G Vajta

Aarhus University, Aarhus, Central Jutland, Denmark

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Publications (185)420.53 Total impact

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    ABSTRACT: Recent studies involving a limited number of patients have indicated a correlation between aneuploidy and various morphokinetic parameters during preimplantation development. The results among different groups, however, have been inconsistent in identifying the parameters that are able to predict chromosomal abnormalities. The aim of this study was to investigate whether aneuploidy of human blastocysts was detectable by specific morphokinetic parameters in patients at increased risk of aneuploidy because of advanced maternal age, history of unsuccessful IVF treatments, or both. A longitudinal cohort study was conducted using 455 blastocysts from 138 patients. Morphokinetic features of preimplantation development were detected in a timelapse incubator. Blastocysts were subjected to trophectodermal biopsy and comprehensive chromosomal screening. Analyses were conducted by means of logistic mixed-effects models, with a subject-specific intercept. No statistical correlation between 16 commonly detected morphokinetic characteristics of in-vitro embryo development and aneuploidy was found. Results suggest that morphokinetic characteristics cannot be used to select euploid blastocysts in poor-prognosis patients regarded as candidates for pre-implantation genetic screening.
    Reproductive biomedicine online 10/2014; · 2.68 Impact Factor
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    ABSTRACT: Recent advances in embryology and related research offer considerable possibilities to accelerate genetic improvement in cattle breeding. Such progress includes optimization and standardization of laboratory embryo production (in vitro fertilization; IVF), introduction of a highly efficient method for cryopreservation (vitrification), and dramatic improvement in the efficiency of somatic cell nuclear transfer (cloning) in terms of required effort, cost and overall outcome. Handmade cloning (HMC), a simplified version of somatic cell nuclear transfer, offers the potential for relatively easy and low cost production of clones. A potentially modified method of vitrification used at a centrally located laboratory facility could result in cloned offspring that are economically competitive with elite animals produced by more traditional means. Apart from routine legal and intellectual property issues, the main obstacle that hampers rapid uptake of these technologies by the beef cattle industry is a lack of confidence from scientific and commercial sources. Once stakeholder support is increased, the combined application of these methods makes a rapid advance toward desirable traits (rapid growth, high quality beef, optimized reproductive performance) a realistic goal. The potential impact of these technologies on genetic advancement in beef cattle herds in which improvement of stock is sought, such as in northern Australia, is hard to overestimate.
    Animal Reproduction Science 08/2014; · 1.58 Impact Factor
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    ABSTRACT: In somatic cell nuclear transfer (SCNT), donor cell reprogramming is considered as a biologically important and vulnerable event. Various donor cell pre-treatments with Xenopus egg extracts can promote reprogramming. Here we investigated if the reprogramming effect of one treatment with Xenopus egg extract on donor cells was maintained for several cell passages. The extract treatment resulted in increased cell-colony formation from early passages in treated porcine fibroblasts (ExTES), and increased development of cloned embryos. Partial dedifferentiation was observed in ExTES cells, shown as a tendency towards upregulation of NANOG, c-MYC and KLF-4 and downregulation of DESMIM compared with ExTES at Passage 2. Compared with our routine SCNT, continuously increased development of cloned embryos was observed in the ExTES group, and ExTES cloned blastocysts displayed hypermethylated DNA patterns and hypermethylation of H3K4me3 and H3K27me3 in ICM compared with TE. All seven recipients became pregnant after transferral of ExTES cloned embryos and gave birth to 7-22 piglets per litter (average 12). In conclusion, our results demonstrate that one treatment of porcine fibroblasts with Xenopus egg extract can result in long-term increased ability of the cells to promote their in vitro function in subsequent SCNT. Finally these cells can also result in successful development of cloned embryos to term.
    Reproduction Fertility and Development 08/2014; 26(7):1017-31. · 2.58 Impact Factor
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    ABSTRACT: Handmade cloning (HMC) has been used to generate transgenic pigs for biomedical research. Recently, we found that parthenogenetic activation (PA) of porcine oocytes and improved HMC efficiency could be achieved by treatment with sublethal high hydrostatic pressure (HHP). However, the molecular mechanism underlying the effects of HHP treatment on embryonic development is poorly understood and so was investigated in the present study. Thus, in the present study, we undertook genome-wide gene expression analysis in HHP-treated and untreated oocytes, as well as in 4-cell and blastocyst stage embryos derived by PA or HMC. Hierarchical clustering depicted stage-specific genomic expression profiling. At the 4-cell and blastocyst stages, 103 and 163 transcripts were differentially expressed between the HMC and PA embryos, respectively (P<0.05). These transcripts are predominantly involved in regulating cellular differentiation, gene expression and cell-to-cell signalling. We found that 44 transcripts were altered by HHP treatment, with most exhibiting lower expression in HHP-treated oocytes. Genes involved in embryonic development were prominent among the transcripts affected by HHP. Two of these genes (INHBB and ME3) were further validated by quantitative reverse transcription-polymerase chain reaction. We also observed that HHP treatment activated expression of the imprinting gene DLX5 in 4-cell PA embryos. In conclusion, our genomic expression profiling data suggest that HHP alters the RNA constitution in porcine oocytes and affects the expression of imprinting genes during embryonic development.
    Reproduction Fertility and Development 03/2014; 26(3):469-84. · 2.58 Impact Factor
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    ABSTRACT: During the past decade, cryopreservation of oocytes and embryos has become one of the most exciting areas in human-assisted reproduction. Improvement of the old techniques and introduction of alternative procedures have radically increased the overall efficiency and extended applicability. In parallel, the development of other laboratory techniques (embryo culture and preimplantation diagnostics) as well as medical, social, legal and cultural demands required the rapid and widespread exploitation of the new possibilities. This review summarizes the main features of the technical development, including both achievements and controversies, and outlines the emerging assisted reproductive technique strategies based partially or predominantly on cryopreservation. Application of these new achievements may substantially modify our approach in many fields and increase the overall efficiency of infertility or fertility preservation treatments.
    Expert Review of Obstetrics &amp Gynecology 01/2014; 8(2).
  • Science translational medicine 07/2013; 5(166):1-10. · 14.41 Impact Factor
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    ABSTRACT: Technology of somatic cell nuclear transfer (SCNT) has been adapted worldwide to generate transgenic animals, although the traditional procedure relies largely on instrumental micromanipulation. In this study, we used the modified handmade cloning (HMC) established in cattle and pig to produce transgenic sheep with elevated levels of omega-3 (n-3) fatty acids. Codon-optimized nematode mfat-1 was inserted into a eukaryotic expression vector and was transferred into the genome of primary ovine fibroblast cells from a male Chinese merino sheep. Reverse transcriptase PCR, gas chromatography, and chromosome analyses were performed to select nuclear donor cells capable of converting omega-6 (n-6) into n-3 fatty acids. Blastocysts developed after 7 days of in vitro culture were surgically transplanted into the uterus of female ovine recipients of a local sheep breed in Xinjiang. For the HMC, approximately 8.9% (n = 925) of reconstructed embryos developed to the blastocyst stage. Four recipients became pregnant after 53 blastocysts were transplanted into 29 naturally cycling females, and a total of 3 live transgenic lambs were produced. Detailed analyses on one of the transgenic lambs revealed a single integration of the modified nematode mfat-1 gene at sheep chromosome 5. The transgenic sheep expressed functional n-3 fatty acid desaturase, accompanied by more than 2-folds reduction of n-6/n-3 ratio in the muscle (p<0.01) and other major organs/tissues (p<0.05). To our knowledge, this is the first report of transgenic sheep produced by the HMC. Compared to the traditional SCNT method, HMC showed an equivalent efficiency but proved cheaper and easier in operation.
    PLoS ONE 02/2013; 8(2):e55941. · 3.53 Impact Factor
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    ABSTRACT: Preimplantation genetic diagnosis and screening are widely accepted for chromosomal abnormality identification to avoid transferring embryos with genetic defects. Massively parallel sequencing (MPS) is a rapidly developing approach for genome analysis with increasing application in clinical practice. The purpose of this study was to use MPS for identification of aneuploidies and unbalanced chromosomal rearrangements after blastocyst biopsy. Trophectoderm (TE) samples of 38 blastocysts from 16 IVF cycles were subjected to analysis. Low-coverage whole genome sequencing was performed using the Illumina HiSeq2000 platform with a novel algorithm purposely created for chromosomal analysis. The efficiency of this MPS approach was estimated by comparing results obtained by Affymetrix single nucleotide polymorphism (SNP) array. Whole Genome Amplification (WGA) products of TE cells were detected by MPS with an average of 0.07X depth and 5.5% coverage of the human genome. Twenty-six of embryos (68.4%) were detected as euploid, while 6 embryos (15.8%) contained uniform aneuploidies. Four of these (10.5%) were with solely unbalanced chromosomal rearrangements, whereas the remaining 2 embryos (5.3%) showed both aneuploidies and unbalanced rearrangements. Almost all these results were confirmed by the SNP array, with the exception of one sample, where different size of unbalanced rearrangements were detected possibly due to chromosomal GC bias in array analysis. Our study demonstrated MPS could be applied to accurate detect embryonic chromosomal abnormality with a flexible and cost-effective strategy and higher potential accuracy.
    Biology of Reproduction 01/2013; · 3.45 Impact Factor
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    ABSTRACT: Lack of animal models with human-like size and pathology hampers translational research in atherosclerosis. Mouse models are missing central features of human atherosclerosis and are too small for intravascular procedures and imaging. Modeling the disease in minipigs may overcome these limitations, but it has proven difficult to induce rapid atherosclerosis in normal pigs by high-fat feeding alone, and genetically modified models similar to those created in mice are not available. D374Y gain-of-function mutations in the proprotein convertase subtilisin/kexin type 9 (PCSK9) gene cause severe autosomal dominant hypercholesterolemia and accelerates atherosclerosis in humans. Using Sleeping Beauty DNA transposition and cloning by somatic cell nuclear transfer, we created Yucatan minipigs with liver-specific expression of human D374Y-PCSK9. D374Y-PCSK9 transgenic pigs displayed reduced hepatic low-density lipoprotein (LDL) receptor levels, impaired LDL clearance, severe hypercholesterolemia, and spontaneous development of progressive atherosclerotic lesions that could be visualized by noninvasive imaging. This model should prove useful for several types of translational research in atherosclerosis.
    Science translational medicine 01/2013; 5(166):166ra1. · 14.41 Impact Factor
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    ABSTRACT: Minipigs have become important biomedical models for human ailments due to similarities in organ anatomy, physiology, and circadian rhythms relative to humans. The homeostasis of circadian rhythms in both central and peripheral tissues is pivotal for numerous biological processes. Hence, biological rhythm disorders may contribute to the onset of cancers and metabolic disorders including obesity and type II diabetes, amongst others. A tight regulation of circadian clock effectors ensures a rhythmic expression profile of output genes which, depending on cell type, constitute about 3-20% of the transcribed mammalian genome. Central to this system is the negative regulator protein Cryptochrome 1 (CRY1) of which the dysfunction or absence has been linked to the pathogenesis of rhythm disorders. In this study, we generated transgenic Bama-minipigs featuring expression of the Cys414-Ala antimorphic human Cryptochrome 1 mutant (hCRY1(AP)). Using transgenic donor fibroblasts as nuclear donors, the method of handmade cloning (HMC) was used to produce reconstructed embryos, subsequently transferred to surrogate sows. A total of 23 viable piglets were delivered. All were transgenic and seemingly healthy. However, two pigs with high transgene expression succumbed during the first two months. Molecular analyzes in epidermal fibroblasts demonstrated disturbances to the expression profile of core circadian clock genes and elevated expression of the proinflammatory cytokines IL-6 and TNF-α, known to be risk factors in cancer and metabolic disorders.
    PLoS ONE 01/2013; 8(10):e76098. · 3.53 Impact Factor
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    Cell Research 12/2012; · 11.98 Impact Factor
  • Gábor Vajta
    Reproduction Fertility and Development 08/2012; · 2.58 Impact Factor
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    ABSTRACT: A recently emerged concept utilizing a controlled environmental impact as a treatment for cells and tissues aims to improve neither the in vitro conditions nor the procedures, but the cell itself. Hydrostatic pressure stress emerged as the most controllable and most effective stressor, proving the principle that controlled stress improves cell performance in in vitro procedures, whereas further studies using different stressors (osmotic, oxidative or mechanic stresses) supported the principle. The present summary reviews studies of various stress treatments to treat oocytes of three species (murine, porcine, human) before vitrification, in vitro maturation, enucleation and somatic cell nuclear transfer. Eventually, cleavage and blastocyst rates and--in cases when hydrostatic pressure was used--blastocyst cell number and birth rates as well were significantly improved compared to untreated controls.
    Reproduction in Domestic Animals 08/2012; 47 Suppl 4:197-206. · 1.18 Impact Factor
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    ABSTRACT: Production of transgenic animals via somatic cell nuclear transfer (SCNT) has been adapted worldwide, but this application is somewhat limited by its relatively low efficiency. In this study, we used handmade cloning (HMC) established previously to produce transgenic pigs that express the functional nematode fat-1 gene. Codon-optimized mfat-1 was inserted into eukaryotic expression vectors, which were transferred into primary swine donor cells. Reverse transcriptase PCR (RT-PCR), gas chromatography, and chromosome analyses were performed to select donor clones capable of converting n-6 into n-3 fatty acids. Blastocysts derived from the clones that lowered the n-6/n-3 ratio to approximately 1:1 were transferred surgically into the uteri of recipients for transgenic piglets. By HMC, 37% (n=558) of reconstructed embryos developed to the blastocyst stage after 7 days of culture in vitro, with an average cell number of 81±36 (n=14). Three recipients became pregnant after 408 day-6 blastocysts were transferred into four naturally cycling females, and a total of 14 live offspring were produced. The nematode mfat-1 effectively lowered the n-6/n-3 ratio in muscle and major organs of the transgenic pig. Our results will help to establish a reliable procedure and an efficient option in the production of transgenic animals.
    Cellular reprogramming. 06/2012; 14(3):258-66.
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    ABSTRACT: An efficient method for cryopreservation of human oocytes may offer solutions to legal and ethical problems in routine infertility programs and may also be used for fertility preservation for medical and social reasons. We conducted an observational longitudinal cohort multicentric study to investigate the efficacy and reproducibility of oocyte cryopreservation outcomes in IVF/ICSI cycles. Moreover, the effects of patient and cycle characteristics on the delivery rate (DR) were analyzed. In 486 cycles performed in 450 couples, 2721 oocytes were warmed and 2304 of them survived cryopreservation (84.7%). Of the 2182 oocytes subjected to ICSI, the rates of fertilization and development to top-quality embryos were 75.2 and 48.1%, respectively. A total of 128 deliveries were obtained (26.3% per cycle and 29.4% per transfer) for 450 patients (28.4%) and 147 babies were live born from 929 embryos transferred (15.8%). The forward logistic regression analysis on a per patient basis showed that female age [odds ratio (OR): 0.93, 95% confidence interval (CI): 0.88-0.98], number of vitrified oocytes (OR: 1.08, 95% CI: 1.01-1.17) and the day of transfer (OR: 1.97, 95% CI: 1.14-3.42) influenced DR. By recursive partitioning analysis, it can be estimated that more than eight oocytes vitrified are required to improve the outcome (22.6 versus 46.4% DR, respectively). When fewer oocytes are available in women aged >38 years, results are dramatically reduced (12.6 versus 27.5% DR, respectively). Conversely, when >8 oocytes are available, blastocyst culture represents the most efficient policy (62.1% DR; data from one center only). Oocyte vitrification is an efficient and reliable approach, with consistent results between centers and predictable DRs. It should be applied routinely for various indications. A predictive model is proposed to help patient counselling and selection.
    Human Reproduction 03/2012; 27(6):1606-12. · 4.59 Impact Factor
  • G Vajta, H Callesen
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    ABSTRACT: Handmade cloning (HMC) is now an established procedure used in several species for somatic cell nuclear transfer, but only applied in two related laboratories for pigs. The aim of this review is to facilitate widespread application by summarizing the process of establishment and explaining the background of the incorporated special approaches. Optimized steps of traditional cloning in pigs (in vitro maturation, activation, embryo culture) were merged with those of the micromanipulation-free HMC that has been modified according to the specific needs of sensitive porcine oocytes (partial zona digestion before enucleation, two-step zona-free fusion with the somatic cell; initiation of activation with the second fusion). The zona-free approach required embryo culture to the blastocyst stage before surgical transfer of embryos to the uterine horns of recipient sows in the proper phase of an unstimulated cycle. Eventually a competitive, inexpensive and reliable alternative to traditional porcine nuclear transfer cloning techniques evolved that is also suitable to produce transgenic offspring containing various genetic modifications to establish models for several human diseases with genetic background. Further improvements and involvement of additional techniques to increase the overall efficiency and facilitate practical applications are expected in the foreseeable future.
    Theriogenology 01/2012; 77(7):1263-74. · 1.85 Impact Factor
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    ABSTRACT: Viability of cloned and transgenic piglets is seriously compromised and one obvious reason could be malformations. The aim of the present study was therefore to describe gross pathological conditions in dead pre-weaned piglets born after transfer to Large White (LW) recipients of cloned (LW donor cells) or transgenic (Yucatan or Göttingen donor cells) embryos. Donor cells were fibroblasts and the Göttingen and Yucatan cells were made transgenic with 1 of 5 genes known to dispose for different human diseases. Handmade cloning was used to produce embryos that after 5 to 6 days of in vitro culture were transferred surgically to 108 LW sows 4 days after their natural heat. Of these, 21 sows delivered cloned LW piglets, whereas 17 and 16 sows, respectively, delivered transgenic Göttingen and Yucatan piglets. Stillborn and dead pre-weaned piglets were necropsied and malformations registered. Data were analysed by Fisher's exact test with a significance level of P<0.05. In the 54 litters, total litter size ranged from 1 to 22 piglets (mean 5.4±0.5) and the overall mortality rate until weaning on day 28 was 59%. Malformations were found in piglets from 38 litters where an average of 35% of the piglets showed malformations (between 8 and 100%). In those litters, 1 to 7 piglets had 1, 2, or several malformations (Table 1). The malformation rate in the autopsied transgenic Göttingen was 58% and in Yucatan 46%; these were significantly higher than in the autopsied cloned LW piglets with 18%. Some of the malformations seemed to be related to breed and/or transgene; for instance, heart malformations were most frequent in Yucatan litters (70%) independent of the transgene, whereas gallbladder and gonad malformations were more frequent in various litters with the same transgene. These results show that the use of cloning in pigs results in a considerable loss of piglets due to malformations and transgenic transformation of the cells used for cloning superimpose on this problem. In combination, these elements could seriously compromise the use of pigs as a model for human diseases and the choice of breeds and also transgenes for this kind of work should be considered carefully. However, further improvements in production of cloned/transgenic embryos may ultimately reduce the incidence of malformations.
    Reproduction Fertility and Development 12/2011; 24(1):123. · 2.58 Impact Factor
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    ABSTRACT: Extract from Xenopus eggs can induce reprogramming in somatic cells. In our previous study, cell colony formation was induced during culture of porcine fetal fibroblasts after a single treatment with Xenopus egg extract and culture for several passages and using these long-term cultured cells for cloning increased the resulting blastocyst rate (Liu et al. 2011 Reprod. Fertil. Dev. 23, 130). However, both colony number and cloned blastocyst rate decreased after Passage 15 and no colonies formed after Passage 18. Therefore, in this study we investigated the effect of a second extract treatment on colony formation and cloned blastocyst formation. Extract-treated (ExT) porcine fetal fibroblasts at Passage 13 (P13) grown on poly-L-lysine-coated coverslips were permeabilized by digitonin (7μgmL(-1), 2min, 4°C) and incubated in extract at 37°C for 30min. After resealing the membrane in DMEM supplemented with 2mM CaCl(2), the remaining cells were cultured in ES medium (Vejlsted et al. 2005 Mol. Reprod. Dev. 70, 445). The treated cells were split onto 2 coverslips on Day 7 after the second extract treatment (2ExT), defined as Passage 1 (2ExT P1, comparable with ExT P14). New subcultures were made every 7 to 8 days when 70 to 80% clusters became colonies (i.e. 2ExT P8). Colony cells from both ExT (P14 and P16) and 2ExT (P1, P3 and P6) were used for handmade cloning and nontreated cells were used as control (Day 0). Blastocyst rates were analysed by chi-square test and colony numbers were analysed by 1-way ANOVA (SAS version 9.2). Colony numbers and cloned blastocyst rates on Day 6 are summarised in Table 1. Colonies continued to form in treated cells from 2ExT P1 to P8. The colony number maintained at a high level (60 to 80) from 2ExT P4 to P8 and it was significantly higher than that of ExT cells at the comparable passage numbers. No colonies formed in control cells. When using 2ExT colony cells at P3 and P6 for cloning, the blastocyst rates increased compared with controls and they were also higher than in the ExT group. Cloned blastocyst rates were not different between 2ExT P1 and ExT P14 groups. In conclusion, a second extract treatment can induce colony formation and increase cloned blastocyst rates, indicating that this repeated extract treatment again could activate the extract-treated cells to an activity level similar to that achieved after the first treatment.
    Reproduction Fertility and Development 12/2011; 24(1):122. · 2.58 Impact Factor
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    ABSTRACT: Treatment with cytoplasmic extracts from Xenopus laevis eggs represents a potential tool for universal cellular reprogramming. However, the biochemical activity and quality of the extract vary from batch to batch. This study aimed to evaluate three different extract batches prepared by the same method based on the colony formation of cells after extract treatment, and subsequent in vitro cloning efficiency using treated cells as chromatin donors. Porcine fetal fibroblasts were treated with each batch of extract, and cultured in embryonic stem cell (ES) medium for 12 days. The number of forming colonies in treated cells was counted on Day 7 after extract treatment and significant variability was detected between different batches of extract. Similarly, when using cells from colonies at Days 7 to 8 after treatment for handmade cloning, increased blastocyst formation rates were observed after the cells were treated with a batch showing higher colony formation. In conclusion, assessment of cell colony formation may be used as selection marker for Xenopus egg extract used for pretreatment of donor cells prior to cloning.
    Cellular reprogramming. 11/2011; 13(6):521-6.
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    ABSTRACT: Animal breeding via Somatic Cell Nuclear Transfer (SCNT) has enormous potential in agriculture and biomedicine. However, concerns about whether SCNT animals are as healthy or epigenetically normal as conventionally bred ones are raised as the efficiency of cloning by SCNT is much lower than natural breeding or In-vitro fertilization (IVF). Thus, we have conducted a genome-wide gene expression and DNA methylation profiling between phenotypically normal cloned pigs and control pigs in two tissues (muscle and liver), using Affymetrix Porcine expression array as well as modified methylation-specific digital karyotyping (MMSDK) and Solexa sequencing technology. Typical tissue-specific differences with respect to both gene expression and DNA methylation were observed in muscle and liver from cloned as well as control pigs. Gene expression profiles were highly similar between cloned pigs and controls, though a small set of genes showed altered expression. Cloned pigs presented a more different pattern of DNA methylation in unique sequences in both tissues. Especially a small set of genomic sites had different DNA methylation status with a trend towards slightly increased methylation levels in cloned pigs. Molecular network analysis of the genes that contained such differential methylation loci revealed a significant network related to tissue development. In conclusion, our study showed that phenotypically normal cloned pigs were highly similar with normal breeding pigs in their gene expression, but moderate alteration in DNA methylation aspects still exists, especially in certain unique genomic regions.
    PLoS ONE 10/2011; 6(10):e25901. · 3.53 Impact Factor

Publication Stats

4k Citations
420.53 Total Impact Points


  • 2007–2014
    • Aarhus University
      • • Department of Animal Science
      • • Department of Genetics and Biotechnology
      Aarhus, Central Jutland, Denmark
  • 2013
    • Central Queensland University
      Rockhampton, Queensland, Australia
  • 2010–2012
    • Beijing Genomics Institute
      Bao'an, Guangdong, China
    • University of Copenhagen
      • Faculty of Life Sciences
      Copenhagen, Capital Region, Denmark
    • James Cook University
      Townsville, Queensland, Australia
  • 2008–2012
    • Szent István University, Godollo
      • • Department of Animal Breeding and Genetics
      • • Clinic for Large Animals
      Gödöllő, Pest megye, Hungary
    • Pivet Medical Centre
      Perth City, Western Australia, Australia
  • 2011
    • IT University of Copenhagen
      København, Capital Region, Denmark
    • BGI Human Genome Center
      Peping, Beijing, China
  • 2009
    • Reproductive Biology Associates
      Atlanta, Georgia, United States
    • Fertility Society of Australia
      Cairns, Queensland, Australia
    • Canadian Food Inspection Agency
      Fredericton, New Brunswick, Canada
  • 2004
    • Reproductive Sciences Center & Genetics Institute
      La Jolla, California, United States
  • 1999–2004
    • Monash University (Australia)
      • Centre for Reproduction and Development
      Melbourne, Victoria, Australia
  • 2002
    • Universidade do Estado de Santa Catarina
      Joinville, Santa Catarina, Brazil
  • 2001
    • Hadassah Medical Center
      • Department of Obstetrics and Gynaecology
      Jerusalem, Jerusalem District, Israel