Cloned Mouse Embryos Research Articles

Incomplete activation of Alyref and Gabpb1 leads to preimplantation arrest in cloned mouse embryos.

Differentiated cell nuclei can be reprogrammed after nuclear transfer (NT) to oocytes and the produced NT embryos can give rise to cloned animals. However, development of NT embryos is often hampered by recurrent reprogramming failures, including the incomplete activation of developmental genes, yet specific genes responsible for the arrest of NT embryos are not well understood. Here, we searched for developmentally important genes among the reprogramming-resistant H3K9me3-repressed genes and identified Alyref and Gabpb1 by siRNA screening. Gene knockout of Alyref and Gabpb1 by the CRISPR/Cas9 system resulted in early developmental arrest in mice. Alyref was needed for the proper formation of inner cell mass by regulating Nanog, whereas Gabpb1 deficiency led to apoptosis. The supplement of Alyref and Gabpb1 mRNA supported efficient preimplantation development of cloned embryos. Alyref and Gabpb1 were silenced in NT embryos partially because of the repressed expression of Klf16 by H3K9me3. Thus, our study shows that the H3K9me3-repressed genes contain developmentally required genes, and the incomplete activation of such genes results in preimplantation arrest of cloned embryos.

Dynamic and aberrant patterns of H3K4me3, H3K9me3, and H3K27me3 during early zygotic genome activation in cloned mouse embryos.

Somatic cell nuclear transfer (NT) is associated with aberrant changes in epigenetic reprogramming that impede the development of embryos, particularly during zygotic genome activation. Here, we characterized epigenetic patterns of H3K4me3, H3K9me3, and H3K27me3 in mouse NT embryos up to the second cell cycle (i.e. four-celled stage) during zygotic genome activation. In vivo fertilized and parthenogenetically activated (PA) embryos served as controls. In fertilized embryos, maternal and paternal pronuclei exhibited asymmetric H3K4me3, H3K9me3, and H3K27me3 modifications, with the paternal pronucleus showing delayed epigenetic modifications. Higher levels of H3K4me3 and H3K9me3 were observed in NT and PA embryos than in fertilized embryos. However, NT embryos exhibited a lower level of H3K27me3 than PA and fertilized embryos from pronuclear stage 3 to the four-celled stage. Our finding that NT embryos exhibited aberrant H3K4me3, H3K9me3, and H3K27me3 modifications in comparison with fertilized embryos during early zygotic genome activation help to unravel the epigenetic mechanisms of methylation changes in early NT reprogramming and provide an insight into the role of histone H3 in the regulation of cell plasticity during natural reproduction and somatic cell NT.

Epigenetic reprogramming in cloned mouse embryos following treatment with DNA methyltransferase and histone deacetylase inhibitors

We examined the effects of DNA methyltransferase inhibitor – RG108, and histone deacetylase inhibitor – SAHA, on the reprogramming parameters of cloned mouse embryos produced by somatic cell nuclear transfer into oocytes. The programming parameters studied included dynamics of histone reacetylation, developmental rate, DNA methylation, and transcript levels of genes, all of which are pivotal to lineage specification and blastocyst formation. At the pronuclear stage, somatic nucleus-transplanted oocytes treated with 5 µM SAHA presented higher histone acetylation at H3K9, H3K14, H4K16 and H4K12, compared to untreated clones (p < 0.05). At the morula stage, cloned embryos treated with 5 μM RG108 or 5 μM SAHA presented lower DNA methylation intensity compared to untreated clones (p < 0.05), resembling the intensity levels of fertilized embryos. However, these effects were not observed when RG108 and SAHA were used in combination. The rate of morula formation was significantly higher in cloned embryos treated with 5 µM SAHA than in untreated clones, whereas treatment with RG108 resulted in no obvious effects on morula formation rates. On the other hand, the combined treatment with RG108 and SAHA resulted in inferior rates of cloned morula formation, compared to untreated clones. At the blastocyst stage, the aberrant expression levels of key developmental genes Oct4 and Cdx2, but not Nanog, were corrected in cloned embryos by the treatment with RG108. This is similar to the intensity levels seen in fertilized embryos. The expression of Rpl7l1 gene was significantly higher in embryos treated with both RG108 and SAHA than in untreated and in control groups. In summary, the present study showed that SAHA and RG108, when applied separately, improve the rate and quality of cloned mouse embryos.

Loss of Slc38a4 imprinting is a major cause of mouse placenta hyperplasia in somatic cell nuclear transferred embryos at late gestation.

SUMMARYPlacenta hyperplasia is commonly observed in cloned animals and is believed to impede the proper development of cloned embryos. However, the mechanism underlying this phenomenon is largely unknown. Here, we show that placenta hyperplasia of cloned mouse embryos occurs in both middle and late gestation. Interestingly, restoring paternal-specific expression of an amino acid transporter Slc38a4, which loses maternal H3K27me3-dependent imprinting and becomes biallelically expressed in cloned placentae, rescues the overgrowth of cloned placentae at late gestation. Molecular analyses reveal that loss of Slc38a4 imprinting leads to over-activation of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway in cloned placentae, which is likely due to the increased amino acids transport by SLC38A4. Collectively, our study not only reveals loss of Slc38a4 imprinting is responsible for overgrowth of cloned placentae at late gestation but also suggests the underlying mechanism involves increased amino acid transport and over-activation of mTORC1.

Genome stabilization by RAD51-stimulatory compound 1 enhances efficiency of somatic cell nuclear transfer-mediated reprogramming and full-term development of cloned mouse embryos.

ObjectivesThe genetic instability and DNA damage arise during transcription factor‐mediated reprogramming of somatic cells, and its efficiency may be reduced due to abnormal chromatin remodelling. The efficiency in somatic cell nuclear transfer (SCNT)‐mediated reprogramming is also very low, and it is caused by development arrest of most reconstituted embryos.Materials and MethodsWhether the repair of genetic instability or double‐strand breaks (DSBs) during SCNT reprogramming may play an important role in embryonic development, we observed and analysed the effect of Rad 51, a key modulator of DNA damage response (DDR) in SCNT‐derived embryos.ResultsHere, we observed that the activity of Rad 51 is lower in SCNT eggs than in conventional IVF and found a significantly lower level of DSBs in SCNT embryos during reprogramming. To address this difference, supplementation with RS‐1, an activator of Rad51, during the activation of SCNT embryos can increase RAD51 expression and DSB foci and thereby increased the efficiency of SCNT reprogramming. Through subsequent single‐cell RNA‐seq analysis, we observed the reactivation of a large number of genes that were not expressed in SCNT‐2‐cell embryos by the upregulation of DDR, which may be related to overcoming the developmental block. Additionally, there may be an independent pathway involving histone demethylase that can reduce reprograming‐resistance regions.ConclusionsThis technology can contribute to the production of comparable cell sources for regenerative medicine.

Visualization of endogenous nuclear F-actin in mouse embryos reveals abnormal actin assembly after somatic cell nuclear transfer.

Actin in the nucleus, referred to as nuclear actin, is involved in a variety of nuclear events. Nuclear actin is present as a globular (G-actin) and filamentous form (F-actin), and dynamic assembly/disassembly of nuclear actin profoundly affects nuclear functions. However, it is still challenging to observe endogenous nuclear F-actin. Here, we present a condition to visualize endogenous nuclear F-actin of mouse zygotes using different fixation methods. Zygotes fixed with paraformaldehyde and treated with fluorescently conjugated phalloidin show both short and long actin filaments in their pronuclei. Short nuclear actin filaments are characteristic of phalloidin staining, rather than the consequence of severing actin filaments by the fixation process, since long nuclear actin filaments probed with the nuclear actin chromobody are not disassembled into short filaments after fixation with paraformaldehyde. Furthermore, we find that nuclear actin assembly is impaired after somatic cell nuclear transfer (SCNT), suggesting abnormal nucleoskeleton structures in SCNT embryos. Taken together, our presented method for visualizing nuclear F-actin with phalloidin can be used to observe the states of nuclear actin assembly, and revealed improper reprogramming of actin nucleoskeleton structures in cloned mouse embryos.

Dux-Mediated Corrections of Aberrant H3K9ac during 2-Cell Genome Activation Optimize Efficiency of Somatic Cell Nuclear Transfer.

Differentiated somatic cells can be reprogrammed to totipotent embryos through somatic cell nuclear transfer (SCNT) with low efficiency. The histone deacetylase inhibitor trichostatin A (TSA) has been found to improve SCNT efficiency, but the underlying mechanism remains undetermined. Here, we examined genome-wide H3K9ac during SCNT embryo development and found that aberrant H3K9ac regions resulted in reduced 2-cell genome activation. TSA treatment largely corrects aberrant acetylation in SCNT embryos with an efficiency that is dictated by the native epigenetic environment. We further identified that the overexpression of Dux greatly improves SCNT efficiency by correcting the aberrant H3K9ac signal at its target sites, ensuring appropriate 2-cell genome activation. Intriguingly, the improvement in development mediated by TSA and Kdm4b is impeded by Dux knockout in SCNT embryos. Together, our study reveals that reprogramming of H3K9ac is important for optimal SCNT efficiency and identifies Dux as a crucial transcription factor in this process.

The effect of Xenopus laevis egg extracts with/without BRG1 on the development of preimplantation cloned mouse embryos.

SummaryMuch effort has been devoted to improving the efficiency of animal cloning. The aim of this study was to investigate the effect of BRG1 contained in Xenopus egg extracts on the development of cloned mouse embryos. The results showed that mouse NIH/3T3 cells were able to express pluripotent genes after treatment with egg extracts, indicating that the egg extracts contained reprogramming factors. After co-injection of Xenopus egg extracts and single mouse cumulus cells into enucleated mouse oocytes, statistically higher pronucleus formation and development rates were observed in the egg Extract- co-injected group compared with those in the no egg extract-injected (NT) group (38-66% vs 18-34%, P&lt;0.001). Removal of BRG1 protein from Xenopus egg extracts was conducted, and the BRG1-depleted extracts were co-injected with single donor cells into recipient oocytes. The results showed that the percentages of pronucleus formation were significantly higher in both BRG1-depleted and BRG1-intact groups than that in the nuclear transfer (NT) group (94, 64% vs 50%, P&lt;0.05). Furthermore, percentages in the BRG1-depleted group were even higher than in the BRG1-intact group (94% vs 64%). More confined expression of Oct4 in the inner cell mass (ICM) was observed in the blastocyst derived from the egg extract-injected groups. However, Nanog expression was more contracted in the ICM of cloned blastocysts in the BRG1-depleted group than in the BGR1-intact group. Based on the present study, BRG1 might not play an essential role in reprogramming, but the factors enhancing pronucleus formation and development of cloned mouse embryos are contained in Xenopus egg extracts.

Kdm6a overexpression improves the development of cloned mouse embryos.

Somatic cell nuclear transfer (SCNT) is an important technique for life science research. However, most SCNT embryos fail to develop to term due to undefined reprogramming defects. Here, we show that abnormal Xi occurs in somatic cell NT blastocysts, whereas in female blastocysts derived from cumulus cell nuclear transfer, both X chromosomes were inactive. H3K27me3 removal by Kdm6a mRNA overexpression could significantly improve preimplantation development of NT embryos, and even reached a 70.2% blastocyst rate of cleaved embryos compared with the 38.5% rate of the control. H3K27me3 levels were significantly reduced in blastomeres from cloned blastocysts after overexpression of Kdm6a. qPCR indicated that rDNA transcription increased in both NT embryos and 293T cells after overexpression of Kdm6a. Our findings demonstrate that overexpression of Kdm6a improved the development of cloned mouse embryos by reducing H3K27me3 and increasing rDNA transcription.

RNAi-mediated knockdown of Parp1 does not improve the development of female cloned mouse embryos.

Somatic cell nuclear transfer is an important technique for life science research, but its efficiency is still extremely low, and most genes that are important during early development, such as X chromosome-linked genes, are not appropriately expressed during this process. Poly (ADP-ribose) polymerase (PARP) is an enzyme that transfers ADP ribose clusters to target proteins. PARP family members such as PARP1 participate in cellular signalling pathways through poly (ADP-ribosylation) (PARylation), which ultimately promotes changes in chromatin structure, gene expression, and the localization and activity of proteins that mediate signalling responses. PARP1 is associated with X chromosome inactivation (Xi). Here, we showed that abnormal Xi occurs in somatic cell nuclear transfer (NT) blastocysts, whereas in female blastocysts derived from cumulus cell nuclear transfer, both X chromosomes were inactive. Parp1 expression was higher in female NT blastocysts than that in intracytoplasmic sperm injection (ICSI) embryos but not in male NT blastocysts. After knocking down Parp1 expression, both the pre-rRNA 47S and X-inactivation-specific transcript (Xist) levels increased. Moreover, the expression of genes on the inactivated X chromosome, such as Magea6 and Msn, were also increased in the NT embryos. However, the development of Parp1si NT embryos was impaired, although total RNA sequencing showed that overall gene expression between the Parp1si NT blastocysts and the control was similar. Our findings demonstrate that increases in the expression of several genes on the X chromosome and of rRNA primary products in NT blastocysts with disrupted Parp1 expression are insufficient to rescue the impaired development of female cloned mouse embryos and could even exacerbate the associated developmental deficiencies.

Mutation of the XIST gene upregulates expression of X-linked genes but decreases the developmental rates of cloned male porcine embryos.

XIST is an X-linked, non-coding gene responsible for the cis induction of X-chromosome inactivation (XCI). Knockout of the XIST allele on an active X chromosome abolishes erroneous XCI and enhances the in vivo development of cloned mouse embryos by more than 10-fold. This study aimed to investigate whether a similar manipulation would improve cloning efficiency in pigs. A male, porcine kidney cell line containing an EGFP insert in exon 1 of the XIST gene, resulting in a knockout allele (XIST-KO), was generated by homologous recombination using transcription activator-like effector nucleases (TALENs). The expression of X-linked genes in embryos cloned from the XIST-KO kidney cells was significantly higher than in male embryos cloned from wild-type (WT) kidney cells, but remained lower than that of in vivo fertilization-produced counterparts. The XIST-KO cloned embryos also had a significantly lower blastocyst rate and a reduced full-term development rate compared to cloned WT embryos. These data suggested that while mutation of a XIST gene can partially rescue abnormal XCI, it cannot improve the developmental efficiency of cloned male porcine embryos-a deficiency that may be caused by incomplete rescue of abnormal XCI and/or by long-term drug selection of the XIST-KO nuclear donor cells, which might adversely affect the developmental efficiency of embryos created from them.

Effects of RNAi-mediated knockdown of Xist on the developmental efficiency of cloned male porcine embryos.

Xist is an X-linked gene responsible for cis induction of X chromosome inactivation. Studies have indicated that Xist is abnormally activated in the active X chromosome in cloned mouse embryos due to loss of the maternal Xist-repressing imprint following enucleation during somatic cell nuclear transfer (SCNT). Inhibition of Xist expression by injecting small interfering RNA (siRNA) has been shown to enhance the in vivo developmental efficiency of cloned male mouse embryos by more than 10-fold. The purpose of this study was to investigate whether a similar procedure can be applied to improve the cloning efficiency in pigs. We first found that Xist mRNA levels at the morula stage were aberrantly higher in pig SCNT embryos than in in vivo fertilization-derived pig embryos. Injection of a preselected effective anti-Xist siRNA into 1-cell-stage male pig SCNT embryos resulted in significant inhibition of Xist expression through the 16-cell stage. This siRNA-mediated inhibition of Xist significantly increased the total cell number per cloned blastocyst and significantly improved the birth rate of cloned healthy piglets. The present study contributes useful information on the action of Xist in the development of pig SCNT embryos and proposes a new method for enhancing the efficiency of pig cloning.

Induction of autophagy improves embryo viability in cloned mouse embryos.

Autophagy is an essential cellular mechanism that degrades cytoplasmic proteins and organelles to recycle their components. Moreover, autophagy is essential for preimplantation development in mammals. Here we show that autophagy is also important for reprogramming in somatic cell nuclear transfer (SCNT). Our data indicate that unlike fertilized oocytes, autophagy is not triggered in SCNT embryos during 6 hours of activation. Mechanistically, the inhibited autophagic induction during SCNT activation is due to the cytochalasin B (CB) caused depolymerization of actin filaments. In this study, we induced autophagy during SCNT activation by rapamycin and pp242, which could restore the expected level of autophagy and significantly enhance the development of SCNT embryos to the blastocyst stage when compared with the control (68.5% and 68.7% vs. 41.5%, P < 0.05). Furthermore, the treatment of rapamycin and pp242 accelerates active DNA demethylation indicated by the conversion of 5 mC to 5 hmC, and treatment of rapamycin improves degradation of maternal mRNA as well. Thus, our findings reveal that autophagy is important for development of SCNT embryos and inhibited autophagic induction during SCNT activation might be one of the serious causes of low efficiency of SCNT.

Morphokinetics of cloned mouse embryos treated with epigenetic drugs and blastocyst prediction.

Time-lapse monitoring of somatic cell nuclear transfer (SCNT) embryos may help to predict developmental success and increase birth and embryonic stem cells (ESC) derivation rates. Here, the development of ICSI fertilized embryos and of SCNT embryos, non-treated or treated with either psammaplin A (PsA) or vitamin C (VitC), was monitored, and the ESC derivation rates from the resulting blastocysts were determined. Blastocyst rates were similar among PsA-treated and VitC-treated SCNT embryos and ICSI embryos, but lower for non-treated SCNT embryos. ESC derivation rates were higher in treated SCNT embryos than in non-treated or ICSI embryos. Time-lapse microscopy analysis showed that non-treated SCNT embryos had a delayed development from the second division until compaction, lower number of blastomeres at compaction and longer compaction and cavitation durations compared with ICSI ones. Treatment of SCNT embryos with PsA further increased this delay whereas treatment with VitC slightly reduced it, suggesting that both treatments act through different mechanisms, not necessarily related to their epigenetic effects. Despite these differences, the time of completion of the third division, alone or combined with the duration of compaction and/or the presence of fragmentation, had a strong predictive value for blastocyst formation in all groups. In contrast, we failed to predict ESC derivation success from embryo morphokinetics. Time-lapse technology allows the selection of SCNT embryos with higher developmental potential and could help to increase cloning outcomes. Nonetheless, further studies are needed to find reliable markers for full-term development and ESC derivation success.

BIX-01294 increases pig cloning efficiency by improving epigenetic reprogramming of somatic cell nuclei.

Accumulating evidence suggests that faulty epigenetic reprogramming leads to the abnormal development of cloned embryos and results in the low success rates observed in all mammals produced through somatic cell nuclear transfer (SCNT). The aberrant methylation status of H3K9me and H3K9me2 has been reported in cloned mouse embryos. To explore the role of H3K9me2 and H3K9me in the porcine somatic cell nuclear reprogramming, BIX-01294, known as a specific inhibitor of G9A (histone-lysine methyltransferase of H3K9), was used to treat the nuclear-transferred (NT) oocytes for 14-16 h after activation. The results showed that the developmental competence of porcine SCNT embryos was significantly enhanced both in vitro (blastocyst rate 16.4% vs 23.2%, P<0.05) and in vivo (cloning rate 1.59% vs 2.96%) after 50 nm BIX-01294 treatment. BIX-01294 treatment significantly decreased the levels of H3K9me2 and H3K9me at the 2- and 4-cell stages, which are associated with embryo genetic activation, and increased the transcriptional expression of the pluripotency genes SOX2, NANOG and OCT4 in cloned blastocysts. Furthermore, the histone acetylation levels of H3K9, H4K8 and H4K12 in cloned embryos were decreased after BIX-01294 treatment. However, co-treatment of activated NT oocytes with BIX-01294 and Scriptaid rescued donor nuclear chromatin from decreased histone acetylation of H4K8 that resulted from exposure to BIX-01294 only and consequently improved the preimplantation development of SCNT embryos (blastocyst formation rates of 23.7% vs 21.5%). These results indicated that treatment with BIX-01294 enhanced the developmental competence of porcine SCNT embryos through improvements in epigenetic reprogramming and gene expression.

Improved development of somatic cell cloned mouse embryos by vitamin C and latrunculin A.

Impaired development of embryos produced by somatic cell nuclear transfer (SCNT) is mostly associated with faulty reprogramming of the somatic nucleus to a totipotent state and can be improved by treatment with epigenetic modifiers. Here we report that addition of 100 μM vitamin C (VitC) to embryo culture medium for at least 16 h post-activation significantly increases mouse blastocyst formation and, when combined with the use of latrunculin A (LatA) during micromanipulation and activation procedures, also development to term. In spite of this, no significant effects on pluripotency (OCT4 and NANOG) or nuclear reprogramming markers (H3K14 acetylation, H3K9 methylation and DNA methylation and hydroxymethylation) could be detected. The use of LatA alone significantly improved in vitro development, but not full-term development. On the other hand, the simultaneous treatment of cloned embryos with VitC and the histone deacetylase inhibitor psammaplin A (PsA), in combination with the use of LatA, resulted in cloning efficiencies equivalent to those of VitC or PsA treatments alone, and the effects on pluripotency and nuclear reprogramming markers were less evident than when only the PsA treatment was applied. These results suggest that although both epigenetic modifiers improve cloning efficiencies, possibly through different mechanisms, they do not show an additive effect when combined. Improvement of SCNT efficiency is essential for its applications in reproductive and therapeutic cloning, and identification of molecules which increase this efficiency should facilitate studies on the mechanism of nuclear reprogramming and acquisition of totipotency.

An intracytoplasmic injection of deionized bovine serum albumin immediately after somatic cell nuclear transfer enhances full-term development of cloned mouse embryos.

In mouse somatic cell nuclear transfer (SCNT), polyvinylpyrrolidone (PVP) is typically included in the nuclear donor injection medium. However, the cytotoxicity of PVP, which is injected into the cytoplasm of oocytes, has recently become a cause of concern. In the present study, we determined whether bovine serum albumin deionized with an ion-exchange resin treatment (d-BSA) was applicable to the nuclear donor injection medium in SCNT as an alternative to PVP. The results obtained showed that d-BSA introduced into the cytoplasm of an enucleated oocyte together with a donor nucleus significantly enhanced the rate of in vitro development of cloned embryos to the blastocyst stage compared with that of a conventional nuclear injection with PVP in SCNT. We also defined the enhancing effects of d-BSA on the blastocyst formation rate when d-BSA was injected into the cytoplasm of oocytes reconstructed using the fusion method with a hemagglutinating virus of Japan envelope before oocyte activation. Furthermore, immunofluorescence experiments revealed that the injected d-BSA increased the acetylation levels of histone H3 lysine 9 and histone H4 lysine 12 in cloned pronuclear (PN) and 2-cell embryos. The injection of d-BSA before oocyte activation also increased the production of cloned mouse offspring. These results suggested that intracytoplasmic injection of d-BSA into SCNT oocytes before oocyte activation was beneficial for enhancing the in vitro and in vivo development of mouse cloned embryos through epigenetic modifications to nuclear reprogramming.

Psammaplin a improves development and quality of somatic cell nuclear transfer mouse embryos.

Faulty reprogramming of the donor somatic nucleus to a totipotent embryonic state by the recipient oocyte is a major obstacle for cloning success. Accordingly, treatment of cloned embryos with epigenetic modifiers, such as histone deacetylase inhibitors (HDACi), enhances cloning efficiency. The purpose of our study was to further explore the potential effect of valproic acid (VPA), used in previous studies, and to investigate the effect of psammaplin A (PsA), a novel HDACi, on the development and quality of cloned mouse embryos. To this aim, cloned embryos were treated with 5, 10, and 20 μM PsA or 2 and 4 mM VPA for 8-9 h (before and during activation) or 16 h or 24 h (during and after activation), and their in vitro developmental potential and blastocyst quality were evaluated. Treatments with 10 μM PsA and 2 mM VPA for 16 h were selected as the most optimal, showing higher blastocyst rates and quality. These treatments had no significant effects on the expression of Nanog, Oct4, and Cdx2 or on global histone and DNA methylation levels at the blastocyst stage, but both increased global levels of histone acetylation at early developmental stages. This was correlated with a two-fold (for VPA) and four-fold (for PsA) increase in full-term development, and a 11.5-fold increase when PsA was combined with the use of latrunculin A instead of cytochalasin B. In conclusion, PsA improves mouse cloning efficiency to a higher extent than VPA.

DNA replication is an integral part of the mouse oocyte's reprogramming machinery.

Many of the structural and mechanistic requirements of oocyte-mediated nuclear reprogramming remain elusive. Previous accounts that transcriptional reprogramming of somatic nuclei in mouse zygotes may be complete in 24–36 hours, far more rapidly than in other reprogramming systems, raise the question of whether the mere exposure to the activated mouse ooplasm is sufficient to enact reprogramming in a nucleus. We therefore prevented DNA replication and cytokinesis, which ensue after nuclear transfer, in order to assess their requirement for transcriptional reprogramming of the key pluripotency genes Oct4 (Pou5f1) and Nanog in cloned mouse embryos. Using transcriptome and allele-specific analysis, we observed that hundreds of mRNAs, but not Oct4 and Nanog, became elevated in nucleus-transplanted oocytes without DNA replication. Progression through the first round of DNA replication was essential but not sufficient for transcriptional reprogramming of Oct4 and Nanog, whereas cytokinesis and thereby cell-cell interactions were dispensable for transcriptional reprogramming. Responses similar to clones also were observed in embryos produced by fertilization in vitro. Our results link the occurrence of reprogramming to a previously unappreciated requirement of oocyte-mediated nuclear reprogramming, namely DNA replication. Nuclear transfer alone affords no immediate transition from a somatic to a pluripotent gene expression pattern unless DNA replication is also in place. This study is therefore a resource to appreciate that the quest for always faster reprogramming methods may collide with a limit that is dictated by the cell cycle.

42 EFFECTS OF TRICHOSTATIN A TREATMENT ON GENE EXPRESSION OF CLONED MOUSE 2-CELL AND BLASTOCYST STAGE EMBRYOS

Trichostatin A (TSA) is a potent inhibitor of histone deacetylases and has been shown to improve cloned embryo pre-implantation and term development. We examined the effects of TSA treatment on cloned mouse embryonic gene expression using microarrays. Cloned mouse embryos were generated using long-term haematopoietic stem cells (LT-HSC) and terminally differentiated granulocytes (Gr-1) as nuclear donors, which have been shown to have significantly different cloning efficiencies (Sung et al. 2006 Nat. Gen. 38, 1323–1328). Late 2-cell and blastocyst stage cloned embryos and control, BDF1 in vivo and IVF embryos (n = 10 from each embryo type and stage, except LT-HSC blastocysts, where n = 5) were snap frozen in liquid nitrogen. Total RNA was isolated from individual embryos and amplified using the TargetAmp 2 round Aminoallyl aRNA amplification kit (Epicentre Biotechnologies, Madison, WI, USA). Amplified RNA from each embryo and a standard reference was labelled with Cy3 or Cy5 and hybridized to the mouse exonic evidence based oligonucleotide (MEEBO) microarray allowing for the interrogation of ~25 000 genes. After Loess normalization, ANOVA with false discovery rate (P &lt; 0.001) was used to identify differentially expressed (DE) genes. A subset of the DE genes was verified by RT-qPCR. These two cell types drastically differed in their potential to give rise to morula/blastocyst stage embryos: LT-HSC: 4.1% v. Gr-1: 38.9%. When treated with 10 nM TSA (Sigma, St. Louis, MO, USA) for 10 h immediately after activation, the morula/blastocyst rate increased to 66.1% for the LT-HSC cloned embryos and to 69.3% for the Gr-1 cloned embryos. At the 2-cell stage, we identified 2172 DE genes between the TSA-treated and untreated LT-HSC embryos. There were 512 DE genes between the Gr-1 and Gr-1 TSA embryos. Interestingly, the cloned embryos were more similar to the in vivo and IVF embryos after TSA treatment at the 2-cell stage, as evidenced by hierarchical clustering and the reduced number of DE genes: LT-HSC v. in vivo = 2622 genes; LT-HSC TSA v. in vivo = 473; Gr-1 v. in vivo = 1448; Gr-1 TSA v. in vivo = 312. By the blastocyst stage, the effect of TSA was considerably less pronounced with 18 and 17 DE genes between the LT-HSC/TSA and Gr-1/TSA embryos, respectively. These data indicate that TSA treatment normalizes 2-cell cloned embryo gene expression, enabling significantly more embryos to develop to the blastocyst stage. Our findings demonstrate that TSA exerted the greatest effect on the LT-HSC embryos, which were the most difficult to reprogram by SCNT.