Development of a monkey model for the study of primate genomic imprinting

Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan.
Molecular Human Reproduction (Impact Factor: 3.75). 07/2005; 11(6):413-22. DOI: 10.1093/molehr/gah180
Source: PubMed


An understanding of the role of imprinted genes in primate development requires the identification of suitable genetic markers that allow analysis of allele-specific expression and methylation status. Four genes, NDN (Necdin), H19, SNRPN and IGF2, known to be imprinted in mice and humans, were selected for study in rhesus monkeys along with two imprinting centres (ICs) associated with the regulation of H19/IGF2, NDN and SNRPN. GAPD was employed as a non-imprinted control gene. Primers designed to amplify polymorphic regions in these genes and ICs were based on human sequences. Genomic DNA was isolated from peripheral blood leukocytes of 93 rhesus macaques of Indian or Chinese-origin. Sequence analysis of amplicons resulted in the identification of 32 unique SNPs. Country-of-origin related differences in SNP distributions were evident. Since disruptions in imprinted gene expression and associated developmental abnormalities may result from in vitro embryo manipulation, we also examined imprinting in NDN, H19, SNRPN and IGF2 in rhesus monkey infants produced by natural mating or by ICSI. Muscle biopsies followed by RT-PCR and sequence analysis were performed in four heterozygous animals produced by natural mating and all four genes were expressed monoallelically supporting the conclusion that these genes are normally imprinted in monkeys. In the case of ICSI, five informative infants were selected based on parental analysis. Allele-specific studies indicated that the expected uniparental expression patterns were retained in animals produced from manipulated embryos. Moreover, methylation analysis revealed that CpG islands within H19/IGF2 and SNURF/SNRPN ICs were differentially methylated. The approach described here will allow examination of imprinting in the embryos and embryonic stem cells of the monkey.

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Available from: Shoukhrat Mitalipov, Mar 26, 2015
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    • "The SNRPN DMR is known to be maternally methylated in juvenile rhesus macaque muscle and ES cell lines (Fujimoto et al. 2005; Mitalipov et al. 2007). Our results in cynomolgus macaque tissues are in agreement, and we further demonstrate that differential methylation is also seen in placenta and umbilical cord (Fig. 3). "
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    ABSTRACT: Genomic imprinting is an epigenetic mechanism resulting in parental allele-specific gene expression. Defects in normal imprinting are found in cancer, assisted reproductive technologies, and several human syndromes. In mouse models, germline-derived DNA methylation is shown to regulate imprinting. Though imprinting is largely conserved between mammals, species- and tissue-specific domains of imprinted expression exist. Using the cynomolgus macaque (Macaca fascicularis) to assess primate-specific imprinting, we present a comprehensive view of tissue-specific imprinted expression and DNA methylation at established imprinted gene clusters. For example, like mouse and unlike human, macaque IGF2R is consistently imprinted, and the PLAGL1, INPP5F transcript variant 2, and PEG3 imprinting control regions are not methylated in the macaque germline but acquire this post-fertilization. Methylome data from human early embryos appear to support this finding. These suggest fundamental differences in imprinting control mechanisms between primate species and rodents at some imprinted domains, with implications for our understanding of the epigenetic programming process in humans and its influence on disease. © 2015 Cheong et al.; Published by Cold Spring Harbor Laboratory Press.
    Preview · Article · Apr 2015 · Genome Research
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    • "Characteristics of the single nucleotide polymorphisms (SNPs) employed for allele-specific expression analysis, PCR primers and conditions were previously described in detail (Fujimoto et al., 2005; Fujimoto et al., 2006). Expressed alleles were designed using Primer 3 software ( "
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    ABSTRACT: Parthenogenetic embryonic stem cells (PESCs) may have future utilities in cell replacement therapies since they are closely related to the female from which the activated oocyte was obtained. Furthermore, the avoidance of parthenogenetic development in mammals provides the most compelling rationale for the evolution of genomic imprinting, and the biological process of parthenogenesis raises complex issues regarding differential gene expression. We describe here homozygous rhesus monkey PESCs derived from a spontaneously duplicated, haploid oocyte genome. Since the effect of homozygosity on PESCs pluripotency and differentiation potential is unknown, we assessed the similarities and differences in pluripotency markers and developmental potential by in vitro and in vivo differentiation of homozygous and heterozygous PESCs. To understand the differences in gene expression regulation between parthenogenetic and biparental embryonic stem cells (ESCs), we conducted microarray analysis of genome-wide mRNA profiles of primate PESCs and ESCs derived from fertilized embryos using the Affymetrix Rhesus Macaque Genome array. Several known paternally imprinted genes were in the highly down-regulated group in PESCs compared with ESCs. Furthermore, allele-specific expression analysis of other genes whose expression is also down-regulated in PESCs, led to the identification of one novel imprinted gene, inositol polyphosphate-5-phosphatase F (INPP5F), which was exclusively expressed from a paternal allele. Our findings suggest that PESCs could be used as a model for studying genomic imprinting, and in the discovery of novel imprinted genes.
    Full-text · Article · Aug 2010 · Human Reproduction
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    • "Since then, specific genetic differences between populations of rhesus macaques from China and India have been characterized based on electrophoretically defined protein polymorphisms [22,23], STR polymorphisms [24-29], major histocompatibility (MHC) alleles [30-32], mtDNA [17,28,33] and, most recently, single nucleotide polymorphism (SNPs) [34-37]. Unfortunately, the number of polymorphisms represented above is far too small, and their distribution throughout the genome too broad, to permit whole genome studies of quantitative trait loci (QTL) and disease association [35]. "
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    ABSTRACT: Rhesus macaques (Macaca mulatta) are the primate most used for biomedical research, but phenotypic differences between Indian-origin and Chinese rhesus macaques have encouraged genetic methods for identifying genetic differences between these two populations. The completion of the rhesus genome has led to the identification of many single nucleotide polymorphisms (SNPs) in this species. These single nucleotide polymorphisms have many advantages over the short tandem repeat (STR) loci currently used to assay genetic variation. However, the number of currently identified polymorphisms is too small for whole genome analysis or studies of quantitative trait loci. To that end, we tested a combination of methods to identify large numbers of high-confidence SNPs, and screen those with high minor allele frequencies (MAF). By testing our previously reported single nucleotide polymorphisms, we identified a subset of high-confidence, high-MAF polymorphisms. Resequencing revealed a large number of regionally specific SNPs not identified through a single pyrosequencing run. By resequencing a pooled sample of four individuals, we reliably identified loci with a MAF of at least 12.5%. Finally, we found that when applied to a larger, geographically variable sample of rhesus, a large proportion of our loci were variable in both populations, and very few loci were ancestry informative. Despite this fact, the SNP loci were more effective at discriminating Indian and Chinese rhesus than STR loci. Pyrosequencing and pooled resequencing are viable methods for the identification of high-MAF SNP loci in rhesus macaques. These SNP loci are appropriate for screening both the inter- and intra-population genetic variation.
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