X-Chromosome Inactivation
Because the five genes analyzed are located on four different chromosomes, we postulated that altered levels of gene expression in clones might be due to chromosomal imbalance and embryo mosaicism rather than to gene regulation after reprogramming. This is based on evidence that the first two mitotic divisions are nondisjunction-prone in mouse zygotes [34, 35] and mammalian clones may be more subject to this predisposition [36]. Four cell–stage embryos were incubated for 8 hours in {alpha}-MEM containing 0.5 µg/ml colcemide to arrest cells at metaphase. Embryos were incubated in hypotonic sodium (Na)-citrate solution (0.9% wt/vol in Milli-Q water) for 10 to 15 minutes. They were then transferred individually onto a glass slide in a minimal volume of Na-citrate and fixed by dropping ice-cold methanol:acetic acid (3:1 volume) on top of the hypotonic solution [37]. Chromosome spreads were air-dried, stained with Hoechst 33342 at a concentration of 1 µg/ml in Sorensen’s buffer (pH 6.8), and mounted in Vectashield H-1000 (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com). A DM-IRBE–inverted fluorescence microscope (Leica Microsystems Inc., Bannockburn, IL, http://www.leica-microsystems.com) equipped with ORCA ER camera (Hamamatsu Corp., Bridgewater, NJ, http://www.hamamatsu.com) and Openlab software (Improvision Inc., Lexington, MA, http://www.improvision.com) was used to provide images of the chromosomes under UV light. Only spreads with >40 chromosomes (referred to as hyperaneuploid) were considered indicative of aneuploidy. The ratio of hyperaneuploid spreads over the total number of embryonic mitoses displaying countable chromosomes was taken as the rate of blastomere aneuploidy.

To determine whether regulation of another epigenetic process, such as X-chromosome inactivation, was affected under conditions that altered Oct4, Bex1, and Gapdh, the expression of Xist [28, 29] was examined in clonal as well as in female and male zygotic blastocysts. Regardless of the culture medium used, Xist RNA levels in clones were in the range of those observed in female zygotes and more than 10-fold higher than those observed in male zygotes (not shown). See Light Cycler Q-RT-PCR: quantification of Oct4, Bex1, Gapdh, Cpt2, and Hprt Gene Transcripts In Blastocysts.

Total Cell Count and Differential Labeling of the Inner Cell Mass and Trophectoderm of Blastocysts
The nuclei of blastocyst inner cell mass (ICM) and trophectoderm (TE) cells were differentially labeled with polynucleotide-specific fluorochromes as described [38] after the modified immunodissection procedure of Handyside and Hunter [39]. Whereas total cell numbers were lower in clonal than in zygotic blastocysts at 96 hours of development (p < .05), total cell numbers in clonal blastocysts were similar (p > .05) across culture media (M16: 45 ± 14; CZB: 36 ± 15; KSOM(aa): 44 ± 19; {alpha}-MEM: 45 ± 18), as were cell numbers in the ICM (M16: 15 ± 5; CZB: 12 ± 5; KSOM(aa): 10 ± 4; {alpha}-MEM: 17 ± 7) (n > 10 blastocysts in each group).

Whole-Mount Riboprobe In Situ Hybridization of Blastocysts
Riboprobe in situ hybridization (RISH) of Oct4 mRNA was performed essentially as previously described [40].

Derivation of ES Cell Lines from Clonal Blastocysts
After digestion of the zonae pellucidae with acidic Tyrode’s solution, groups of 8 to 10 blastocysts were transferred on to STO feeder cell layers in four-well dishes and were left undisturbed for 6 to 7 days without passage. The ICMs were selectively detached (picked with a polished glass Pasteur pipette) from the trophoblastic outgrowths in the presence of 0.25% trypsin/1 mM EDTA (Invitrogen catalog no. 25200-056) and replated onto a 96-well dish covered with mitotically arrested mouse embryo fibroblast (MEF) feeders. The ES cells were grown to subconfluency and gradually expanded by serial trypsinization, dissociation, and replating (3 days) onto freshly prepared MEF feeders, scaling up to larger well sizes. The medium for blastocyst outgrowth formation and ES cell culture consisted of DMEM high glucose (Invitrogen, catalog no. 11960-044), 15% fetal calf serum (ANF190A9; HyClone, Logan, UT, http://www.hyclone.com), 0.1 mM MEM nonessential amino acids (catalog no. 11140-050; Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 100 U/ml Pen-Strep, 2 mM L-glutamine, 10 µM ß-mercaptoethanol, and 104 U/ml leukemia inhibitory factor. STO or MEF feeder cells were mitotically arrested with 10 µg/ml mitomycin C (catalog No. M4287; Sigma, St. Louis, http://www.sigmaaldrich.com) in DMEM for 2 hours.

In Vivo Development of Embryos
Transfer of zygotic and clonal blastocysts to the genital tract was performed essentially as previously described [40]. When required, the cavities of zygotic blastocysts (B6C3F1 x ICR) at 96 hours of development were microinjected with 10 to 15 clonal ES cells and then transferred into the uterus of 2.5 days post coitum (dpc) pseudopregnant ICR females. Development rate and developmental contribution were assessed at 10.5 and 13.5 dpc for clones and injected blastocysts, respectively.

Data Analysis
Means, SDs, analysis of variance (ANOVA), and correlation analysis were performed using JMP 5.1 (Statistical Analysis System, SAS Institute, Cary, NC, http://www.sas.com). Differences were considered significant when p < .05.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Note Added in Proof
 References
 
Quality Control for Cumulus Cell Nuclear Transfer
In principle, the differences observed in clones are possible consequences of either manipulation or faulty reprogramming, although Wakayama and colleagues [41, 42] showed that manipulation and culture are not as detrimental to clonal mouse embryos as often assumed. The fact that cloning results are very variable, while originating from research in a limited number of laboratories, demonstrates the complexity of the technology used and exemplifies the above concern. In this study, possible variations in micromanipulation that might cause differences of development and gene transcription in the clones were neutralized by manipulating the prospective six-media clones using the same setup and only then allocated to the different culture media. We previously showed that oocyte micromanipulation per se does not cause significant reduction of ATP content in clones within 48 hours compared with zygotes [23]. The effect of micro-injection, as measured by development after intracytoplasmic sperm injection (ICSI) into metaphase II oocytes, was negligible in that 87% of the oocytes survived the sperm head injection (n = 1,991/2,301), 91% and 86% of these cleaved to two and four cells, respectively (n = 1714 and 1,816/1,991), 49% formed blastocysts at 96 hours (n = 969/1,991), and 32% of the embryos transferred in vivo at various stages developed to term (n = 40/125). ICSI is routinely performed in our laboratory: data just presented were the average of a 1-year period that encompasses the present study (Boiani, unpublished data). To further assess whether micromanipulation damages the integrity of the transplanted genome, the karyotype of clonal embryos was analyzed (Fig. 1Go). Karyotype analysis of clonal four-cell stage embryos detected hyperaneuploid sets of chromosomes (>40) in 10.8% ± 3.2% of blastomeres (n = 93). This did not differ significantly (p > .05) from the rate of hyperaneuploidy observed in blastomeres of parthenogenetic (10.1% ± 3.4%; n = 79) and zygotic (8.3% ± 2.4%; n = 133) embryos cultured under the same conditions (Boiani, unpublished data). Gene/allele regulation, rather than physical absence of loci, would therefore seem to be at the root of the different patterns of gene expression expected to be found in clones. Clones and zygotes had an equivalent ooplasmic composition, B6C3, thus preventing strain-specific ooplasmic modifiers from having an unequal effect on the incoming nucleus [43].

Culture Conditions Affect the Rate of Blastocyst Formation of Genetically Identical Mouse Clones
As long as the clones are generated with the same experimental setup and only subsequently allocated to different culture media, as was the case in this study, differences among clones depend on the culture media only. We assessed the developmental program of clonal and zygotic mouse embryos by culturing them in parallel up to the blastocyst stage. To this end, strain B6C3 mouse oocytes were enucleated and transplanted with B6C3 cumulus cell nuclei to derive the clones or were fertilized in vivo with B6C3 sperm to recover one-cell zygotes from the oviducts.

At 96 hours of development in vitro (108 hours after human chorionic gonadotropin), zygotes had formed blastocysts at similarly high rates (80%–85%) in all media tested except DMEM (8%; see Fig. 2AGo for absolute rates). The rate of clonal blastocyst formation progressively increased (multiple t-tests, p < .05) from DMEM through G1/G2, CZB, KSOM(aa), M16 to {alpha}-MEM (1% ± 0.7%, 10% ± 3.5%, 26% ± 2.3%, 32% ± 2.5%, 38% ± 2.5%, and 52% ± 2.6%, respectively; see Fig. 2BGo for absolute rates). Diploid parthenogenetic B6C3 embryos activated by the same stimulus as that used for clones formed blastocysts at nearly the same rate across the different media (not shown), as reported for B6D2 parthenogenetic embryos [44] and B6C3 zygotic embryos [45, 46]. These parthenogenetic and zygotic mouse embryos demonstrate an inherent ability to maintain homeostasis, which seems lacking in clonal embryos. Therefore, the transplanted somatic genome may be more susceptible to environmental cues than a parthenogenetic or zygotic genome. Because culture protocols were identical with respect to the origin of the donor nuclei, the nuclear transfer procedure, and the activation method, the observed difference in clonal development was attributed to an unequal ability of the different culture environments to support reprogramming.



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  		Figure 2. Rates of blastocyst formation for (A) zygotes and (B) cumulus cell–derived clones cultured side by side in DMEM, G1/G2, CZB, KSOM (aa), M16, and {alpha}-MEM. Each data point is an average of measurements obtained from 15 independent experiments (number of starting one-cell embryos: 42, 16, 93, 91, 82, 91 zygotes; 246, 90, 490, 418, 415, 443 clones allocated to DMEM, G1/G2, CZB, KSOM(aa), M16, and {alpha}-MEM, respectively). Error bars indicate the SDs. Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; MEM, minimal essential medium.

 
Culture Conditions Modulate Gene Expression in Clones Differently Than in Zygotes
The developmental program that guides the progression of clones through preimplantation stages may result in the formation of either genuine blastocysts or seeming blastocysts that have only the morphological appearance of normal blastocysts (phenocopies [32]). These phenocopies can be distinguished by their aberrant gene expression that may correlate with subsequent developmental failure, in vivo and also in part in vitro. Although as many as 70% of cumulus cell–derived mouse clones can develop up to the blastocyst stage in vitro, most fail to reproduce the level and spatial pattern of Oct4 gene expression present during zygotic development that confers pluripotency on blastomeres of the morula and the ICM [32, 40, 47]. Therefore, we investigated whether cumulus-cell clones cultured in different media uniformly failed to reactivate the Oct4 gene from the silent somatic state [32].

The expression level of Oct4 and other genes was analyzed in individual blastocysts by real-time Q-RT-PCR as follows: Oct4 (chromosome [chr] 17), Bex1 a.k.a. Rex3 (chr X), Gapdh (chr 6), Cpt2 (chr 4), and Hprt (chr X). These genes were chosen for the following reasons: Bex1 is a TE-restricted gene in mouse blasto-cyst development [48] whereas Oct4 is not [47], and Gapdh and Cpt2 are metabolic genes that are relevant to the health status of the embryo in culture [49, 50]. Hprt was used for normalization purposes. In theory, differences in the gene expression levels between clonal and zygotic blastocysts might simply reflect differences in the numbers of cells in these two types of blastocysts [38] or may be due to differences in the state of activity of the genes at the time of nuclear transfer rather than to differential gene regulation due to reprogramming. Hprt was used for normalization purposes after finding it not to be altered significantly in clonal and zygotic blastocysts within the culture medium groups (see ANOVA below). Hprt expression levels are also conserved in male and female zygotic mouse blastocysts [51].

Five single clonal blastocysts and five single zygotic blastocysts grown in each medium were analyzed by Q-RT-PCR. This approach relies on the buildup of the amplification product with each PCR cycle, which increases SYBR green I light emission until it crosses a threshold; hence, the lower the template amount, the higher the number of cycles. Raw Ct and SDs of clonal and zygotic blastocysts are shown in Table 2Go. One-way ANOVA of Ct values for Hprt by type of embryo produces nearly identical distributions for clonal and zygotic blastocysts in concert with Hprt as a normalizer (F1,71 = 0.0274, P > F 0.8689). The raw Q-RT-PCR data indicate that Oct4 mRNA amplifies above threshold 4.11 cycles earlier than Hprt in the {alpha}-MEM medium, whereas it amplifies 3.54 cycles earlier than Hprt in the CZB medium. Thus, there seems to be less Oct4 mRNA in the CZB than in the {alpha}-MEM sample on an absolute basis, in agreement with the RISH data. However, Q-RT-PCR data presented in raw format are not acceptable as they may merely reflect the amount of total mRNA extracted.


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  		Table 2. Amounts of clonal and zygotic blastocyst mRNAs expressed by the crossover threshold (Ct) number
 
Therefore, values were normalized to that of Hprt mRNA, and a clone-zygote comparison of Hprt-normalized transcript levels (Table 3Go) was conducted with the {Delta} {Delta}Ct method (ABI Prism 7.700 User Bulletin 2; PerkinElmer, Wellesley, MA, http://www.perkinelmer.com) [52]. Zygotes assumed a value of 1, and clones that exhibited a higher or lower gene transcript level than zygotes were given a value greater than or less than 1, respectively. Oct4 transcripts were detected in all embryos analyzed, and in general, based on the normalized values, Oct4 was expressed at higher levels in clones than in zygotes (1 to 5.14, +414%). There were also notable variations in Oct4 transcript levels in CZB-cultured clones (1 to 14.96, +1,396%). These results were consistent with the lower Bex1 expression levels (1 to 0.27, –73%) based on opposite regional regulation of Oct4 and Bex1 genes in the blastocyst (Oct4 expression in the ICM, Bex1 in the TE). The level of Cpt2 relative to Hprt mRNA was similar in clones and zygotes across all media (1 to 0.86, –14%), whereas Gapdh mRNA levels fluctuated. Overall, Gapdh exhibited 185% higher (1 to 2.85, +185%) expression in clones compared with zygotes, with 118% of the total increase contributed by mRNA levels in one medium—CZB. As determined by karyotype and Xist analysis, gene/allele regulation rather than physical gain or loss of chromosomes, or X-chromosome inactivation status, seemed to cause the different patterns of gene expression (see Quality Control for Cumulus Cell Nuclear Transfer and Materials and Methods).


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  		Table 3. Use of the {Delta} {Delta}Ct method (PerkinElmer ABI Prism 7.700 User bulletin n.2) to calculate deviations of clonal versus zygotic blastocysts in transcripts amounts
 
The expression patterns of the five genes in clones were compared across the different media. This was done by calculating the correlation coefficient (r) between the medium series of gene expression (Table 3Go) that were paired in all possible ways leaving out reciprocal duplicates (e.g., correlation between Cpt2 and Oct4 expression levels in G1/G2, CZB, KSOM(aa), M16, and {alpha}-MEM), based on the formula r = covariance [series1, series2]/std dev[series1]*std dev [series2]. Correlation coefficients were as follows: Bex1-Oct4, r = 0.004; Gapdh-Bex1, r = 0.136; Gapdh-Cpt2, r = 0.248; Cpt2-Oct4, r = 0.286; Cpt2-Bex1, r = 0.492; Gapdh-Oct4, r = 0.953 (Gapdh-Gapdh, r = 1). Oct4 and Bex1 patterns (r = 0.004) were actually inversely related (Oct4 and 1/Bex1, r = 0.893). This demonstrated that genes among the set analyzed in clones respond in a variable manner to different culture conditions, particularly Oct4. This seems contradictory to the critical requirement of Oct4 for the establishment of the pluripotent founder cell population in mouse embryos [47], to the tight Oct4 regulation in mouse ES cells [53], and to the observation that Oct4 mRNA levels are rather conserved in individual ICM cells of human blastocysts [54].

Functional, But Not Spatial, Definition of the Blastocyst ICM Is Altered by Culture Conditions
Until recently, quantitative analysis of high-resolution two-dimensional protein gels [55], mRNA differential display [56], analysis of expressed sequence tags derived from libraries of various preimplantation stages [57, 58], analysis of selected genes [59], and microarray techniques [52, 60, 61] have been used in gene expression studies. Although these approaches have shed some light on the molecular basis of preimplantation development, they are based on pooled embryos and thus offer limited insight into clonal embryo development wherein embryo-embryo variability occurs, even within the same batch of clones.

Alterations in the transcript levels of genes that are locally/regionally modulated cannot be detected by any mRNA profiling method, Q-RT-PCR, or microarray technology, unless each individual cell is profiled in situ. For instance, higher Oct4 expression levels (+414% on average; Table 3Go) and lower Bex1 expression levels (–73% on average) in clones relative to zygotes may indicate an expansion of the ICM compartment relative to a shrunken TE. The fact that all embryos tested positive for Oct4 transcript by Q-RT-PCR where as previous studies (using riboprobes and conventional RT-PCR) found that some tested negative as well [40] reflects, in part, the different sensitivity of these assays. Furthermore, it suggests that the amount of Oct4 mRNA may be so high but in only a few cells or so minute overall as to be functionally equivalent to zero when assessed spatially in situ.

Clonal blastocysts (n = 234 in total from the various media) were analyzed individually for the presence of an ICM, as defined by localized Oct4 mRNA RISH. Differences were observed between CZB, KSOM(aa), M16, and {alpha}-MEM culture media (DMEM and G1/G2 did not yield enough blastocysts to compensate for losses incurred by RISH). Hybridized blastocysts were categorized [38] into four groups: having strong Oct4 signal restricted to the ICM as opposed to weak signal; having unrestricted signal (ICM + TE); or lacking signal in both (Fig. 3AGo). In general, clonal blastocysts had consistently lower rates of strong, ICM-restricted distribution of Oct4 mRNA than zygotic blastocysts cultured in the same medium (p < .05, except for KSOM(aa); Fig. 3BGo), although intensity levels were also lower (weaker signal) for zygotes after culture in CZB (not shown). Clones cultured in CZB and KSOM(aa) media exhibited rare and limited Oct4 expression, respectively; whereas a higher proportion of clonal blastocysts exhibited a restricted pattern of Oct4 expression (Fig. 3AGo, type D) in M16 and {alpha}-MEM media. Thus, the spatial distribution of Oct4 transcript in clonal blastocysts is neither constant nor stochastic but varies with the culture conditions used, depicting a progressive increase in the rate of Oct4 mRNA restriction to the ICM (CZB < KSOM(aa) < M16 < {alpha}-MEM; {alpha}-MEM != CZB at p < .05; Fig. 3BGo). The differences between genetically identical clones cultured in different media are overt in Figure 3CGo. We would like to stress that because RISH is orders of magnitude less sensitive than Q-RT-PCR, the lack of detectable signal in situ is not equivalent to lack of mRNA. Contrast between the poor spatial localization of Oct4 mRNA (Figs. 3A, 3BGo) and its higher relative amount (Table 3Go), particularly in the CZB-cultured clones, reflects the normalization that was introduced after the Q-RT-PCR, whereby the RISH data stand as absolute values, whereas the Q-RT-PCR data are relative to Hprt mRNA. There might also be a possibility that Q-RT-PCR detects not only the parent Oct4 transcript but also transcripts originating from Oct4 pseudogene(s) [62].



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  		Figure 3. Expression patterns of Oct4 in clonal and zygotic blastocysts detected by RISH. (A): General patterns of Oct4 mRNA distribution in blastocysts. (B): Frequency of pattern A (worst) and D (best) is dependent on the culture medium used for clones. A total of 234 clonal blastocysts (n = 64, 21, 55, 89, 5 in M16, CZB, KSOM(aa), {alpha}-MEM, and G1/G2, respectively) were examined by RISH. Control blastocysts were obtained in parallel (n = 41, 35, 30, 32, 35) after culture in G1/G2, CZB, KSOM(aa), M16, and {alpha}-MEM media, respectively. Each data point is an average of measurements from five independent experiments. Error bars indicate SDs. (C): Clonal blastocysts obtained in CZB and {alpha}-MEM, from the same experiment, documented after simultaneous processing in the RISH reaction and 3 hours of color reaction. Bar = 100 µm. Abbreviations: ICM, inner cell mass; MEM, minimal essential medium; RISH, riboprobe in situ hybridization.

 
Because Oct4 becomes restricted to the ICM during normal blastocyst formation, differences in the regional distribution of the transcript among clonal and between clonal and zygotic blastocysts may simply reflect differences in cell numbers and allocations to ICM/TE [38] in these two types of blastocysts rather than differences in gene regulation. Variability in Oct4 mRNA distribution/restriction to the ICM observed in different culture environments was found to coexist with a similar number of embryonic cells allocated to the ICM, that is, 12 ± 5 ICM cells in CZB and 17 ± 7 ICM cells in {alpha}-MEM (p > .05) (see Materials and Methods). It follows that reinstatement of pluripotency after cumulus cell nuclear transfer is a primary effect of culture conditions on reprogramming rather than a side effect of cell number allocation or faulty chromosome segregation during cleavage of clonal embryos.

Consequence of the Culture Medium–Dependent Oct4 Gene Reprogramming
Altered Oct4 levels affect the pluripotency of ES cells [53], and lack of Oct4 in the ICM negates the further development of mouse blastocysts [47]. Therefore, it seems likely that differences in Oct4 mRNA levels in clonal blastocysts, observed more frequently than in zygotic blastocysts, also have an impact on clonal development and ES cell derivation. To track this, we compared the reprogramming of genetically identical cumulus-cell nuclei in oocytes cultured in different media by measuring Oct4-GFP expression in those nuclei (Fig. 4Go, see legend for absolute rates). The normal timing of maternal-zygotic transition to Oct4 gene expression occurs in mice at the eight-cell stage [63]. Although the onset of Oct4-GFP expression in zygotes at the eight-cell stage was equally frequent across all media (p > .05), the proportion of eight-cell clones having Oct4-GFP expression at 55 hours was lower in CZB and G1/G2 and higher in {alpha}-MEM (p < .05). This different penetrance is notable in view of the fact that CZB and {alpha}-MEM clones had been activated synchronously and formed four cells at similar rates (Fig. 2Go). Yet, after imaging, clones formed blastocysts at variable rates, as described. ES cell rates were determined as a measure of whether formation of an ICM endowed with more or less pluripotential character (Oct4 mRNA RISH) correlated with developmental outcomes, both in vitro and in vivo (Fig. 5Go).



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  		Figure 4. Viable monitoring of nuclear reprogramming during clonal embryo cleavage. Onset and frequency of Oct4-GFP expression in eight-cell clonal and zygotic embryos cultured in M16, CZB, KSOM(aa), {alpha}-MEM, and G1/G2 media; base of data for each bar, 25 embryos. Error bars indicate SDs. Asterisks indicate significant differences in rates of Oct4-GFP expression between zygotes and clones for the particular culture medium used. Abbreviation: MEM, minimal essential medium.

 


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  		Figure 5. Stem cell and fetal consequences of clonal embryo epigenetic reprogramming. Culture conditions conducive to clonal blastocyst formation are associated with differences in the potential of the inner cell mass and trophectoderm to give rise to ES cells and fetal implants, respectively. (A): Clonal blastocysts seeded on feeder cells. (A’): Clonal ES cell colony expressing Oct4-GFP at passage 6. (A"): Oct4-GFP clonal ES cells after injection in zygotic blastocysts. (A"’): Germ cell contribution of Oct4-GFP clonal ES cells that were injected into blastocysts to form 13.5 dpc chimeric fetuses. (B): Summary of ES cell and fetal rates of clones formed in CZB and {alpha}-MEM. Bar = 100 µm. Abbreviations: ES, embryonic stem; MEM, minimal essential medium.

 
To this end, we generated and assayed for ES cell potential an additional batch of clones (n = 400) under the worst and best reprogramming conditions for Oct4 (Fig. 3Go): Half of the clones were cultured in CZB and half in {alpha}-MEM, in parallel, from the one-cell stage up to the blastocyst stage, followed by derivation of ES cells. These blastocysts were plated in parallel onto feeder cells (Fig. 5AGo), using a standard method for derivation and culture of ES cells. Cell lines were identified as ES cells by observing the expression of Oct4-GFP after six passages (Fig. 5AGo’) and by blastocyst injection (see below). The rates of cell line derivation were found to be 8.2% for the CZB-cultured clones (n = 5 cell lines/61 blastocysts) and 39.3% for the {alpha}-MEM–cultured clones (n = 33 cell lines/84 blastocysts) (p < .05). Routine derivation from F2 (B6C3xB6C3) zygotic blastocysts in our laboratory yields 20% cell lines, regardless of the embryo culture medium used. To extend this observation, more clonal blastocysts were produced (n = 225), half in CZB and half in {alpha}-MEM. These were transferred at 96 hours of development into 11 recipient females, alongside zygotic blastocysts (n = 175) transferred in 8 separate but synchronous recipients. All recipients became pregnant, and fetal development was assessed at 10.5 dpc. Fetal rates for clones did not stand up to the projections of their blastocyst rates. Clones formed in {alpha}-MEM (52% blastocysts) exhibited modest implantation (18.6%) and fetal developmental (9.1%) rates, whereas clones cultured up to the blastocyst stage in CZB (26% blastocysts) exhibited higher rates of implantation and fetal development (61.4% and 25.9%, respectively). Zygotes cultured in CZB and {alpha}-MEM, while not resulting in higher implantation rates (24%–47%) than the clones, resulted in significantly higher fetal rates (50%–83%).

Altogether, these data show that the patterns of Oct4 gene expression in blastocysts cannot only be assessed as done previously [40] but also arise nonrandomly according to culture medium patterns and that higher rates of blastocyst formation and ES cell potential of clones in vitro did not translate into higher rates of fetal development (Fig. 5BGo). To determine whether clonal ES cells would be impaired under conditions that impaired fetal development from precursor blastocysts, clonal ES cells from {alpha}-MEM were injected into the cavity of blastocysts of zygotic embryos and then assessed for germline contribution at 13.5 dpc. Nine out of 13 lines tested contributed to the formation of germ cells (Figs. 5A", 5A"’Go). Considering ES cells as the equivalent of the ICM, these results suggest that the reprogrammability of the donor nucleus to give rise to an ICM lineage is different from that required to give rise to a TE lineage.


   DISCUSSION
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Note Added in Proof
 References
 
Contrary to the expectation that the reprogrammability of somatic nuclei in clones is highly error-prone while being influenced by the type of donor cell [3] and/or limited by size, age, and composition of the ooplasm, in our current studies we show for cumulus cell nuclei that reprogrammability is contingent on the culture environment of the clone. While clonal development appears to be autonomous to the donor nucleus source (primordial germ cells, thymocytes, cumulus cells, and ES cells are not, rarely, infrequently, and quite supportive of full clonal development, respectively [64]), there seems to be an environmental component as well. This new concept in mouse cloning should not be discounted as aberrant, because Doherty and colleagues [65] showed that the expression of the H19 gene in mouse zygotes could be experimentally manipulated by in vitro culture conditions (Whitten’s versus KSOM(aa) medium). They also found a correlation between promoter DNA methylation and H19 expression in these media.

Clones derived from the same cluster of cumulus cells, activated simultaneously in the same medium, and differing only in the postactivation medium used for culture form blastocysts and express mRNA of the stem cell marker Oct4 at different rates. Discrepancy between the success rates presented here and those previously published is due in part to the scoring criteria (rigorously blastocysts at 96 hours postactivation in this study, blastocysts up to 120 hours in Chung et al. [66], or morulae-blastocysts in Wakayama et al. [67]). Regarding the differential expression of Oct4, although the formal possibility exists that that the epigenotype of the nucleus donor cells was mixed [3] and that the proportion of each epigenotype allocated to the groups of clones did not reflect the original proportions in the donor cell population, this possibility is deemed very unlikely in view of the number of clones allocated to each group and the number of replicates performed. The clonal phenotype could not be as variable (albeit not randomly) if the cell type and the epigenotype of the nucleus donor cells were solely responsible for developmental outcome. Rather, heterogeneity would be possible if reprogramming events occurred or continued to occur subsequent to the first cell division.

Examination of Oct4-GFP fluorescence during cleavage foreshadows differences seen in clonal versus zygotic embryos after culture to the blastocyst stage in various media. Aneuploidy and cell number progression of clones resembled those typical of zygotes up to the morula stage [38] (Boiani, unpublished observations), and clonal blastocysts were composed of about the same number of cells at the blastocyst stage across culture media. Therefore, chromosome malsegregation, nonuniform retardation of cleavage, and different health status among the clones in different media are discounted as the basis for the differential Oct4 as well as other gene expression. Rather, the activation of the silent, somatic Oct4-GFP alleles unfolds differently in different media, as detected 55 hours after nuclear transfer (29%–71% penetrance), with overt differences seen in blastocysts at 96 hours. Reactivation of the silent, thymocytic Oct4-GFP took approximately 36 to 48 hours to occur, at nearly 100% penetrance, after cell hybridization with ES cells [68]. Although the use of different, not interchangeable media for oocytes and ES cells makes it hard to compare these nuclear reprogramming systems, observations suggest that the erasure of the somatic cell–specific epigenotype either follows different paths in oocytes and hybridized ES cells (possibly related to the absence/presence of a companion nucleus [69]) or is simply delayed in the former. Subcellular compartments and pools of molecules are known to reallocate within the cell in response to environmental cues [13], and interactive processes between the cytoplasm and the nucleus stretch over a longer distance in an oocyte compared with an ES cell. This may account for response delay of reprogramming in the oocyte-based system. Concerning how the nucleus-cytoplasm interaction may sense the extracellular environment, leading to different patterns of gene expression in different media, this remains to be determined. Here we speculate that the enzymatic activity of Gapdh in the cytoplasm is engaged in a metabolic feedback loop with culture medium components (e.g., glucose and pyruvate), thereby affecting ATP and ATP-dependent chromatin remodeling activities [70] that are pivotal to the reactivation of embryonic genes silent in cumulus cells, such as Oct4. Variability in Gapdh gene expression among cumulus cell mouse clones was noted earlier [49]. Unlike that of Gapdh, mRNA of other housekeeping genes was not (Hprt) or marginally (Cpt2) altered by culture conditions.

Analysis of Oct4 gene expression in clones has relevance beyond the immediate question of how the clone regulates metabolism and gene expression, as the Oct4 transcription factor is essential for the establishment of ES cells from the ICM [47] and their maintenance. An increase in the levels of Oct4 expression in mouse ES cells by approximately 50%, by experimental manipulations, leads to differentiation of these cells into endoderm and mesoderm; reduced levels result in the development to the TE, and intermediate levels preserve the pluripotent stem cell status [53]. Thus, small changes in the expression of a transcription factor could lead to widespread and dramatic changes in gene expression associated with various phenotypes. In clonal blastocysts formed in different media, the variations in Oct4 mRNA levels were not well correlated with the size of the ICM; i.e., 14.96-fold the Hprt mRNA level over the control in blastocysts with 12 ± 5 ICM cells (CZB) and 3.58-fold the Hprt mRNA level in blastocysts with 17 ± 7 ICM cells ({alpha}-MEM). According to these observations, the level of Oct4 mRNA in each individual ICM cell would appear to be rather dissimilar. Although the formal possibility exists that variation is present also in the zygotes used as normalizers, this possibility is deemed unlikely in view of their degree of homogeneity in terms of developmental rates (high) and restriction of Oct4 mRNA to the ICM (frequent).

Although this assumption is now being contended [71], ES cells are often assumed to be the equivalent of ICM cells. Therefore, we attempted to derive ES cells from clonal blastocysts cultured in CZB and {alpha}-MEM media and to transfer clonal blastocysts to females, anticipating a positive correlation between the rates and quality of blastocyst ICM formation and subsequent development. Clones grown in {alpha}-MEM were superior to those grown in CZB with respect to ES cell derivation rates (39.3% versus 8.2%), in accord with the prevalence of ICM-restricted Oct4 mRNA distribution in the two culture media. This is notable because recent experiments using embryo carcinoma (EC) cells as donors for nuclear cloning have resulted in derivative clonal ES cells that retained the traits of the donor cells—that is, no epigenetic changes [72]. Previously, epigenetic heterogeneity of ES cell–derived mouse clones was ascribed to pre-existing heterogeneity and epigenetic instability of ES cells [73]. In contrast, we show that genetically identical cumulus cell nuclei have multiple and distinct ES cell potentials, suggestive of the implementation of different epigenetic traits depending on the extracellular environment of the clone undergoing reprogramming. The rate of ES cell derivation from the {alpha}-MEM cumulus cell clones is an unprecedented high reported to date for somatic cell donors (39.3%), only lower than the rate of derivation after cloning from ES and EC cells, albeit an MEK1 inhibitor was used there to facilitate derivation [72]. Because EC and ES cells already express Oct4 and therefore derivative clones do not depend on the reinstatement of Oct4 expression, it will be of interest to test the response of these clones to different culture conditions. It will also be of interest to challenge the reprogramming-efficient {alpha}-MEM environment with hard-to-clone cell types. Although it has been shown that nuclear transfer can in principle reprogram terminally differentiated B lymphocytes, this was highly inefficient [74]. In fact, the recent discovery that the olfactory receptor gene choice does not involve genetic alterations [75], unlike the case for immunoglobulin genes [74], did not rest on advances in understanding of clonal developmental biology but on the incorporation and expansion of the rare, viably reprogrammed nuclear states into ES cells.

Surprisingly, the success in ES cell formation contrasted fetal development in both media (9% and 26% for {alpha}-MEM and CZB, respectively). Definitely, the efficiency of ES cell derivation does not predict the potential of clonal blastocysts to develop into fetuses. The critical requirement of Oct4 for the establishment of the pluripotent cell population precursor of germ layers in early mouse development may seem contradictory to the dual outcome observed, that is, loss of the embryo but survival of ES cells ({alpha}-MEM) and survival of the embryo but loss of ES cells (CZB). Albeit in a different scenario, Oct4 has been shown to exert a dual role: cell fate determinant before embryo gastrulation and cell survival factor (for germ cells) after gastrulation [76]. The lack of a positive association between ES cell potential and fetal development of {alpha}-MEM clonal blastocysts can be explained by the following possibly superimposed possibilities.

First, blastocysts may form at high rates due to efficient reprogramming up to that stage, but genes required thereafter are not reprogrammed properly. It is not clear why genes not yet expressed at the blastocyst stage would have been reprogrammed differently in the different media. However, ooplasmic epigenetic modifiers may tag genes expressed after implantation. Modifier genes may cause blastocysts with normal gene expression to express such tagged genes abnormally, thereby causing embryos to fail at midgestation [77].

Second, reprogramming errors affect all genes but may be less detrimental to the development of a preimplantation embryo than for subsequent stages due to the increasing number of functions required. This is consistent with the observation that ES cells could still be derived even if the conceptus suffered a condition that became overt at a stage following that of ES cell derivation [78, 79]; this situation is not equivalent to the ability of parthenogenetic and androgenetic mouse embryos to give rise to ES cells [80, 81]. One fundamental difference is that these uniparental embryos originate with an imprinting imbalance that per se dooms fetal development, unlike somatic cell clones that are derived from biparental nuclei.

Third, the blastocysts formed may contain a functional ICM but an impaired TE (reprogramming errors would be at genes that affect the TE but not the ICM, e.g., E-type cyclins [82] and retinoblastoma [83]), or both the ICM and the TE are impaired, but a subset of functional ICM cells is sufficient to derive ES cells whereas a subset of functional trophectodermal cells is not sufficient for successful implantation. This scenario is consistent with the lower levels of Bex1 mRNA observed in G1/G2 and {alpha}-MEM clones compared with CZB and KSOM(aa) that correlate with the low rates of development up to 10.5 dpc observed in the former group. Recently published work showed that imprinted gene expression (H19 and Snrpn) was mostly preserved in the mouse embryo proper but not in the extraembryonic tissues after culture in vitro and assessment in vivo at 9.5 dpc [84]. Previous work [3] showed that bovine blastocysts produced by cloning fail to establish epigenetic asymmetry between ICM and TE for either DNA or H3-K9 histone methylation, with the TE as highly methylated as the ICM. This may underlie the substantial proportion of genes whose expression is downregulated in placentae of clonal embryos, as well as the characteristic placental abnormalities of mouse clones [85]. The trophoblast, although essential for development in utero, can hinder ICM growth during culture and make ES cell line establishment quite problematic [86].

Fourth, the lack of a positive association between ES cell potential and fetal development of {alpha}-MEM clonal blastocysts is because ES cells do not correspond to ICM cells but to an early germ cell state [71]. Therefore, the ability to derive ES cells does not necessarily predict the fetal competence of the ICM.

In conclusion, although the Oct4 expression has been used by several groups as a marker of reprogramming in the strict sense that its silent state must be reversed after somatic cell nuclear transfer, this change unfolds in a variable manner depending on the embryonic environment, and development to the blastocyst stage in the presence of Oct4 is not sufficient for a broader definition of reprogramming (also Oct4–/– embryos form blastocyst-like structures [47]). Although epigenetic errors of gene expression arise very early in clonal development [49, 87], they have no inevitable ending but are tractable experimentally, as shown by the zygotic-like Oct4 gene expression obtained after clone-clone embryo aggregation [38]. Although the precise causal mechanisms of the nucleus-oocyte-medium interaction described here in have not been determined, epigenetic marks at the Oct4 locus are labile and can be experimentally manipulated in embryo-derived cell lines [88], raising the possibility that molecules active in chromatin remodeling may hijack this process if they are present in the culture medium and internalized by the embryo. Thus, new ways of reprogramming cell fate before or without transplanting nuclei into oocytes may be envisioned [89], as seen in myotubes reverting to myoblasts after exposure to myoseverin [90] or myoblasts forming osteoblasts after exposure to reversine [91]. Among the soluble, cell-permeable molecules active in chromatin remodeling, which may play a role in (clonal) embryos, the inositol precursor of nuclear inositol phosphates [92, 93] is present in the {alpha}-MEM and is internalized by the mouse embryo via a sodium transporter [94]. The molecular size of inositol phosphates is compatible with a postulated role of small molecules in nuclear reprogramming after the observation that Oct4 can be activated from the silent somatic state in HeLa cells placed in connection with mouse embryo blastomeres [95].


   NOTE ADDED IN PROOF
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
References
 
That reprogramming after somatic cell nuclear transfer may indeed occur subsequent to the metaphase II oocyte stage was reported by Eckardt et al. [96] after this manuscript was submitted.


   ACKNOWLEDGMENTS
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 
M.B. and L.G. contributed equally to this work. This work was supported by the Marion Dilley and David George Jones Funds and the Commonwealth and General Assembly of Pennsylvania and by grant NIH 1RO1HD42011-01 to H.R.S. The authors would like to thank Drs. Rabindranath DeLaFuente, Maria Viveiros, John McLaughlin, Alexey Tomilin, James Kehler, and Ulrike Nuber for critical reading of the manuscript. We also thank Dr. Jeff Mann (Beckman Institute, City of Hope, CA) for the gift of OG2 mice and Rose Kessler (Roche Bioscience) and Nicola Marziliano (Applied Biosystems) for advice on real-time RT-PCR. Special thanks to Leah Kauffman for editing the final version of the manuscript.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Note Added in Proof
 References
 

  1. Santos F, Dean W. Epigenetic reprogramming during early development in mammals. Reproduction 2004;127:643–651.[Abstract/Free Full Text]

  2. Vickaryous N, Whitelaw E. The role of early embryonic environment on epigenotype and phenotype. Reprod Fertil Dev 2005;17:335–340.[CrossRef][Medline]

  3. Santos F, Zakhartchenko V, Stojkovic M et al. Epigenetic marking correlates with developmental potential in cloned bovine preimplantation embryos. Curr Biol 2003;13:1116–1121.[CrossRef][Medline]

  4. Gurdon JB, Byrne JA. The first half-century of nuclear transplantation. Proc Natl Acad Sci U S A 2003;100:8048–8052.[Abstract/Free Full Text]

  5. Eggan K, Akutsu H, Loring J et al. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc Natl Acad Sci U S A 2001;98:6209–6214.[Abstract/Free Full Text]

  6. Rideout WM 3rd, Wakayama T, Wutz A et al. Generation of mice from wild-type and targeted ES cells by nuclear cloning. Nat Genet 2000;24:109–110.[CrossRef][Medline]

  7. Ogonuki N, Inoue K, Yamamoto Y et al. Early death of mice cloned from somatic cells. Nat Genet 2002;30:253–254.[CrossRef][Medline]

  8. Tamashiro KL, Wakayama T, Akutsu H et al. Cloned mice have an obese phenotype not transmitted to their offspring. Nat Med 2002;8:262–267.[CrossRef][Medline]

  9. Morgan HD, Sutherland HG, Martin DI et al. Epigenetic inheritance at the agouti locus in the mouse. Nat Genet 1999;23:314–318.[CrossRef][Medline]

  10. Rakyan VK, Chong S, Champ ME et al. Transgenerational inheritance of epigenetic states at the murine Axin(Fu) allele occurs after maternal and paternal transmission. Proc Natl Acad Sci U S A 2003;100:2538–2543.[Abstract/Free Full Text]

  11. Waterland RA, Jirtle RL. Transposable elements: targets for early nutritional effects on epigenetic gene regulation. Mol Cell Biol 2003;23:5293–5300.[Abstract/Free Full Text]

  12. Hadi T, Hammer MA, Algire C et al. Similar effects of osmolarity, glucose, and phosphate on cleavage past the 2-cell stage in mouse embryos from outbred and F1 hybrid females. Biol Reprod 2005;72:179–187.[Abstract/Free Full Text]

  13. Gajewski CD, Yang L, Schon EA et al. New insights into the bioenergetics of mitochondrial disorders using intracellular ATP reporters. Mol Biol Cell 2003;14:3628–3635.[Abstract/Free Full Text]

  14. Gao S, Latham KE. Maternal and environmental factors in early cloned embryo development. Cytogenet Genome Res 2004;105:279–284.[CrossRef][Medline]

  15. Hardy K, Spanos S, Becker D et al. From cell death to embryo arrest: mathematical models of human preimplantation embryo development. Proc Natl Acad Sci U S A 2001;98:1655–1660.[Abstract/Free Full Text]

  16. Szollosi D, Czolowska R, Soltynska MS et al. Remodelling of thymocyte nuclei in activated mouse oocytes: an ultrastructural study. Eur J Cell Biol 1986;42:140–151.[Medline]

  17. Czolowska R, Modlinski JA, Tarkowski AK. Behaviour of thymocyte nuclei in non-activated and activated mouse oocytes. J Cell Sci 1984;69:19–34.[Abstract]

  18. Dyban AP, Lee K, O’Neill GT et al. Cytogenetic study of silver-staining NOR in 8-cell-stage mouse blastomeres fused to 1-cell-stage embryos. Development 1988;104:453–463.[Abstract/Free Full Text]

  19. Bordignon V, Clarke HJ, Smith LC. Factors controlling the loss of immunoreactive somatic histone H1 from blastomere nuclei in oocyte cytoplasm: a potential marker of nuclear reprogramming. Dev Biol 2001;233:192–203.[CrossRef][Medline]

  20. Gao S, Chung YG, Parseghian MH et al. Rapid H1 linker histone transitions following fertilization or somatic cell nuclear transfer: evidence for a uniform developmental program in mice. Dev Biol 2004;266:62–75.[CrossRef][Medline]

  21. Szabo PE, Hubner K, Schöler H et al. Allele-specific expression of imprinted genes in mouse migratory primordial germ cells. Mech Dev 2002;115:157–160.[CrossRef][Medline]

  22. Boiani M, Kehler J, Schöler HR. Activity of the germline-specific Oct4-GFP transgene in normal and clone mouse embryos. Methods Mol Biol 2004;254:1–34.[Medline]

  23. Boiani M, Gambles V, Schöler H. ATP levels in clone mouse embryos. Cytogenet Genome Res 2004;105:270–278.[CrossRef]

  24. Nagy A, Gertsenstein M, Vintersten K et al. Manipulating the mouse embryo, 3rd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 2002:164.

  25. Chatot CL, Ziomek CA, Bavister BD et al. An improved culture medium supports development of random-bred 1-cell mouse embryos in vitro. J Reprod Fertil 1989;86:679–688.

  26. Barnes FL, Crombie A, Gardner DK et al. Blastocyst development and birth after in-vitro maturation of human primary oocytes, intracytoplasmic sperm injection and assisted hatching. Hum Reprod 1995;10:3243–3247.[Abstract/Free Full Text]

  27. Abbondanzo SJ, Gadi I, Stewart CL. Derivation of embryonic stem cell lines. Methods Enzymol 1993;225:803–823.[Medline]

  28. Hartshorn C, Rice JE, Wangh LJ. Developmentally-regulated changes of Xist RNA levels in single preimplantation mouse embryos, as revealed by quantitative real-time PCR. Mol Reprod Dev 2002;61:425–436.[CrossRef][Medline]

  29. Hartshorn C, Rice JE, Wangh LJ. Differential pattern of Xist RNA accumulation in single blastomeres isolated from 8-cell stage mouse embryos following laser zona drilling. Mol Reprod Dev 2003;64:41–51.[CrossRef][Medline]

  30. Steuerwald N, Cohen J, Herrera RJ et al. Analysis of gene expression in single oocytes and embryos by real-time rapid cycle fluorescence monitored RT-PCR. Mol Hum Reprod 1999;5:1034–1039.[Abstract/Free Full Text]

  31. Steuerwald N, Cohen J, Herrera RJ et al. Quantification of mRNA in single oocytes and embryos by real-time rapid cycle fluorescence monitored RT-PCR. Mol Hum Reprod 2000;6:448–453.[Abstract/Free Full Text]

  32. Bortvin A, Eggan K, Skaletsky H et al. Incomplete reactivation of Oct4-related genes in mouse embryos cloned from somatic nuclei. Development 2003;130:1673–1680.[Abstract/Free Full Text]

  33. Greenlee AR, Krisher RL, Plotka ED. Rapid sexing of murine preim-plantation embryos using a nested, multiplex polymerase chain reaction (PCR). Mol Reprod Dev 1998;49:261–267.[CrossRef][Medline]

  34. Bean CJ, Hassold TJ, Judis L et al. Fertilization in vitro increases non-disjunction during early cleavage divisions in a mouse model system. Hum Reprod 2002;17:2362–2367.[Abstract/Free Full Text]

  35. Bean CJ, Hunt PA, Millie EA et al. Analysis of a malsegregating mouse Y chromosome: evidence that the earliest cleavage divisions of the mammalian embryo are non-disjunction-prone. Hum Mol Genet 2001;10:963–972.[Abstract/Free Full Text]

  36. Simerly C, Dominko T, Navara C et al. Molecular correlates of primate nuclear transfer failures. Science 2003;300:297.[Free Full Text]

  37. Zuccotti M, Boiani M, Garagna S et al. Analysis of aneuploidy rate in antral and ovulated mouse oocytes during female aging. Mol Reprod Dev 1998;50:305–312.[CrossRef][Medline]

  38. Boiani M, Eckardt S, Leu AN et al. Pluripotency deficit in mouse clones overcome by clone-clone aggregation: epigenetic complementation? EMBO J 2003;22:5304–5312.[CrossRef][Medline]

  39. Handyside AH, Hunter S. A rapid procedure for visualising the inner cell mass and trophectoderm nuclei of mouse blastocysts in situ using poly-nucleotide-specific fluorochromes. J Exp Zool 1984;231:429–434.[CrossRef][Medline]

  40. Boiani M, Eckardt S, Schöler HR et al. Oct4 distribution and level in mouse clones: consequences for pluripotency. Genes Dev 2002;16:1209–1219.[Abstract/Free Full Text]

  41. Kishikawa H, Wakayama T, Yanagimachi R. Comparison of oocyte-activating agents for mouse cloning. Cloning 1999;1:153–159.

  42. Wakayama T, Yanagimachi R. Effect of cytokinesis inhibitors, DMSO and the timing of oocyte activation on mouse cloning using cumulus cell nuclei. Reproduction 2001;122:49–60.[Abstract]

  43. Sapienza C, Paquette J, Tran TH et al. Epigenetic and genetic factors affect transgene methylation imprinting. Development 1989;107:165–168.[Abstract]

  44. Gao S, Czirr E, Chung YG et al. Genetic variation in oocyte phenotype revealed through parthenogenesis and cloning: correlation with differences in pronuclear epigenetic modification. Biol Reprod 2004;70:1162–1170.[Abstract/Free Full Text]

  45. Leppens-Luisier G, Sakkas D. Development, glycolytic activity, and viability of preimplantation mouse embryos subjected to different periods of glucose starvation. Biol Reprod 1997;56:589–596.[Abstract]

  46. Scott L, Whittingham DG. Role of facilitative glucose uptake in the glucose-inorganic phosphate-mediated retardation and inhibition of development in different strains of mouse embryos. Reproduction 2002;123:691–700.

  47. Nichols J, Zevnik B, Anastassiadis K et al. Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 1998;95:379–391.[CrossRef][Medline]

  48. Williams JW, Hawes SM, Patel B et al. Trophectoderm-specific expression of the X-linked Bex1/Rex3 gene in preimplantation stage mouse embryos. Mol Reprod Dev 2002;61:281–287.[CrossRef][Medline]

  49. Mann MR, Chung YG, Nolen LD et al. Disruption of imprinted gene methylation and expression in cloned preimplantation stage mouse embryos. Biol Reprod 2003;69:902–914.[Abstract/Free Full Text]

  50. Gentile L, Monti M, Sebastiano V et al. Single-cell quantitative RT-PCR analysis of Cpt1b and Cpt2 gene expression in mouse antral oocytes and in preimplantation embryos. Cytogenet Genome Res 2004;105:215–221.[CrossRef][Medline]

  51. Latham KE, Patel B, Bautista FD et al. Effects of X chromosome number and parental origin on X-linked gene expression in preimplantation mouse embryos. Biol Reprod 2000;63:64–73.[Abstract/Free Full Text]

  52. Zeng F, Baldwin DA, Schultz RM. Transcript profiling during preimplantation mouse development. Dev Biol 2004;272:483–496.[CrossRef][Medline]

  53. Niwa H, Miyazaki J, Smith AG. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000;24:372–376.[CrossRef][Medline]

  54. Hansis C, Grifo JA, Krey LC. Oct-4 expression in inner cell mass and trophectoderm of human blastocysts. Mol Hum Reprod 2000;6:999–1004.[Abstract/Free Full Text]

  55. Latham KE, Garrels JI, Chang C et al. Quantitative analysis of protein synthesis in mouse embryos. I. Extensive reprogramming at the one- and two-cell stages. Development 1991;112:921–932.[Abstract]

  56. Ma J, Svoboda P, Schultz RM et al. Regulation of zygotic gene activation in the preimplantation mouse embryo: global activation and repression of gene expression. Biol Reprod 2001;64:1713–1721.[Abstract/Free Full Text]

  57. Ko MS, Kitchen JR, Wang X et al. Large-scale cDNA analysis reveals phased gene expression patterns during preimplantation mouse development. Development 2000;127:1737–1749.[Abstract]

  58. Sharov AA, Piao Y, Matoba R et al. Transcriptome analysis of mouse stem cells and early embryos. PLoS Biol 2003;1:E74.[Medline]

  59. Ho Y, Wigglesworth K, Eppig JJ et al. Preimplantation development of mouse embryos in KSOM: augmentation by amino acids and analysis of gene expression. Mol Reprod Dev 1995;41:232–238.[CrossRef][Medline]

  60. Hamatani T, Carter MG, Sharov AA et al. Dynamics of global gene expression changes during mouse preimplantation development. Dev Cell 2004;6:117–131.[CrossRef][Medline]

  61. Rinaudo P, Schultz RM. Effects of embryo culture on global pattern of gene expression in preimplantation mouse embryos. Reproduction 2004;128:301–311.[Abstract/Free Full Text]

  62. Pain D, Chirn GW, Strassel C et al. Multiple retropseudogenes from pluripotent cell-specific gene expression indicates a potential signature for novel gene identification. J Biol Chem 2005;280:6265–6268.[Abstract/Free Full Text]

  63. Yoshimizu T, Sugiyama N, De Felice M et al. Germline-specific expression of the Oct-4/green fluorescent protein (GFP) transgene in mice. Dev Growth Differ 1999;41:675–684.[CrossRef][Medline]

  64. Wakayama T, Yanagimachi R. Mouse cloning with nucleus donor cells of different age and type. Mol Reprod Dev 2001;58:376–383.[CrossRef][Medline]

  65. Doherty AS, Mann MR, Tremblay KD et al. Differential effects of culture on imprinted H19 expression in the preimplantation mouse embryo. Biol Reprod 2000;62:1526–1535.[Abstract/Free Full Text]

  66. Chung YG, Mann MR, Bartolomei MS et al. Nuclear-cytoplasmic "tug of war" during cloning: effects of somatic cell nuclei on culture medium preferences of preimplantation cloned mouse embryos. Biol Reprod 2002;66:1178–1184.[Abstract/Free Full Text]

  67. Wakayama T, Perry AC, Zuccotti M et al. Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 1998;394:369–374.[CrossRef][Medline]

  68. Tada M, Takahama Y, Abe K et al. Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells. Curr Biol 2001;11:1553–1558.[CrossRef][Medline]

  69. Do JT, Schöler HR. Nuclei of embryonic stem cells reprogram somatic cells. STEM CELLS 2004;22:941–949.[Abstract/Free Full Text]

  70. Lusser A, Kadonaga JT. Chromatin remodeling by ATP-dependent molecular machines. Bioessays 2003;25:1192–1200.[CrossRef][Medline]

  71. Zwaka TP, Thomson JA. A germ cell origin of embryonic stem cells? Development 2005;132:227–233.[Abstract/Free Full Text]

  72. Blelloch RH, Hochedlinger K, Yamada Y et al. Inaugural article: nuclear cloning of embryonal carcinoma cells. Proc Natl Acad Sci U S A 2004;101:13985–13990.[Abstract/Free Full Text]

  73. Humpherys D, Eggan K, Akutsu H et al. Epigenetic instability in ES cells and cloned mice. Science 2001;293:95–97.[Abstract/Free Full Text]

  74. Hochedlinger K, Jaenisch R. Monoclonal mice generated by nuclear transfer from mature B and T donor cells. Nature 2002;415:1035–1038.[CrossRef][Medline]

  75. Li J, Ishii T, Feinstein P et al. Odorant receptor gene choice is reset by nuclear transfer from mouse olfactory sensory neurons. Nature 2004;428:393–399.[CrossRef][Medline]

  76. Kehler J, Tolkunova E, Koschorz B et al. Oct4 is required for primordial germ cell survival. EMBO Rep 2004;5:1078–1083.[CrossRef][Medline]

  77. Pickard B, Dean W, Engemann S et al. Epigenetic targeting in the mouse zygote marks DNA for later methylation: a mechanism for maternal effects in development. Mech Dev 2001;103:35–47.[CrossRef][Medline]

  78. Morin-Kensicki EM, Faust C, LaMantia C et al. Cell and tissue requirements for the gene eed during mouse gastrulation and organogenesis. Genesis 2001;31:142–146.[CrossRef][Medline]

  79. Wilson V, Rashbass P, Beddington RS. Chimeric analysis of T (Brachyury) gene function. Development 1993;117:1321–1331.[Abstract]

  80. Allen ND, Barton SC, Hilton K et al. A functional analysis of imprinting in parthenogenetic embryonic stem cells. Development 1994;120:1473–1482.[Abstract]

  81. Mann JR, Gadi I, Harbison ML et al. Androgenetic mouse embryonic stem cells are pluripotent and cause skeletal defects in chimeras: implications for genetic imprinting. Cell 1990;62:251–260.[CrossRef][Medline]

  82. Parisi T, Beck AR, Rougier N et al. Cyclins E1 and E2 are required for endoreplication in placental trophoblast giant cells. EMBO J 2003;22:4794–4803.[CrossRef][Medline]

  83. Wu L, de Bruin A, Saavedra HI et al. Extra-embryonic function of Rb is essential for embryonic development and viability. Nature 2003;421:942–947.[CrossRef][Medline]

  84. Mann MR, Lee SS, Doherty AS et al. Selective loss of imprinting in the placenta following preimplantation development in culture. Development 2004;131:3727–3735.[Abstract/Free Full Text]

  85. Ogura A, Inoue K, Ogonuki N et al. Phenotypic effects of somatic cell cloning in the mouse. Cloning Stem Cells 2002;4:397–405.[CrossRef][Medline]

  86. Brook FA, Gardner RL. The origin and efficient derivation of embryonic stem cells in the mouse. Proc Natl Acad Sci U S A 1997;94:5709–5712.[Abstract/Free Full Text]

  87. Chung YG, Ratnam S, Chaillet JR et al. Abnormal regulation of DNA methyltransferase expression in cloned mouse embryos. Biol Reprod 2003;69:146–153.[Abstract/Free Full Text]

  88. Hattori N, Nishino K, Ko YG et al. Epigenetic control of mouse Oct-4 gene expression in embryonic stem cells and trophoblast stem cells. J Biol Chem 2004;279:17063–17069.[Abstract/Free Full Text]

  89. Ding S, Schultz PG. A role for chemistry in stem cell biology. Nat Biotechnol 2004;22:833–840.[CrossRef][Medline]

  90. Rosania GR, Chang YT, Perez O etal. Myoseverin, a microtubule-binding molecule with novel cellular effects. Nat Biotechnol 2000;18:304–308.[CrossRef][Medline]

  91. Chen S, Zhang Q, Wu X et al. Dedifferentiation of lineage-committed cells by a small molecule. J Am Chem Soc 2004;126:410–411.[CrossRef][Medline]

  92. Shen X, Xiao H, Ranallo R et al. Modulation of ATP-dependent chromatin-remodeling complexes by inositol polyphosphates. Science 2003;299:112–114.[Abstract/Free Full Text]

  93. Steger DJ, Haswell ES, Miller AL et al. Regulation of chromatin remodeling by inositol polyphosphates. Science 2003;299:114–116.[Abstract/Free Full Text]

  94. Higgins BD, Kane MT. Inositol transport in mouse oocytes and preimplantation embryos: effects of mouse strain, embryo stage, sodium and the hexose transport inhibitor, phloridzin. Reproduction 2003;125:111–118.[Abstract]

  95. Burnside AS, Collas P. Induction of Oct-3/4 expression in somatic cells by gap junction-mediated cAMP signaling from blastomeres. Eur J Cell Biol 2002;81:585–591.[CrossRef][Medline]

  96. Eckardt S, Leu NA, Kurosaka S et al. Differential reprogramming of somatic cell nuclei after transfer into mouse cleavage stage blastomeres. Reproduction 2005;129:547–556.[Abstract/Free Full Text]

Received on December 10, 2004; accepted for publication on April 20, 2005.




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