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a Germline Development Group, Center for Animal Transgenesis and Germ Cell Research, New Bolton Center, University of Pennsylvania, Kennett Square, Pennsylvania, USA;
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" C8 G2 F, K6 R; g- V0 X% pb Laboratory of Developmental Biology, Department of Animal Biology, University of Pavia, Pavia, Italy( ^6 b8 ^+ ?) I$ Z5 O
8 C0 o% l2 i1 y" zKey Words. Embryo ? Embryonic stem cell ? Nuclear transfer ? Oct4 ? Reprogramming
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Correspondence: Michele Boiani, Ph.D., Max Planck Institute for Molecular Biomedicine, Mendelstra?e 7, D-48149 Münster, Germany. Telephone: 0049-0251-980-2864; Fax: 0049-0251-980-2992; e-mail: mboiani@mpi-muenster.mpg.de! f6 J& G& h) I6 _! }; h, _
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ABSTRACT6 H2 }5 @ o2 Z3 F" F j5 Z+ u4 o, I
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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 . Although these epigenetic changes always take place to some extent in clonal embryos, they are not always consequential in terms of gene expression . Therefore, reprogramming at the DNA template level demands phenotypic assessment through studies of gene expression and cellular function in clones.
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3 `* C( {. M, ?5 ~, ^/ hThe 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 . This phenomenon is notably evident in mouse cloning experiments using donor nuclei from somatic cells and pluripotent embryonic stem (ES) cells . After birth, Sertoli cell–cloned mice tend to die prematurely of hepatic failure or tumors and cumulus cell–cloned mice tend to grow obese , whereas ES cell–cloned mice survive in larger numbers〞provided they originate from F1 nucleus donors . 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 .
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+ q" D" G' _* ?, w7 e8 a% qAlthough 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 , and diet can alter the methylation status of the retrotransposon and cause the phenotype change . 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 . In somatic cells, cytoplasmic compartments and pools of molecules are able to reallocate in response to changes in the culture medium composition . 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 . Environmental cues seem to merely modulate the developmental competence of already established zygotes , 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 , albeit not to the extent observed when donor nuclei are exposed to metaphase II ooplasms . 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.7 |& O! Z* U. F1 Y: [8 k2 U
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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.# |8 _4 n- V _7 g; _" _
, u2 g S6 p, @; E+ e1 k5 a% `+ `( lMATERIALS AND METHODS
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+ t$ ^ z0 E. R) P+ S3 ]- {& y- kQuality Control for Cumulus Cell Nuclear Transfer4 n4 h2 V, w: f; o
$ D! s# v. n( M+ zIn principle, the differences observed in clones are possible consequences of either manipulation or faulty reprogramming, although Wakayama and colleagues 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 . 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. 1). 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 .
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Culture Conditions Affect the Rate of Blastocyst Formation of Genetically Identical Mouse Clones% P- a& q A4 }# |, d% n
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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.$ x. t% \, c4 u
' h9 x8 s2 }5 V1 M+ rAt 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. 2A for absolute rates). The rate of clonal blastocyst formation progressively increased (multiple t-tests, p
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0 C( `5 r8 Z/ f( o! \" S! B9 TFigure 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 -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 -MEM, respectively). Error bars indicate the SDs. Abbreviations: DMEM, Dulbecco’s modified Eagle’s medium; MEM, minimal essential medium.
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3 v& o. c* Y+ z: |& \; s) tCulture Conditions Modulate Gene Expression in Clones Differently Than in Zygotes( a c W: r4 m% {5 ~% L$ z
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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 ). 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 . Therefore, we investigated whether cumulus-cell clones cultured in different media uniformly failed to reactivate the Oct4 gene from the silent somatic state .' g9 m. d/ J1 [6 v
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The expression level of Oct4 and other genes was analyzed in individual blastocysts by real-time Q-RT-PCR as follows: Oct4 (chromosome 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 whereas Oct4 is not , and Gapdh and Cpt2 are metabolic genes that are relevant to the health status of the embryo in culture . 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 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 ./ C) s% j, W' W- o+ P6 S% r- a4 K
" k7 U N7 B" z/ ^0 IFive 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 2. 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 -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 -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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7 x4 _. F) y( DTable 2. Amounts of clonal and zygotic blastocyst mRNAs expressed by the crossover threshold (Ct) number, e$ p$ \* z% d I0 m5 V n2 X5 M8 A5 w
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Therefore, values were normalized to that of Hprt mRNA, and a clone-zygote comparison of Hprt-normalized transcript levels (Table 3) was conducted with the Ct method (ABI Prism 7.700 User Bulletin 2; PerkinElmer, Wellesley, MA, http://www.perkinelmer.com) . 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).- Q4 q( |7 A1 K8 l2 m! u$ J5 j, g
* F* _' X( g9 ?0 _7 ~: uTable 3. Use of the Ct method (PerkinElmer ABI Prism 7.700 User bulletin n.2) to calculate deviations of clonal versus zygotic blastocysts in transcripts amounts
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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 3) 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 -MEM), based on the formula r = covariance /std dev*std dev . 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 , to the tight Oct4 regulation in mouse ES cells , and to the observation that Oct4 mRNA levels are rather conserved in individual ICM cells of human blastocysts .
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Functional, But Not Spatial, Definition of the Blastocyst ICM Is Altered by Culture Conditions& `! }) E |7 N* n4 K1 Z0 b/ F
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Until recently, quantitative analysis of high-resolution two-dimensional protein gels , mRNA differential display , analysis of expressed sequence tags derived from libraries of various preimplantation stages , analysis of selected genes , and microarray techniques 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.- ?/ L" G( Y0 ^( X+ p, b- q
8 n$ }2 I5 E3 g" T) L: K7 OAlterations 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 3) 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 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.
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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 -MEM culture media (DMEM and G1/G2 did not yield enough blastocysts to compensate for losses incurred by RISH). Hybridized blastocysts were categorized 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. 3A). 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 : y. [* f; O5 I; G1 U( v
$ X) F0 {$ i: Z& k. _+ K1 O+ q: J5 w1 N0 TFigure 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), -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 -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 -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.
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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 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 -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.
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Consequence of the Culture Medium–Dependent Oct4 Gene Reprogramming
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! i: O0 d6 }4 c2 UAltered Oct4 levels affect the pluripotency of ES cells , and lack of Oct4 in the ICM negates the further development of mouse blastocysts . 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. 4, see legend for absolute rates). The normal timing of maternal-zygotic transition to Oct4 gene expression occurs in mice at the eight-cell stage . 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 -MEM (p
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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), -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 -MEM. Bar = 100 μm. Abbreviations: ES, embryonic stem; MEM, minimal essential medium.) m! E* @" L% \6 \& D/ X- h5 W' X
) r0 O* z; z/ b! q5 q2 YTo 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. 3): Half of the clones were cultured in CZB and half in -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. 5A), 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. 5A’) 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 -MEM–cultured clones (n = 33 cell lines/84 blastocysts) (p
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Altogether, these data show that the patterns of Oct4 gene expression in blastocysts cannot only be assessed as done previously 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. 5B). To determine whether clonal ES cells would be impaired under conditions that impaired fetal development from precursor blastocysts, clonal ES cells from -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"’). 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.
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DISCUSSION( o8 G# W" @4 I2 D4 s
2 A! G4 B; }6 U6 K7 K. iThat reprogramming after somatic cell nuclear transfer may indeed occur subsequent to the metaphase II oocyte stage was reported by Eckardt et al. after this manuscript was submitted.
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