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Heiner Niemann - One of the best experts on this subject based on the ideXlab platform.

  • Efficient Knock-in of a Point Mutation in Porcine Fibroblasts Using the CRISPR/Cas9-GMNN Fusion Gene
    MDPI AG, 2018
    Co-Authors: Max Gerlach, Björn Petersen, Heiner Niemann, Theresia Kraft, Bernhard Brenner, Judith Montag
    Abstract:

    During CRISPR/Cas9 mediated genome editing, site-specific double strand breaks are introduced and repaired either unspecific by non-homologous end joining (NHEJ) or sequence dependent by homology directed repair (HDR). Whereas NHEJ-based generation of gene knock-out is widely performed, the HDR-based knock-in of specific mutations remains a bottleneck. Especially in primary cell lines that are essential for the generation of cell culture and animal models of inherited human diseases, knock-in efficacy is insufficient and needs significant improvement. Here, we tested two different approaches to increase the knock-in frequency of a specific point mutation into the MYH7-gene in porcine fetal fibroblasts. We added a small molecule inhibitor of NHEJ, SCR7 (5,6-bis((E)-benzylideneamino)-2-mercaptopyrimidin-4-ol), during genome editing and screened cell cultures for the point mutation. However, this approach did not yield increased knock-in rates. In an alternative approach, we fused humanized Cas9 (hCas9) to the N-terminal peptide of the Geminin gene (GMNN). The fusion protein is degraded in NHEJ-dominated cell cycle phases, which should increase HDR-rates. Using hCas9-GMNN and point mutation-specific real time PCR screening, we found a two-fold increase in genome edited cell cultures. This increase of HDR by hCas9-GMNN provides a promising way to enrich specific knock-in in porcine fibroblast cultures for Somatic Cloning approaches

  • Somatic Cloning and Epigenetic Reprogramming in Mammals
    Principles of Regenerative Medicine, 2011
    Co-Authors: Heiner Niemann, Andrea Lucas-hahn, Wilfried A. Kues, Joseph Wallace Carnwath
    Abstract:

    This chapter provides a comprehensive review of the present state of Somatic cell nuclear transfer (SCNT)-based Cloning, including potential areas of application, with emphasis on the epigenetic reprogramming of the transferred Somatic cell nucleus. Common Somatic Cloning protocols involve several technical steps such as collection and enucleation of the recipient oocyte, preparation and subzonal transfer of the donor cell, fusion of the two components, activation of the reconstructed complex, temporary culture of the reconstructed embryo, and transfer to a foster mother or storage in liquid nitrogen. Basic epigenetic mechanisms include DNA methylation that plays a crucial role in suppressing the activities of parasitic promoters and is thus part of the gene-silencing system in eukaryotic cells. Methylation is associated with silencing of a given gene, but an increasing number of genes are found to be activated by methylation, particularly tumor-suppressor gene. Imprinting represents a specific function of DNA methylation. A typical feature of genomic imprinting is that the two alleles of a given gene are expressed differently. Usually one allele, either the maternal or the paternal, is silenced throughout development by covalent addition of methyl groups to cytosine residues in CpG dinucleotides. DNA demethylation is a first step in epigenetic reprogramming and is essential for Oct4 transcription. The failure of demethylation is associated with impaired development in cloned mice embryos.

  • Development and validation of a highly efficient protocol of porcine Somatic Cloning using preovulatory embryo transfer in peripubertal gilts.
    Cloning and stem cells, 2008
    Co-Authors: Björn Petersen, Andrea Lucas-hahn, Erika Lemme, N. Hornen, P. Hassel, Marianne Oropeza, A. L. Queisser, Heiner Niemann
    Abstract:

    The efficiency of porcine Somatic nuclear transfer (born piglets/transferred embryos) is low. Here, we report a highly efficient protocol using peripubertal gilts as recipients synchronized to ovulate approximately 24 h after transfer of cloned embryos. Retrospectively, we compared the efficiency of two different synchronization protocols: In group 1, recipient animals were synchronized to ovulate approximately 6 h prior to surgical embryo transfer while in group 2 the animals were treated to ovulate 24 h after embryo transfer. In total, 1562 cloned embryos were transferred to 12 recipients in group 1; two of them became pregnant (16.7%). One pregnancy was lost on day 32, the second pregnancy went to term, and led to the birth of one healthy piglet after Cesarean section. In group 2, 1531 cloned embryos were transferred to 12 recipients. Nine recipients (75.0%) became pregnant as determined by ultrasound scanning on day 25. All pregnancies went to term and delivered a total of 47 live-born piglets. The Cloning efficiency of both groups differed significantly (group 1: 0.1%, group 2: 3.1%, p < 0.05). This modified protocol was then applied in subsequent experiments using different types of transgenic and nontransgenic donor cells with similar success rates. Results show that this protocol is robust and highly reproducible, and can thus be employed for routine production of cloned pigs.

  • 76 PREOVULATORY EMBRYO TRANSFER INCREASES SUCCESS OF PORCINE Somatic Cloning
    Reproduction Fertility and Development, 2007
    Co-Authors: Björn Petersen, Andrea Lucas-hahn, Erika Lemme, N. Hornen, P. Hassel, Heiner Niemann
    Abstract:

    Somatic Cloning has emerged as a valuable tool for the production of transgenic animals. However, the technology still suffers from low efficiencies (

  • From fibroblasts and stem cells: implications for cell therapies and Somatic Cloning
    Reproduction Fertility and Development, 2005
    Co-Authors: Wilfried A. Kues, Joseph Wallace Carnwath, Heiner Niemann
    Abstract:

    Pluripotent embryonic stem cells (ESCs) from the inner cell mass of early murine and human embryos exhibit extensive self-renewal in culture and maintain their ability to differentiate into all cell lineages. These features make ESCs a suitable candidate for cell-replacement therapy. However, the use of early embryos has provoked considerable public debate based on ethical considerations. From this standpoint, stem cells derived from adult tissues are a more easily accepted alternative. Recent results suggest that adult stem cells have a broader range of potency than imagined initially. Although some claims have been called into question by the discovery that fusion between the stem cells and differentiated cells can occur spontaneously, in other cases Somatic stem cells have been induced to commit to various lineages by the extra- or intracellular environment. Recent data from our laboratory suggest that changes in culture conditions can expand a subpopulation of cells with a pluripotent phenotype from primary fibroblast cultures. The present paper critically reviews recent data on the potency of Somatic stem cells, methods to modify the potency of Somatic cells and implications for cell-based therapies.

X. Cindy Tian - One of the best experts on this subject based on the ideXlab platform.

  • Reprogramming of epigenetic inheritance by Somatic cell nuclear transfer.
    Reproductive biomedicine online, 2004
    Co-Authors: X. Cindy Tian
    Abstract:

    Somatic Cloning by nuclear transfer returns a differentiated cell to a totipotent stage, a process termed nuclear reprogramming. During this de-differentiation process, genes inactivated during tissue differentiation are re-activated in a temporal and spatial special manner. It is believed that tissue differentiation occurs through epigenetic mechanisms, genetic inheritance that does not involve changes in DNA sequences. Developmental abnormalities and a high mortality rate in cloned offspring have frequently been observed and probably result from incomplete nuclear reprogramming. In this review, the reprogramming of two epigenetic mechanisms, imprinting and X chromosome inactivation, as well as recent attempts to modify pre-existing epigenetic marks in donor cells to improve nuclear transfer efficacy, are discussed.

  • Cloning animals by Somatic cell nuclear transfer – biological factors
    Reproductive Biology and Endocrinology, 2003
    Co-Authors: X. Cindy Tian, Chikara Kubota, Brian Enright, Xiangzhong Yang
    Abstract:

    Cloning by nuclear transfer using mammalian Somatic cells has enormous potential application. However, Somatic Cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the Somatic nuclei by the Cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in Cloning cattle than any other specie, this review will be focused on Somatic cell Cloning of cattle.

  • Cloning Animals by Somatic Cell Nuclear Transfer--Biological Factors
    Reproductive biology and endocrinology : RB&E, 2003
    Co-Authors: X. Cindy Tian, Chikara Kubota, Brian P. Enright, Xiangzhong Yang
    Abstract:

    Cloning by nuclear transfer using mammalian Somatic cells has enormous potential application. However, Somatic Cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the Somatic nuclei by the Cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in Cloning cattle than any other specie, this review will be focused on Somatic cell Cloning of cattle.

Xiangzhong Yang - One of the best experts on this subject based on the ideXlab platform.

  • the cell agglutination agent phytohemagglutinin l improves the efficiency of Somatic nuclear transfer Cloning in cattle bos taurus
    Theriogenology, 2006
    Co-Authors: Perng Chih Shen, Liying Sung, Seon B Jeong, T L Nedambale, J W Riesen, Cindy X Tian, Winston T K Cheng, Shan Nan Lee, Xiangzhong Yang
    Abstract:

    One of the several factors that contribute to the low efficiency of mammalian Somatic Cloning is poor fusion between the small Somatic donor cell and the large recipient oocyte. This study was designed to test phytohemagglutinin (PHA) agglutination activity on fusion rate, and subsequent developmental potential of cloned bovine embryos. The toxicity of PHA was established by examining its effects on the development of parthenogenetic bovine oocytes treated with different doses (Experiment 1), and for different durations (Experiment 2). The effective dose and duration of PHA treatment (150 microg/mL, 20 min incubation) was selected and used to compare membrane fusion efficiency and embryo development following Somatic cell nuclear transfer (Experiment 3). Cloning with Somatic donor fibroblasts versus cumulus cells was also compared, both with and without PHA treatment (150 microg/mL, 20 min). Fusion rate of nuclear donor fibroblasts, after phytohemagglutinin treatment, was increased from 33 to 61% (P < 0.05), and from 59 to 88% (P < 0.05) with cumulus cell nuclear donors. The nuclear transfer (NT) efficiency per oocyte used was improved following PHA treatment, for both fibroblast (13% versus 22%) as well as cumulus cells (17% versus 34%; P < 0.05). The cloned embryos, both with and without PHA treatment, were subjected to vitrification and embryo transfer testing, and resulted in similar survival (approximately 90% hatching) and pregnancy rates (17-25%). Three calves were born following vitrification and embryo transfer of these embryos; two from the PHA-treated group, and one from non-PHA control group. We concluded that PHA treatment significantly improved the fusion efficiency of Somatic NT in cattle, and therefore, increased the development of cloned blastocysts. Furthermore, within a determined range of dose and duration, PHA had no detrimental effect on embryo survival post-vitrification, nor on pregnancy or calving rates following embryo transfer.

  • Cloning animals by Somatic cell nuclear transfer – biological factors
    Reproductive Biology and Endocrinology, 2003
    Co-Authors: X. Cindy Tian, Chikara Kubota, Brian Enright, Xiangzhong Yang
    Abstract:

    Cloning by nuclear transfer using mammalian Somatic cells has enormous potential application. However, Somatic Cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the Somatic nuclei by the Cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in Cloning cattle than any other specie, this review will be focused on Somatic cell Cloning of cattle.

  • Cloning Animals by Somatic Cell Nuclear Transfer--Biological Factors
    Reproductive biology and endocrinology : RB&E, 2003
    Co-Authors: X. Cindy Tian, Chikara Kubota, Brian P. Enright, Xiangzhong Yang
    Abstract:

    Cloning by nuclear transfer using mammalian Somatic cells has enormous potential application. However, Somatic Cloning has been inefficient in all species in which live clones have been produced. High abortion and fetal mortality rates are commonly observed. These developmental defects have been attributed to incomplete reprogramming of the Somatic nuclei by the Cloning process. Various strategies have been used to improve the efficiency of nuclear transfer, however, significant breakthroughs are yet to happen. In this review we will discuss studies conducted, in our laboratories and those of others, to gain a better understanding of nuclear reprogramming. Because cattle are a species widely used for nuclear transfer studies, and more laboratories have succeeded in Cloning cattle than any other specie, this review will be focused on Somatic cell Cloning of cattle.

Eckhard Wolf - One of the best experts on this subject based on the ideXlab platform.

  • Quantitative Monitoring of Pluripotency Gene Activation after Somatic Cloning in Cattle
    Biology of reproduction, 2007
    Co-Authors: Annegret Wuensch, Felix A. Habermann, Satoshi Kurosaka, Regina Klose, Valeri Zakhartchenko, Horst-dieter Reichenbach, Fred Sinowatz, K. John Mclaughlin, Eckhard Wolf
    Abstract:

    The development of Somatic cell nuclear transfer (SCNT) embryos critically depends on appropriate reprogramming and expression of pluripotency genes, such as Pou5f1/POU5F1 (previously known as Oct4/OCT4). To study POU5F1 transcription activation in living bovine SCNT embryos without interference by maternal POU5F1 mRNA, we generated chromosomally normal fetal fibroblast donor cells stably carrying a mouse Pou5f1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene at a single integration site without detectable EGFP expression. Morphologic and quantitative analyses of whole-mount SCNT embryos by confocal microscopy revealed robust initial activation of the Pou5f1 reporter gene during the fourth cell cycle. In Day 6 SCNT embryos EGFP expression levels were markedly higher than in Day 4 embryos but varied substantially between individual embryos, even at comparable cell numbers. Embryos with low EGFP levels had far more morphologically abnormal cell nuclei than those with high EGFP levels. Our data strongly suggest that bovine SCNT embryos consistently start activation of the POU5F1 promoter during the fourth cell cycle, whereas later in development the expression level substantially differs between individual embryos, which may be associated with developmental potential. In fibroblasts from phenotypically normal SCNT fetuses recovered on Day 34, the Pou5f1 reporter promoter was silent but was activated by a second round of SCNT. The restoration of pluripotency can be directly observed in living cells or SCNT embryos from such Pou5f1-EGFP transgenic fetuses, providing an attractive model for systematic investigation of epigenetic reprogramming in large mammals.

  • epigenetic reprogramming in mammalian nuclear transfer
    Differentiation, 2003
    Co-Authors: Valeri Zakhartchenko, Eckhard Wolf
    Abstract:

    With the exception of lymphocytes, the various cell types in a higher multicellular organism have basically an identical genotype but are functionally and morphologically different. This is due to tissue-specific, temporal, and spatial gene expression patterns which are controlled by genetic and epigenetic mechanisms. Successful Cloning of mammals by transfer of nuclei from differentiated tissues into enucleated oocytes demonstrates that these genetic and epigenetic programs can be largely reversed and that cellular totipotency can be restored. Although these experiments indicate an enormous plasticity of nuclei from differentiated tissues, Somatic Cloning is a rather inefficient and unpredictable process, and a plethora of anomalies have been described in cloned embryos, fetuses, and offspring. Accumulating evidence indicates that incomplete or inappropriate epigenetic reprogramming of donor nuclei is likely to be the primary cause of failures in nuclear transfer. In this review, we discuss the roles of various epigenetic mechanisms, including DNA methylation, chromatin remodeling, imprinting, X chromosome inactivation, telomere maintenance, and epigenetic inheritance in normal embryonic development and in the observed abnormalities in clones from different species. Nuclear transfer represents an invaluable tool to experimentally address fundamental questions related to epigenetic reprogramming. Understanding the dynamics and mechanisms underlying epigenetic control will help us solve problems inherent in nuclear transfer technology and enable many applications, including the modulation of cellular plasticity for human cell therapies.

Michel Guillomot - One of the best experts on this subject based on the ideXlab platform.

  • Analysis of 5-methyl cytosine and 5-hydromethyl cytosine content and distribution in bovine placental and fetal tissues after Somatic nuclear reprogramming
    2014
    Co-Authors: Michel Guillomot, Evelyne Campion, Audrey Prezelin, Hélène Kiefer, Nathalie Beaujean, Tiphaine Aguirre Lavin, Helene Jammes
    Abstract:

    Objectives were to determine whether these early alterations are maintained during pregnancy. For that purpose, the content and distribution of 5-mC and 5-hmC were analyzed in different bovine cell line- ages during development. Bovine fetal (brain, liver, heart), extraembryonic (allantois, amnion) and placental (chorion and cotyledon) tissues were collected at day 60 of gestation from SCNT or from artificial insemination pregnancies. Methods: The global 5-mC content was determined by LUminometric Methylation Analysis, an approach using DNA genomic, enzyme cleavages (HpaII/MsepI/EcoRI) and pyrosequencing. The 5-hmC or 5-mC content was also measured using specific 5-hmC or 5-mC immunoassays. The tissular distribution of 5-mC and 5-hmC was analyzed by immunostaining with 5- mC and 5-hmC antibodies on paraffin sections. Result: Our data provide the first overview of the distribution of 5-mC and 5-hmC during the development in bovine. We observed a lower level of 5- mC in placental tissues compared with the embryonic and extraembryonic tissues from 30% to 90% of methylation (chorionic villi < chorion < liver < heart < brain < amnion < allantois). Somatic Cloning induced an increase of the methylation level in chorionic villi only in late pregnancy stages. However, only the mesenchymal cells of the chorionic villi were immunostained for 5-mC and was involved in the methylation increase. In contrast, the 5hm-C content and the staining were more stable between the different lineages, suggesting a role of this epigenetic mark in all lineages in bovine. Conclusion: SCNT alters in a different manner the DNA methylation content in the cell lineages during the development.

  • spatial and temporal changes of decorin type i collagen and fibronectin expression in normal and clone bovine placenta
    Placenta, 2014
    Co-Authors: Michel Guillomot, Christophe Richard, Evelyne Campion, Audrey Prezelin, Olivier Sandra, F H Biase, C Rabel, R Wallace, Le D Bourhis, Harris A Lewin
    Abstract:

    Abstract Introduction Alteration of expression of various genes including extracellular matrix components, have been suggested to play major role in the placental pathologies after Somatic Cloning in mammals. The objectives of the present study were to analyze pattern of expression (mRNA and protein) of the small leucine-rich proteoglycan, Decorin in association with Type I Collagen and Fibronectin in bovine placental tissues from normal and clone pregnancies. Methods Genotyping and allelic expression of Decorin were determined by Sanger sequencing. The expression patterns of Decorin , Type I collagen and Fibronectin 1 were analyzed by quantitative RT-qPCR and combined in situ hybydization (ISH) and immunohistochemistry (IHC) in endometrial and placental tissues from D18 to term from artificially inseminated and Somatic Cloning pregnancies. Results The expression levels of DCN increased in the AI endometrial stroma and chorionic mesenchyme during implantation and declined during placentome growth until term. Combined ISH and IHC revealed an unexpected discrepancy mRNA and protein tissue distribution. Moreover, Decorin was maintained in the placentome tissues from SCNT pregnancies while both mRNA and protein were absent in AI derived placenta. Discussion In bovine, the pattern of expression of Decorin exhibits significant changes during placental formation. Downregulation of Decorin is associated with proliferation, remodeling and vascularization of placental tissues. These observations reinforces the putative role of Decorin in these processes. Conclusions These observations suggest that Decorin is involved in placental growth and that dysregulation of its expression is associated with placental abnormalities in SCNT derived pregnancy.

  • Spatial and temporal changes of Decorin, Type I collagen and Fibronectin expression in normal and clone bovine placenta
    Placenta, 2014
    Co-Authors: Michel Guillomot, Christophe Richard, Evelyne Campion, Audrey Prezelin, Olivier Sandra, Isabelle Hue, Daniel Le Bourhis, F H Biase, C Rabel, R Wallace
    Abstract:

    Alteration of expression of various genes including extracellular matrix components, have been suggested to play major role in the placental pathologies after Somatic Cloning in mammals. The objectives of the present study were to analyze pattern of expression (mRNA and protein) of the small leucine-rich proteoglycan, Decorin in association with Type I Collagen and Fibronectin in bovine placental tissues from normal and clone pregnancies. Genotyping and allelic expression of Decorin were determined by Sanger sequencing. The expression patterns of Decorin, Type I collagen and Fibronectin 1 were analyzed by quantitative RT-qPCR and combined in situ hybydization (ISH) and immunohistochemistry (IHC) in endometrial and placental tissues from D18 to term from artificially inseminated and Somatic Cloning pregnancies. The expression levels of DCN increased in the AI endometrial stroma and chorionic mesenchyme during implantation and declined during placentome growth until term. Combined ISH and IHC revealed an unexpected discrepancy mRNA and protein tissue distribution. Moreover, Decorin was maintained in the placentome tissues from SCNT pregnancies while both mRNA and protein were absent in AI derived placenta. In bovine, the pattern of expression of Decorin exhibits significant changes during placental formation. Downregulation of Decorin is associated with proliferation, remodeling and vascularization of placental tissues. These observations reinforces the putative role of Decorin in these processes. These observations suggest that Decorin is involved in placental growth and that dysregulation of its expression is associated with placental abnormalities in SCNT derived pregnancy.

  • Abnormal expression of the imprinted gene Phlda2 in cloned bovine placenta
    Placenta, 2010
    Co-Authors: Michel Guillomot, Pascale Chavatte-palmer, Isabelle Hue, Geraldine Taghouti, Fabienne Constant, Severine Degrelle, Helene Jammes
    Abstract:

    Cloning in mammals suffers from high rates of pregnancy losses associated with abnormal placentation, mainly placentomegaly, leading to fetal death. Placental growth is dependent on the regulated expression of many genes of which imprinted genes play a fundamental role. Among them, the Phlda2 gene is expressed from the maternal allele and acts to limit placental growth in mouse and human. Here we used Northern blots, quantitative RT-PCR and in situ hybridization to analyze the expression patterns of bovine PHLDA2 and to compare its expression levels in normal and Somatic cell nuclear transfer (SCNT) placentas over a range of gestational stages. PHLDA2 is not expressed in extra-embryonic tissues before d32 of gestation but the level of expression increases throughout pregnancy until term in the placental villi collected from pregnancy obtained by artificial insemination (AI). At all stages of pregnancy, PHLDA2 mRNA are specifically localized in the trophoblast mononucleated cells contrasting with lack of expression in the binucleated cells and uterine tissues. In SCNT placentas, a similar pattern of expression was observed during early pregnancy. In contrast the level of expression is significantly reduced around d200 of gestation in the placental villi from pathological clones. The reduced expression of PHLDA2 was obvious particularly in the placental villi anchored within the uterine crypts with expression confined to the trophoblast of the chorionic plate. Altogether, these results highlight a similarity in expression patterns for PHLDA2 bovine and human where expression is localized to the trophoblast throughout pregnancy and parallels the continuous growth of the placenta. Moreover, the lack of expression in the fetal villi from oversized bovine cloned placenta is consistent with the function of PHLDA2 in restraining placental growth and underlines an aberrant expression of this gene after Somatic Cloning.

  • 28 EXPRESSION OF MHC CLASS I IN THE PLACENTA AND THE LYMPHOCYTES OF BOVINE Somatic CLONES
    Reproduction Fertility and Development, 2007
    Co-Authors: Pascale Chavatte-palmer, Yvan Heyman, Michel Guillomot, Isabelle Hue, J. Roïz, J.l. Servely, S.a. Ellis
    Abstract:

    A major limitation for the development of Somatic Cloning in cattle is the low efficiency for producing live offspring because only 5 to 25% of the reconstructed embryos reach full-term development, with variation between the genotypes. Hill et al. (2002 Biol. Reprod. 67, 55–63) reported that early fetal losses were due to inappropriate expression of trophoblast major histocompatibility complex Class 1 (MHC1) antigens on the placenta of bovine Somatic clones. This abnormal expression was present in all clones, with more cells expressing MHC1 in those with growth retardation. These clones, however, originated from only one genotype, and only one antibody recognizing a monomorphic determinant of bovine MHCI was used. In the present study, MHC1 expression was examined throughout gestation in bovine clone placentas originating from 3 different genotypes. Placentas were collected at Days 32, 62, 180, 260, and 280 (term) in clones (n = 8) and in controls obtained by artificial insemination (n = 6). Four different monoclonal antibodies that recognize different determinants of MHC1 molecules (ILA-A88, IL-A19, H58A, and the β2-microglobulin, i.e. the MHC1 light chain) were used. Results showed MHC1 expression in the maternal tissue but not on the fetal side, regardless of group, gestational age, or fetal growth, although patchy expression of β2-microglobulin could be found in both clones and controls in the fetal mesenchyme in some but not all samples. In conclusion, MHC1 protein expression appears normally down-regulated in the placenta of clones, but the antibodies used do not discriminate between classical and non-classical expression. The mRNA expression of classical and non-classical MHC1 genes is currently being explored. These results cast doubt on the hypothesis that the observed fetal losses would be the result of an immunologic rejection due to inappropriate expression of some placental MHC1 genes. Although early observations suggest that immune functions appear normal (Lanza et al. 2001 Science 294, 1893–1894; Chavatte-Palmer et al. 2006 Reprod. Fert. Dev. 18, 121 abst), immunological deficiencies have been reported in some bovine Somatic clones after birth (Renard et al. 1999 The Lancet 353, 1489–1491), and they may be related to an abnormal expression of MHC1 genes. We therefore determined the MHC1 haplotype of cell donors from 4 genotypes and 3 of their healthy clones using the Reference Strand Conformation Analysis method with DNA extracted from lymphocytes. Results showed that the MHC1 type was identical, as could be expected in clones. Animals that died of immune deficiencies, however, may have had abnormal MHC1 expression. Further work is therefore being performed on tissues obtained postmortem from cloned animals having been diagnosed with thymic atrophy.