Generation of Primordial Germ Cell-like Cells from iPSCs Derived from Turner Syndrome Patients.

Turner syndrome (TS) is a genetic disorder in females with X Chromosome monosomy associated with highly variable clinical features, including premature primary gonadal failure leading to ovarian dysfunction and infertility. The mechanism of development of primordial germ cells (PGCs) and their connection with ovarian failure in TS is poorly understood. An in vitro model of PGCs from TS would be beneficial for investigating genetic and epigenetic factors that influence germ cell specification. Here we investigated the potential of reprogramming peripheral mononuclear blood cells from TS women (PBMCs-TS) into iPSCs following in vitro differentiation in hPGCLCs. All hiPSCs-TS lines demonstrated pluripotency state and were capable of differentiation into three embryonic layers (ectoderm, endoderm, and mesoderm). The PGCLCs-TS recapitulated the initial germline development period regarding transcripts and protein marks, including the epigenetic profile. Overall, our results highlighted the feasibility of producing in vitro models to help the understanding of the mechanisms associated with germ cell formation in TS.

Overcoming bioprocess bottlenecks in the large-scale expansion of high-quality hiPSC aggregates in vertical-wheel stirred suspension bioreactors.

BACKGROUND: Human induced pluripotent stem cells (hiPSCs) hold enormous promise in accelerating breakthroughs in understanding human development, drug screening, disease modeling, and cell and gene therapies. Their potential, however, has been bottlenecked in a mostly laboratory setting due to bioprocess challenges in the scale-up of large quantities of high-quality cells for clinical and manufacturing purposes. While several studies have investigated the production of hiPSCs in bioreactors, the use of conventional horizontal-impeller, paddle, and rocking-wave mixing mechanisms have demonstrated unfavorable hydrodynamic environments for hiPSC growth and quality maintenance. This study focused on using computational fluid dynamics (CFD) modeling to aid in characterizing and optimizing the use of vertical-wheel bioreactors for hiPSC production. METHODS: The vertical-wheel bioreactor was modeled with CFD simulation software Fluent at agitation rates between 20 and 100 rpm. These models produced fluid flow patterns that mapped out a hydrodynamic environment to guide in the development of hiPSC inoculation and in-vessel aggregate dissociation protocols. The effect of single-cell inoculation on aggregate formation and growth was tested at select CFD-modeled agitation rates and feeding regimes in the vertical-wheel bioreactor. An in-vessel dissociation protocol was developed through the testing of various proteolytic enzymes and agitation exposure times. RESULTS: CFD modeling demonstrated the unique flow pattern and homogeneous distribution of hydrodynamic forces produced in the vertical-wheel bioreactor, making it the opportune environment for systematic bioprocess optimization of hiPSC expansion. We developed a scalable, single-cell inoculation protocol for the culture of hiPSCs as aggregates in vertical-wheel bioreactors, achieving over 30-fold expansion in 6 days without sacrificing cell quality. We have also provided the first published protocol for in-vessel hiPSC aggregate dissociation, permitting the entire bioreactor volume to be harvested into single cells for serial passaging into larger scale reactors. Importantly, the cells harvested and re-inoculated into scaled-up vertical-wheel bioreactors not only maintained consistent growth kinetics, they maintained a normal karyotype and pluripotent characterization and function. CONCLUSIONS: Taken together, these protocols provide a feasible solution for the culture of high-quality hiPSCs at a clinical and manufacturing scale by overcoming some of the major documented bioprocess bottlenecks.

Routine Karyotyping Reveals Frequent Mosaic Reciprocal Chromosome Translocations in Swine: Prevalence, Pedigree, and Litter Size.

In the routine commercial karyotype analysis on 5,481 boars, we identified 32 carriers of mosaic reciprocal translocations, half of which were carrying a specific recurrent translocation, mos t(7;9). An additional 7 mosaic translocations were identified through lymphocyte karyotype analysis from parents and relatives of mosaic carriers (n = 45), a control group of non-carrier boars (n = 73), and a mitogen assessment study (n = 20), bringing the total number of mosaic carriers to 39 cases. Mosaic translocations in all carriers were recognized to be confined to hematopoietic cells as no translocations were identified in fibroblasts cells of the carriers. In addition, negative impact on reproduction was not observed as the fertility of the carriers and their relatives were comparable to breed averages, and cryptic mosaicism was not detected in the family tree. This paper presents the first study of mosaic reciprocal translocations identified in swine through routine screening practices on reproductively unproven breeding boars while presenting evidence that these type of chromosome abnormalities are not associated with any affected phenotype on the carrier animals. In addition, the detection of recurrent mosaic translocations in this study may emphasize the non-random nature of mosaic rearrangements in swine and the potential role of genomic elements in their formation.

Optimized serial expansion of human induced pluripotent stem cells using low-density inoculation to generate clinically relevant quantities in vertical-wheel bioreactors.

Human induced pluripotent stem cells (hiPSCs) have generated a great deal of attention owing to their capacity for self-renewal and differentiation into the three germ layers of the body. Their discovery has facilitated a new era in biomedicine for understanding human development, drug screening, disease modeling, and cell therapy while reducing ethical issues and risks of immune rejection associated with traditional embryonic stem cells. Bioreactor-based processes have been the method of choice for the efficient expansion and differentiation of stem cells in controlled environments. Current protocols for the expansion of hiPSCs use horizontal impeller, paddle, or rocking wave mixing method bioreactors which require large static cell culture starting populations and achieve only moderate cell fold increases. This study focused on optimizing inoculation, agitation, oxygen, and nutrient availability for the culture of hiPSCs as aggregates in single-use, low-shear, vertical-wheel bioreactors. Under optimized conditions, we achieved an expansion of more than 30-fold in 6 days using a small starting population of cells and minimal media resources throughout. Importantly, we showed that that this optimized bioreactor expansion protocol could be replicated over four serial passages resulting in a cumulative cell expansion of 1.06E6-fold in 28 days. Cells from the final day of the serial passage were of high quality, maintaining a normal karyotype, pluripotent marker staining, and the ability to form teratomas in vivo. These findings demonstrate that a vertical-wheel bioreactor-based bioprocess can provide optimal conditions for efficient, rapid generation of high-quality hiPSCs to meet the demands for clinical manufacturing of therapeutic cell products.

Androgen Receptor Gene Variants in New Cases of Equine Androgen Insensitivity Syndrome.

In the domestic horse; failure of normal masculinization and virilization due to deficiency of androgenic action leads to a specific disorder of sexual development known as equine androgen insensitivity syndrome (AIS). Affected individuals appear to demonstrate an incoherency between their genetic sex and sexual phenotype; i.e., XY-sex chromosome constitution and female phenotypic appearance. AIS is well documented in humans. Here we report the finding of two novel genetic variants for the AR-gene identified in a Tennessee Walking Horse and a Thoroughbred horse mare; each in individual clinical cases of horse AIS syndrome.

Non-Random Distribution of Reciprocal Translocation Breakpoints in the Pig Genome.

Balanced chromosome rearrangements are one of the main etiological factors contributing to hypoprolificacy in the domestic pig. Amongst domestic animals, the pig is considered to have the highest prevalence of chromosome rearrangements. To date over 200 unique chromosome rearrangements have been identified. The factors predisposing pigs to chromosome rearrangements, however, remain poorly understood. Nevertheless, here we provide empirical evidence which sustains the notion that there is a non-random distribution of chromosomal rearrangement breakpoints in the pig genome. We sought to establish if there are structural chromosome factors near which rearrangement breakpoints preferentially occur. The distribution of rearrangement breakpoints was analyzed across three level, chromosomes, chromosome arms, and cytogenetic GTG-bands (G-banding using trypsin and giemsa). The frequency of illegitimate exchanges (e.g., reciprocal translocations) between individual chromosomes and chromosome arms appeared to be independent of chromosome length and centromere position. Meanwhile chromosome breakpoints were overrepresented on some specific G-bands, defining chromosome hotspots for ectopic exchanges. Cytogenetic band level factors, such as the length of bands, chromatin density, and presence of fragile sites, were associated with the presence of translocation breakpoints. The characteristics of these bands were largely similar to that of hotspots in the human genome. Therefore, those hotspots are proposed as a starting point for future molecular analyses into the genomic landscape of porcine chromosome rearrangements.

Meiotic Synapsis and Gene Expression Altered by a Balanced Y-Autosome Reciprocal Translocation in an Azoospermic Pig.

Meiotic sex chromosome silencing (MSCS) has been argued as a prerequisite for normal meiotic cell division progression during the synaptic prophase I stage. Furthermore, irregular asynapsis of autosomal axes at meiosis may be encompassing the lack of transcriptional activity normally observed for the X and Y sex chromosomes. Therefore, any chromosomal rearrangement compromising the normal mechanism of MSCS and/or the contrary, the normal meiotic transcriptional activity of autosomal chromosomes, may be observed as a meiotic and concomitant spermatogenesis arrest. Previously, we have described a Y-autosome translocation t(Y;13)(p1.3;q3.3) in an azoospermic boar. Its chromosome synapsis behavior by synaptonemal complex immunostaining and FISH analyses is documented here. Histone γH2AX protein foci appeared to be located at unsynapsed chromosomal segments (e.g., X chromosome univalents or unpaired multivalent segments), although interestingly a high proportion of primary spermatocytes showed full paired synaptonemal complex-multivalent configurations which were devoid of a γH2AX focus signal, indicating meiotic chromosome silencing. RT-qPCR analysis of testicular expression showed downregulation of 3 SSC13 genes (MLH1, SOX2, UBE2B) and upregulation of SSCY genes (ZFY, SRY). The irregularity of the normal transcription pattern in case of these genes with proven roles in the testis is in agreement with the cytological observations and could contribute to the observed phenotype.

In vivo and in vitro ageing results in accumulation of de novo copy number variations in bulls.

We have identified de novo copy number variations (CNVs) generated in bulls as they age. Blood samples from eight bulls were collected and SNP arrayed in a prospective design over 30 months allowing us to differentiate de novo CNVs from constant CNVs that are present throughout the sampling period. Quite remarkably, the total number of CNVs doubled over the 30-month period, as we observed an almost equal number of de novo and constant CNVs (107 and 111, respectively, i.e. 49% and 51%). Twice as many de novo CNVs emerged during the second half of the sampling schedule as in the first part. It suggests a dynamic generation of de novo CNVs in the bovine genome that becomes more frequent as the age of the animal progresses. In a second experiment de novo CNVs were detected through in vitro ageing of bovine fibroblasts by sampling passage #5, #15 and #25. De novo CNVs also became more frequent, but the proportion of them was only ~25% of the total number of CNVs (21 out of 85). Temporal generation of de novo CNVs resulted in increasing genome coverage. Genes and quantitative trait loci overlapping de novo CNVs were further investigated for ageing related functions.

Highly dynamic temporal changes of TSPY gene copy number in aging bulls.

The Y-chromosomal TSPY gene is one of the highest copy number mammalian protein coding gene and represents a unique biological model to study various aspects of genomic copy number variations. This study investigated the age-related copy number variability of the bovine TSPY gene, a new and unstudied aspect of the biology of TSPY that has been shown to vary among cattle breeds, individual bulls and somatic tissues. The subjects of this prospective 30-month long study were 25 Holstein bulls, sampled every six months. Real-time quantitative PCR was used to determine the relative TSPY copy number (rTSPY CN) and telomere length in the DNA samples extracted from blood. Twenty bulls showed an altered rTSPY CN after 30 months, although only 9 bulls showed a significant change (4 significant increase while 5 significant decrease, P<0.01). The sequential sampling provided the flow of rTSPY CN over six observations in 30 months and wide-spread variation of rTSPY CN was detected. Although a clear trend of the direction of change was not identifiable, the highly dynamic changes of individual rTSPY CN in aging bulls were observed here for the first time. In summary we have observed a highly variable rTSPY CN in bulls over a short period of time. Our results suggest the importance of further long term studies of the dynamics of rTSPY CN variablility.

Androgen Insensitivity Syndrome in a Family of Warmblood Horses Caused by a 25-bp Deletion of the DNA-Binding Domain of the Androgen Receptor Gene.

Testicular feminization, an earlier term coined for describing a syndrome resulting from failure of masculinization of target organs by androgen secretions during embryo development, has been well documented not only in humans but also in the domestic horse. The pathology, actually referred to as androgen insensitivity syndrome (AIS), has been proposed to follow an X-linked recessive pattern of inheritance in some horse breeds already investigated. Affected individuals are characterized by a female phenotype but with a stallion genotype of 64,XY SRY+ constitution. We identified a Warmblood horse pedigree segregating AIS, where the molecular analyses of the androgen receptor gene in the family provided evidences that a 25-bp deletion of the DNA-binding domain is causative of this equine syndrome.

Azoospermia and Testicular Hypoplasia in a Boar Carrier of a Novel Y-Autosome Translocation.

Few sex-autosome chromosome abnormalities have been documented in domestic animal species. In humans, Y-autosome chromosome abnormalities may occur at a rate of 1/2,000 live births, whereas in the domestic pig only 2 Y-autosome reciprocal translocations have been previously described. During a routine cytogenetic screening of young boars, we identified a new Y-autosome translocation carrier, which after puberty showed semen devoid of sperm and testicular hypoplasia with spermatogenesis arrest. Whole chromosome painting by FISH analysis corroborated the reciprocal nature of the chromosomal exchanges between the Y chromosome and SSC13. The possible causes for the observed meiotic arrest of the carrier are reviewed.

Prevalence and consequences of chromosomal abnormalities in Canadian commercial swine herds.

BACKGROUND: Structural chromosome abnormalities are well known as factors that reduce fertility rate in domestic pigs. According to large-scale national cytogenetic screening programs that are implemented in France, it is estimated that new chromosome abnormalities occur at a rate of 0.5 % in fertility-unproven boars. RESULTS: This work aimed at estimating the prevalence and consequences of chromosome abnormalities in commercial swine operations in Canada. We found pig carriers at a frequency of 1.64 % (12 out of 732 boars). Carrier pigs consistently showed lower fertility values. The total number of piglets born for litters from carrier boars was between 4 and 46 % lower than the herd average. Similarly, carrier boars produced litters with a total number of piglets born alive that was between 6 and 28 % lower than the herd average. A total of 12 new structural chromosome abnormalities were identified. CONCLUSIONS: Reproductive performance is significantly reduced in sires with chromosome abnormalities. The incidence of such abnormal sires appears relatively high in populations without routine cytogenetic screening such as observed for Canada in this study. Systematic cytogenetic screening of potential breeding boars would minimise the risk of carriers of chromosome aberrations entering artificial insemination centres. This would avoid the large negative effects on productivity for the commercial sow herds and reduce the risk of transmitting abnormalities to future generations in nucleus farms.

Somatic Mosaicism in Bulls Estimated from Genome-Wide CNV Array and TSPY Gene Copy Numbers.

Somatic mosaicism has become a focus in human research due to the implications of individual genetic variability in disease. Here, we assessed somatic copy number variations (CNVs) in Holstein bulls in 2 respects. We estimated genome-wide CNVs and assayed CNVs of the TSPY gene, the most variable bovine gene from the Y chromosome. Somatic tissues (blood, lung, heart, muscle, testis, and brain) of 4 bulls were arrayed on the Illumina Bovine SNP50k chip and qPCR tested for TSPY copy numbers. Our results showed extensive copy number divergence in tissues within the same animal as well as significant copy number alterations of TSPY. We detected a mean of 31 CNVs per animal among which 14 were of germline origin, as they were constantly present in all investigated tissues of the animal, while 18 were specific to 1 tissue. Thus, 57% of the total number of detected CNVs was the result of de novo somatic events. Further, TSPY copy number was found to vary significantly among tissues as well as among the same tissue type from different animals in a wide range from 7 to 224% of the calibrator. Our study shows significant autosomal and Y-chromosomal de novo somatic CNV in bulls.

Ovarian tumour growth is characterized by mevalonate pathway gene signature in an orthotopic, syngeneic model of epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is the most lethal gynecological cancer and often is not detected until late stages when cancer cells transcoelomically metastasize to the abdomen and typically become resistant to therapy resulting in very low survival rates. We utilize an orthotopic, syngeneic mouse model to study late stage disease and have discovered that the tumor cells within the abdominal ascites are irreversibly re-programmed, with an increased tumorigenicity and resistance to apoptosis. The goal of this study was to characterize the reprogramming that occurred in the aggressive ascites-derived cells (28-2 cells) compared to the original cell line used for tumor induction (ID8 cells). Microarray experiments showed that the majority of genes upregulated in the 28-2 cells belonged to the mevalonate pathway, which is involved in cholesterol biosynthesis, protein prenylation, and activation of small GTPases. Upregulation of mevalonate appeared to be associated with the acquisition of a p53 mutation in the ascites-derived cells. Treatment with simvastatin to inhibit HMG CoA reductase, the rate limiting enzyme of this pathway, induced apoptosis in the 28-2 cell line. Rescue experiments revealed that mevalonate, but not cholesterol, could inhibit the simvastatin-mediated effects. In vivo, daily intraperitoneal simvastatin treatment significantly regressed advanced stage disease and induced death of metastatic tumor cells. These data suggest that ovarian cancer cells become reprogrammed, with genetic mutations, and upregulation of the mevalonate pathway, which facilitates the development of advanced stage disease. The use of statins to inhibit HMGCR may provide novel therapeutic opportunities for the treatment of advanced stage EOC.

Missense Mutation in the Ligand-Binding Domain of the Horse Androgen Receptor Gene in a Thoroughbred Family with Inherited 64,XY (SRY+) Disorder of Sex Development.

Disorders of sex development (DSD) have long been documented in domestic animal species including horses. However, there is only a single report of an androgen receptor (AR) mutation causative of such a DSD syndrome in a horse pedigree. Here, we present a new familial AR mutation in horses. A missense mutation (c.2042G>C) at AR exon 4 explains the segregation of the DSD in a Thoroughbred horse pedigree. The mutation, expected to affect the ligand-binding domain of the AR protein, led to complete androgen insensitivity of 64,XY SRY+, testicular DSD individuals. Additionally, the design of a PCR-RFLP technique provided an accurate molecular test for the identification of horses carrying the mutation.

Thyroid hormones alter the transcriptome of in vitro-produced bovine blastocysts.

Thyroid hormones (THs) have been shown to improve in vitro embryo production in cattle by increasing blastocyst formation rate, and the average cell number of blastocysts and by significantly decreasing apoptosis rate. To better understand those genetic aspects that may underlie enhanced early embryo development in the presence of THs, we characterized the bovine embryonic transcriptome at the blastocyst stage, and examined differential gene expression profiles using a bovine-specific microarray. We found that 1212 genes were differentially expressed in TH-treated embryos when compared with non-treated controls (>1.5-fold at P < 0.05). In addition 23 and eight genes were expressed uniquely in control and treated embryos, respectively. The expression of genes specifically associated with metabolism, mitochondrial function, cell differentiation and development were elevated. However, TH-related genes, including those encoding TH receptors and deiodinases, were not differentially expressed in treated embryos. Furthermore, the over-expression of 52 X-chromosome linked genes in treated embryos suggested a delay or escape from X-inactivation. This study highlights the significant impact of THs on differential gene expression in the early embryo; the identification of TH-responsive genes provides an insight into those regulatory pathways activated during development.

The Porcine TSPY Gene Is Tricopy but Not a Copy Number Variant.

The testis-specific protein Y-encoded (TSPY) gene is situated on the mammalian Y-chromosome and exhibits some remarkable biological characteristics. It has the highest known copy number (CN) of all protein coding genes in the human and bovine genomes (up to 74 and 200, respectively) and also shows high individual variability. Although the biological function of TSPY has not yet been elucidated, its specific expression in the testis and several identified binding domains within the protein suggests roles in male reproduction. Here we describe the porcine TSPY, as a multicopy gene with three copies located on the short arm of the Y-chromosome with no variation at three exon loci among 20 animals of normal reproductive health from four breeds of domestic pigs (Piétrain, Landrace, Duroc and Yorkshire). To further investigate the speculation that porcine TSPY is not a copy number variant, we have included five Low-fertility boars and five boars with exceptional High-fertility records. Interestingly, there was no difference between the High- and Low-fertile groups, but we detected slightly lower TSPY CN at all three exons (2.56-2.85) in both groups, as compared to normal animals, which could be attributed to technical variability or somatic mosaicism. The results are based on both relative quantitative real-time PCR (qPCR) and droplet digital PCR (ddPCR). Chromosomal localization of the porcine TSPY was done using fluorescence in situ hybridization (FISH) with gene specific PCR probes.

Copy number variations in high and low fertility breeding boars.

BACKGROUND: In this study we applied the extreme groups/selective genotyping approach for identifying copy number variations in high and low fertility breeding boars. The fertility indicator was the calculated Direct Boar Effect on litter size (DBE) that was obtained as a by-product of the national genetic evaluation for litter size (BLUP). The two groups of animals had DBE values at the upper (high fertility) and lower (low fertility) end of the distribution from a population of more than 38,000 boars. Animals from these two diverse phenotypes were genotyped with the Porcine SNP60K chip and compared by several approaches in order to prove the feasibility of our CNV analysis and to identify putative markers of fertility. RESULTS: We have identified 35 CNVRs covering 36.5 Mb or ~1.3% of the porcine genome. Among these 35 CNVRs, 14 were specific to the high fertility group, while 19 CNVRs were specific to the low fertility group which overlap with 137 QTLs of various reproductive traits. The identified 35 CNVRs encompassed 50 genes, among them 40 were specific to the low fertility group, seven to the high fertility group, while three were found in regions that were present in both groups but with opposite gain/loss status. A functional analysis of several databases revealed that the genes found in CNVRs from the low fertility group have been significantly enriched in members of the innate immune system, Toll-like receptor and RIG-I-like receptor signaling and fatty acid oxidation pathways. CONCLUSIONS: We have demonstrated that our analysis pipeline could identify putative CNV markers of fertility, especially in case of low fertility boars.

Interleukin 11 expression in the normal canine eye.

OBJECTIVES: The purpose of this study was to characterize the expression of interleukin-11 (IL-11), a cytokine with anti-inflammatory, cytoprotective, and immune-modulating characteristics, in the canine eye. PROCEDURES: Normal canine eyes were collected from clinically healthy dogs that had been euthanized for reasons unrelated to this study. The distribution of IL-11 expression in the different ocular layers was evaluated by immunofluorescence (eight eyes). Expression levels were quantified (based on fluorescence intensity) using pixel density analysis. Primary cell cultures were derived from all three corneal cell layers. IL-11 mRNA expression was assessed in these cultures using quantitative RT-PCR before and after treatment with TGF-ß1, a known inducer of IL-11 expression. IL-11 protein expression was also assessed in the media of these cells by Western blot analysis. RESULTS: IL-11 protein was detected in the corneal epithelium, keratocytes, and the corneal endothelium of the normal canine eyes examined using immunofluorescence. Baseline IL-11 mRNA expression was noted in the corneal epithelium, fibroblasts, and endothelium using quantitative RT-PCR. Treatment of canine corneal cell lines with TGF-ß1 resulted in statistically significant increases in IL-11 expression in the corneal epithelium, endothelial and fibroblast cell lines with strongest induction noted in the fibroblasts and endothelium. CONCLUSION: This is the first description of IL-11 expression in the canine eye. The protein and mRNA appear to be constitutively present throughout all layers of the cornea and are increased by TGF-ß1, a cytokine important in ocular inflammation and disease.

Effect of histone deacetylase inhibitor valproic acid treatment on donor cell growth characteristics, cell cycle arrest, apoptosis, and handmade cloned bovine embryo production efficiency.

In this study, we tested the effects of valproic acid (VPA), a known histone deacetylase inhibitor (HDACi), on the growth characteristics, apoptosis, and cell cycle stages distribution of donor cells, as well as cloning efficiency, embryo development, and histone methylation. Our results showed that treatment of donor cells with VPA (2.5 mM, 5.0 mM, 7.5 mM, or 10 mM) for 24 h resulted in altered cell proliferation, extent of apoptosis and necrosis, and cell cycle stage distribution, whereas no changes in cell viability and chromosomal complements were observed. Measurement of relative gene expression using real-time PCR of a few developmentally important genes in treated donor cells showed decreased expression of HDAC1 and increased expression of BAX (p<0.05). No change in relative expression of HDAC2 and Bcl2 was noticed. Treatment of donor cells with VPA for 24 h before electrofusion significantly (p<0.05) increased the blastocyst formation rate of somatic cell nuclear transfer (SCNT) embryos compared to the control embryos. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive nuclei in SCNT blastocysts derived from VPA-treated donor cells were significantly decreased compared to the control blastocysts (p<0.05). Immunolocalization studies revealed that the levels of histone H3 at lysine 9 (H3K9me3) were lower in VPA-treated donor cells derived cloned blastocysts than nontreated cloned embryos, and was at the level of in vitro fertilization (IVF) counterparts, although no effects of treatments were found in donor cells. Our study demonstrates that the use of VPA in SCNT has been beneficial for efficient reprogramming of donor cells. Its effect on histone methylation in cloned embryos correlates with their developmental potential and may be a useful epigenetic marker to predict the efficiency of SCNT.