40 VARIOUS DNA METHYLATION LEVELS OF IMPRINTED GENES IN CLONED COWS FROM THE SAME DONOR CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 126
Author(s):  
M. Kaneda ◽  
S. Watanabe ◽  
S. Akagi ◽  
T. Somfai ◽  
S. Haraguchi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Environmental Changes ◽  
Monozygotic Twins ◽  
Preimplantation Embryos ◽  
Imprinted Genes ◽  
Imprinted Gene ◽  
Donor Cells ◽  
Pcr Products ◽  
Epigenetic Patterns

Somatic cell nuclear transferred (SCNT) animals are genetically identical to the donors; however, because of epigenetic abnormalities caused by incomplete reprogramming during nuclear transfer, the efficiency of SCNT is still very low. Monozygotic twins are also genetically identical, but it is reported that their epigenetic patterns on the genome, the so-called epigenome, are different. The epigenome is easily influenced by aging, environmental changes and nutrients, therefore these effects can be predicted by comparing epigenetic differences between genetically identical animals. Here we analysed DNA methylation levels of imprinted genes, which express in a parent-of-origin specific manner, in various tissues of cloned cows derived from the same donor cells. Imprinted gene expression is controlled by DNA methylation and other epigenetic modifications and abnormal expression/methylation patterns of imprinted genes have been observed in cloned animals. These alterations also occur during in vitro development of preimplantation embryos, which suggests that imprinted genes are easily influenced by environmental changes. Therefore, we chose H19 and PEG3 imprinted genes for the analysis to determine the epigenetic differences between individual cloned cows derived from the same donor cells. From 5 cloned and 5 non-cloned cows, we isolated DNA from 8 tissues (heart, lung, liver, kidney, spleen, intestine, muscle, and spinal cord) and analysed DNA methylation levels by bisulfite sequencing method. Briefly, genomic DNA was isolated by QIAGEN DNeasy Blood & Tissue Kit and bisulfite converted by QIAGEN EpiTect Bisulfite Kits (Qiagen, Valencia, CA). After amplification, the PCR products were cloned into TA vector and at least 10 clones were sequenced in each gene/sample. In every tissue analysed, the methylation levels largely differ among tissues and individuals. On average, the paternally imprinted gene H19 was 9.4 to 47.9% methylated (average 27.6 ± 10.3%) in clones and 0.5 to 69.8% methylated (average 29.0 ± 16.8%) in non-clones. The maternally imprinted gene PEG3 was 18.8 to 82.2% methylated (average 43.5 ± 15.8%) in clones and 8.0 to 98.7% (average 48.2 ± 18.8%) in non-clones. Even though there were large variations in DNA methylation levels, the variability tends to be low in clones compared to non-clones. More specifically, the variabilities of H19 methylation levels in spleen and intestine were significantly lower in clones than those in non-clones (32.3 ± 5.4% v. 27.0 ± 19.0% and 25.1 ± 4.2% v. 45.1 ± 14.3%, respectively, F-test; P < 0.05). These results suggest for the first time that epigenetic patterns in some tissues of both clones and non-clones are influenced by genetic background; however, mostly they are varied depending on non-genetic factors.

scholarly journals 26ABERRANT REPROGRAMMING OF IMPRINTED GENE EXPRESSION IN ENLARGED PLACENTAS OF MICE CLONED FROM ES CELLS TREATED WITH TSA OR 5AZAC

2004 ◽  
Vol 16 (2) ◽  
pp. 135
Author(s):  
S.G. Baqir ◽  
Q. Zhou ◽  
A. Jouneau ◽  
J.-P. Renard ◽  
D.H. Betts ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Histone Acetylation ◽  
Nuclear Transfer ◽  
Es Cells ◽  
Expression Patterns ◽  
Gene Expression Patterns ◽  
Imprinted Genes ◽  
Imprinted Gene ◽  
Donor Cells

The success rate of producing cloned animals is very low, and in many cases is associated with the formation of enlarged placentas. Increasing evidence has pointed towards epigenetic deregulation of imprinted genes due to incomplete or abnormal resetting of DNA methylation and/or histone acetylation patterns during development. It has previously been shown that drugs that alter DNA methylation (5AzaC) and histone acetylation (TSA) over-express imprinted genes in mouse ES cells (Baqir and Smith, 2001, Theriogenology 55, 410). Our objective in this study was to determine whether nuclear transfer is able to reprogram imprinted gene expression patterns in the placenta of mice cloned from ES donor nuclei exposed to 5AzaC and TSA. ES donor cells were treated with either TSA or 5AzaC prior to injection into enucleated oocytes. Total RNA was extracted from placentas of day 14–15 fetus clones, and reversed transcribed; the expression pattern of imprinted genes (Ipl, Mash2, Igf2, H19, Igf2r, p57, Peg1), non-imprinted placental-specific genes (Esx1, Dlx3, Tpbp) and a housekeeping gene (Gapdh) was examined by Real Time PCR. Samples were standardized with an exogenous control (Globin) and expressed as fold changes in relation to placentas of cloned fetus derived from non-treated donor cells. Data were analyzed by ANOVA and mean gene expression values were compared using the Tukey-Kramer test. Our results show that several imprinted genes (Mash2, H19, Ipl) and placenta-specific genes (Esx1 and Dlx1) were properly reprogrammed in non-enlarged (71mg) placentas of fetus clones derived from the TSA and 5AzaC treated ES donor cells. Although Gapdh expression did not differ among normal and enlarged 210mg) placenta groups, the expression level of Igf2 and Mash2 was higher in enlarged placentas from fetus clones produced from TSA-treated ES donor cells (4.6 and 3.5 fold) compared to non-enlarged placentas from non-treated ES cells (1 fold). Conversely, oversized placentas from cloned fetuses derived from TSA-treated donor ES cells under-expressed Peg1, H19 and Ipl (0.5, 0.2 and 0.2 fold, respectively) compared to control placentas (1 fold). In addition, enlarged placentas from the TSA- and 5AzaC-treated group displayed down-regulation of placenta specific genes Esx1 and Dlx3 and up-regulation of Tpbp, suggesting the presence of abnormal distribution of placental layers. These results indicate that while several imprinted and non-imprinted placenta specific genes were correctly expressed in normal size placentas of fetus clones derived from TSA and 5AzaC treated donor ES cells, enlarged placentas displayed aberrant gene expression patterns, suggesting that improper resetting of the epigenetic program after nuclear transfer is directly related to altered DNA methylation and histone acetylation patterns. Funded by NSERC &amp; CIHR.

scholarly journals Identification of Putative Virulence Genes by DNA Methylation Studies in the Cereal Pathogen Fusarium graminearum

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1192
Author(s):  
Francesco Tini ◽  
Giovanni Beccari ◽  
Gianpiero Marconi ◽  
Andrea Porceddu ◽  
Micheal Sulyok ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Fusarium Graminearum ◽  
Environmental Changes ◽  
Virulent Strain ◽  
Fungal Virulence ◽  
Wheat Seedlings ◽  
Wide Range ◽  
Conidia Production ◽  
Natural Hosts

DNA methylation mediates organisms’ adaptations to environmental changes in a wide range of species. We investigated if a such a strategy is also adopted by Fusarium graminearum in regulating virulence toward its natural hosts. A virulent strain of this fungus was consecutively sub-cultured for 50 times (once a week) on potato dextrose agar. To assess the effect of subculturing on virulence, wheat seedlings and heads (cv. A416) were inoculated with subcultures (SC) 1, 23, and 50. SC50 was also used to re-infect (three times) wheat heads (SC50×3) to restore virulence. In vitro conidia production, colonies growth and secondary metabolites production were also determined for SC1, SC23, SC50, and SC50×3. Seedling stem base and head assays revealed a virulence decline of all subcultures, whereas virulence was restored in SC50×3. The same trend was observed in conidia production. The DNA isolated from SC50 and SC50×3 was subject to a methylation content-sensitive enzyme and double-digest, restriction-site-associated DNA technique (ddRAD-MCSeEd). DNA methylation analysis indicated 1024 genes, whose methylation levels changed in response to the inoculation on a healthy host after subculturing. Several of these genes are already known to be involved in virulence by functional analysis. These results demonstrate that the physiological shifts following sub-culturing have an impact on genomic DNA methylation levels and suggest that the ddRAD-MCSeEd approach can be an important tool for detecting genes potentially related to fungal virulence.

53 IMPACTS OF USING PROCAINE AS A DNA-DEMETHYLATING AGENT IN IN VITRO CULTURE OF BOVINE CELLS

2011 ◽  
Vol 23 (1) ◽  
pp. 132
Author(s):  
V. A. Michalczechen-Lacerda ◽  
F. C. Rodrigues ◽  
R. V. de Sousa ◽  
R. Rumpf ◽  
M. M. Franco
Keyword(s):  
Dna Methylation ◽  
Cell Viability ◽  
In Vitro Culture ◽  
Cytogenetic Analysis ◽  
Imprinted Genes ◽  
Demethylating Agent ◽  
Chromosome Integrity ◽  
Methylation Patterns ◽  
Number Of Cells

Euchromatin and heterochromatin organisation define the specificity of each cell type. This structure is controlled by epigenetic modifications and the DNA methylation is one of the best known for inducing transcriptional repression. Recently, procaine was uncovered as a DNA-demethylating agent, but there are few reports about its dynamic epigenetic action on somatic cells. Mono-allelic expression of imprinted genes is controlled by DNA methylation and inherited to somatic tissues of a sex-specific manner. The aim was to investigate the effects of using procaine, a DNA-demethylating agent, in in vitro culture of bovine (Bos taurus indicus) fibroblast for 72 h (passage 4). We have evaluated cell viability, chromosome integrity, and DNA methylation patterns. To evaluate cell viability, we have used trypan blue 0.4%. To evaluate chromosome integrity, we have used conventional cytogenetic analysis. To investigate DNA methylation patterns, we have analysed 2 differentially methylated regions (DMR) located into the exon 10 of IGF2 and exon 1 of XIST imprinted genes, using the bisulfite sequencing method (EZ DNA methylation kit, Zymo Research, Orange, CA, USA). After bisulfite treatment and nested-PCR, the amplicons were separated in agarose gel electrophoresis, purified with GenClean III kit (MP Biomedicals, Irvine, CA, USA), cloned in a pGEM-T easy vector system (Promega, Madison, WI), and sequenced. The DNA sequences were analysed using the BiQ Analyzer v. 2.0 (2008) software. The cell viability data were analysed using ANOVA and Tukey or Kruskal-Wallis and Mann-Whitney tests, and the methylation status were analysed using Student’s t-test or Mann-Whitney tests in the Prophet software (BBN Systems and Technologies). Cell culture using 0.1 mM or 0.5 mM of procaine were viable and the number of cells with intact membrane was higher than the control and 2.0 mM of procaine groups (P ≤ 0.05). The total number of cells was lower in the group with 2.0 mM of procaine (P ≤ 0.01). Cytogenetic analysis showed no differences among the groups, with no chromosome abnormalities detected. The methylation pattern was not different for both DMR evaluated among the groups. We have observed that there was a beneficial effect to the cells that have received supplementation with 0.1 mM or 0.5 mM of procaine, because there was an increase in the number of viable cells without chromosomal abnormalities. We cannot ignore that a global DNA demethylation may have occurred, which was not detected in the specific analysed regions. The results obtained here may contribute to improving the efficiency of animal cloning, transgenic animal production, and the knowledge about stem cells. Supported by Embrapa Genetic Resources and Biotechnology and CAPES.

Effect of vitrification on in vitro development and imprinted gene Grb10 in mouse embryos

Reproduction ◽  
2017 ◽  
Vol 154 (3) ◽  
pp. 197-205 ◽  
Author(s):  
Jianfeng Yao ◽  
Lixia Geng ◽  
Rongfu Huang ◽  
Weilin Peng ◽  
Xuan Chen ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cpg Island ◽  
Formation Rate ◽  
Mouse Embryos ◽  
Major Type ◽  
Hatching Rate ◽  
Preimplantation Embryo Development ◽  
Imprinted Gene

Vitrification of embryos is a routine procedure in IVF (in vitrofertilization) laboratories. In the present study, we aimed to investigate the effect of vitrification on mouse preimplantation embryo developmentin vitro, and effect on the epigenetic status of imprinted geneGrb10in mouse embryos. The blastocyst formation rate for vitrified 8-cell embryos was similar to the non-vitrified 8-cell embryos, whereas the blastocyst hatching rate was lower than that of the non-vitrified group. The expression level ofGrb10major-type transcript decreased significantly in vitrified blastocysts compared with non-vitrified andin vivoblastocysts. Moreover, the global DNA methylation level in 8-cell embryos and blastocysts, and the DNA methylation at CpG island 1 (CGI1) ofGrb10in blastocysts were also significantly decreased after vitrification.In vitroculture condition had no adverse effect, except for on the DNA methylation inGrb10CGI1. These results suggest that vitrification may reduce thein vitrodevelopment of mouse 8-cell embryos and affect the expression and DNA methylation of imprinted geneGrb10.

75 EPIGENETIC EFFECTS OF VITRIFICATION ON PRONUCLEAR DOMESTIC CAT EMBRYOS

2015 ◽  
Vol 27 (1) ◽  
pp. 131 ◽  
Author(s):  
J. H. Galiguis ◽  
C. E. Pope ◽  
M. N. Biancardi ◽  
C. Dumas ◽  
G. Wang ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Genetic Material ◽  
Domestic Cat ◽  
Blastocyst Development ◽  
In Vitro Development ◽  
Epigenetic Effects ◽  
Donor Cells ◽  
Vitro Development

Vitrification remains a promising technique in the preservation of valuable genetic material; however, in the cat, success has varied. Live kittens have been produced from embryos vitrified at early cleavage stages, but phenotypic abnormalities in some kittens suggest possible epigenetic effects of the vitrification process. It has been reported that cryopreservation alters epigenetic events in somatic donor cells, which indirectly influences physical status of cloned offspring. However, extending post-warming in vitro culture of donor cells corrects these epigenetic modifications, resulting in normal embryos/clones. Accordingly, in the present study, vitrification was performed at the pronuclear stage to lengthen pretransfer culture time, and vitrified cat zygotes were assessed by analysing (1) histone acetylation/methylation, (2) global DNA methylation, (3) pluripotent gene expression, (4) in vitro development, and () in vivo viability. In vivo matured/IVF oocytes were vitrified in 15% dimethyl sulfoxide, 15% ethylene glycol, and 0.5 M sucrose at 16 h post-insemination (PI). After warming in 1.0 M sucrose at 38°C, embryos were fixed at 18 h or 40 h PI, and the nuclear intensity of either acetyl/dimethyl-H3K9 or 5-methylcytosine was determined by immunofluorescence. Results showed that at 18 h PI, mean H3K9ac intensity of vitrified embryos (11.8; n = 6) was higher than that of corresponding nonvitrified (fresh) controls (4.5; n = 6) and the fresh (3.2; n = 11) and vitrified (0.6; n = 7) 40-h groups (2-way ANOVA; P < 0.05). H3K9me2 in the fresh (36.9) and vitrified (32.5) 18-h embryos was similar but increased relative to both fresh (10.7) and vitrified (9.2) 40-h groups (P < 0.05). Mean DNA methylation (5MeC) in the fresh (31.6; n = 1) and vitrified (24.7; n = 3) 18-h groups was similar to that of the fresh 40-h group (19.8; n = 4) but higher than that of the vitrified 40-h group (15.0; n = 5; P < 0.05). To assess expression of POU5F1 and Nanog, qRT-PCR was performed on Day 8 blastocysts. Relative to controls (n = 9), mean POU5F1 and Nanog levels in vitrified blastocysts (n = 24) were 1.38- and 1.98-fold higher, respectively (one-way ANOVA; P > 0.05). In terms of in vitro development, Day 2 cleavage of vitrified zygotes (59%; n = 508) was similar to that of controls (66%; n = 340), but Day 8 blastocyst formation was reduced (9 v. 31%; t-test; P < 0.05). In vivo viability was assessed by oviducal transfer of 41 Day 1 embryos into 2 recipients. One pregnancy was established (50%), with 3 live kittens weighing 70, 79, and 131 g delivered without assistance on Day 65 of gestation. The 2 smaller kittens died within a few hours of birth, with the smallest exhibiting an umbilical hernia and organ exteriorization. The third kitten developed into a normal, healthy adult. In summary, mean H3K9me2, 5MeC, and POU5F1/Nanog expression of vitrified zygotes was similar to corresponding controls. H3K9ac increased at 18h PI as a result of vitrification, but was reduced after culture to 40 h PI. Although vitrified zygotes cleaved in vitro at rates similar to controls, blastocyst development was reduced. In vivo viability was demonstrated; however, postnatal survival of kittens produced was low.

400 KARYOTYPIC ANALYSIS AND EPIGENETIC MODIFICATIONS OF CULTURED PORCINE ADIPOSE TISSUE-DERIVED STEM CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 356
Author(s):  
K. J. Williams ◽  
K. R. Bondioli ◽  
R. A. Godke
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Stem Cells ◽  
Chromatin Remodeling ◽  
Primary Cultures ◽  
Donor Cell ◽  
Global Methylation ◽  
Histone 3 ◽  
Donor Cells

The introduction of genetic modifications in donor cells for NT requires a significant number of population doublings (PD), and the deleterious effects, which may be attributed to aneuploidy or changes in DNA methylation and histone acetylation, are difficult at this time to circumvent. We hypothesize that the identification of a donor cell that is genetically stable for a long period of time in vitro such as somatic stem cells or those cells that demonstrate stem-like characteristics may be reprogrammed more completely, thus providing the key to increasing the efficiency of NT. Regulators of development in undifferentiated cells are suggested to be silenced by the presence of a bivalent domain modification pattern in which a large region of repressive histone 3 lysine 27 trimethylation (H3K27me3) contains smaller regions of activating histone 3 lysine 4 trimethylation (H3K4me3).The dual marks work to silence developmental genes in embryonic stem cells while simultaneously keeping them receptive to activation. The objectives of the current study were to determine the chromosomal stability of porcine adipose tissue-derived adult stem cells (pASC) through in vitro culture, to analyze pASC alongside fetal porcine fibroblasts (FPF) for gene expression profiles of chromatin remodeling proteins and global methylation and acetylation patterns, and to determine the presence of a co-enrichment of H3K27me3 and H3K4me3 within the promoter regions of developmentally important transcription factors. Metaphase spreads were prepared, and the presence of H3K27me3 and H3K4me3 was investigated in each of 3 individual pASC primary cultures for each analysis; whereas, gene expression and global methylation and acetylation were analyzed in each of 4 individual pASC and FPF primary cultures. Of 714 metaphases analyzed, 509 (71.3%) were aneuploid and only 205 (28.7%) were normal diploid porcine cells. For each cell population, we found a remarkable percentage of aneuploidies (43.7, 48.9, and 47.3, with a 46.6 ± 1.5 average) present immediately after the cultures were established. Chi-square analysis indicated that the percent of aneuploid cells during PD 1-10 was significantly less than that for PD 11-20 and PD 21-30. Also, porcine ASC demonstrated a consistently lower level of DNA methylation and histone acetylation through passages 2 through 7; whereas, the patterns for FPF varied. The expression levels of chromatin remodeling transcripts remained lower in pASC throughout culture when compared with FPF. Finally, porcine ASC possess a co-enrichment of H3K27me3 and H3K4me3 on the promoter region of the developmentally important transcription factor OCT-4. In vitro-cultured porcine ASC used as donor cells for NT should be chosen from early PD because of increased levels of aneuploidy at later PD. With a more complete characterization of porcine ASC, a donor cell population that can be more efficiently reprogrammed following fusion with the oocyte might be identified.

45 The role of TRIM28 in porcine somatic cell nuclear transfer embryo development

2019 ◽  
Vol 31 (1) ◽  
pp. 148
Author(s):  
Y. H. Zhai ◽  
X. L. An ◽  
Z. R. Zhang ◽  
S. Zhang ◽  
Z. Y. Li
Keyword(s):  
Dna Methylation ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Chromatin Modification ◽  
Blastocyst Stage ◽  
Imprinted Genes ◽  
Cell Stage ◽  
Genomic Methylation

During fertilization, the parental genome undergoes extensive demethylation. Global DNA demethylation is a hallmark of epigenetic reprogramming. Embryos engage non-canonical DNA methylation maintenance mechanisms to ensure inheritance of exceptional germline features. However, the mechanisms ensuring demethylation resistance in light of global reprogramming remain poorly understood. TRIM28 is a maternal-effect factor that controls genomic imprinting during early embryonic reprogramming. In this study, cytoplasmic injections of siRNA were performed into oocytes matured in vitro for 26h to interfere with the expression of TRIM28 in oocytes. The injected oocytes were continually matured in vitro until 42h and used to construct somatic cell nuclear transfer (SCNT) embryos. During 2-cell to blastocyst stages, the expression of development-related genes (NANOG, POU5F1, CDX2, BAX, and BCL2), maternal imprinting genes (IGF2, DIO3, PLAGL1, and DLK1), paternal imprinting genes (H19 and PEG3), TRIM28-recruitment complex-associated genes (ZFP57, PGC7, SETDB1, and DNMT), and epigenetic chromatin modification enzymes were detected by quantitative PCR in the constructed TRIM28-interfered SCNT embryos. The DNA methylation levels in the promoter regions of the imprinted genes (H19 and IGF2) and chromatin repeats (PRE-1 and SATELLITE) were analysed by sodium bisulfite genomic sequencing. The results showed that the TRIM28-interfered SCNT embryos had significantly lower cleavage and blastocyst rates (53.9±3.4% and 12.1±4.3%, respectively) than those in control SCNT embryos (64.8±2.7% and 18.8±1.9%, respectively). The expression levels of development-related genes (NANOG and POU5F1) and TRIM28-recruited transcriptional repression complex-associated genes (PGC7, ZFP57, and DNMT1) in the 4-cell stage were significantly reduced (P&lt;0.05). The imprinted genes were significantly up-regulated (P&lt;0.05) from the 2-cell to blastocyst stage in constructed TRIM28-interfered SCNT embryos, except H19 at the 2-cell and blastocyst stage decreased remarkably (P&lt;0.05). The DNA methylation levels of IGF2 decreased 2-fold from the 2-cell to blastocyst stage in TRIM28-interfered SCNT embryos. The PRE-1 and SATELLITE had a remarkably lower (P&lt;0.05) methylation levels in the TRIM28-interfered 2-cell embryos than in control SCNT embryos. The cluster analysis showed some of the chromatin modification enzymes had abnormal expression in the TRIM28-interfered SCNT embryos, especially in the 8-cell stage, where 48 enzymes were significantly decreased (P&lt;0.05). The down-regulation enzymes were mainly clustered in the histone H3K4 methyl transferase and histone acetylase. These results indicate that down-regulation of maternal TRIM28 breaks the steady-state of genomic methylation at a particular locus of the imprinted gene, disrupts the expression of imprinted gene and epigenetic modifications enzymes, and is detrimental to normal development of SCNT embryos. Maternal TRIM28 is needed in maintaining a stable state of genomic methylation and epigenetic modification state during SCNT embryo development.

Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells

Development ◽  
2002 ◽  
Vol 129 (8) ◽  
pp. 1807-1817 ◽  
Author(s):  
Jiyoung Lee ◽  
Kimiko Inoue ◽  
Ryuichi Ono ◽  
Narumi Ogonuki ◽  
Takashi Kohda ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Genomic Imprinting ◽  
Nuclear Transfer ◽  
Monoallelic Expression ◽  
Parental Origin ◽  
Imprinted Genes ◽  
Specific Expression ◽  
Male And Female ◽  
Imprinted Gene ◽  
Methylation Patterns

Genomic imprinting is an epigenetic mechanism that causes functional differences between paternal and maternal genomes, and plays an essential role in mammalian development. Stage-specific changes in the DNA methylation patterns of imprinted genes suggest that their imprints are erased some time during the primordial germ cell (PGC) stage, before their gametic patterns are re-established during gametogenesis according to the sex of individuals. To define the exact timing and pattern of the erasure process, we have analyzed parental-origin-specific expression of imprinted genes and DNA methylation patterns of differentially methylated regions (DMRs) in embryos, each derived from a single day 11.5 to day 13.5 PGC by nuclear transfer. Cloned embryos produced from day 12.5 to day 13.5 PGCs showed growth retardation and early embryonic lethality around day 9.5. Imprinted genes lost their parental-origin-specific expression patterns completely and became biallelic or silenced. We confirmed that clones derived from both male and female PGCs gave the same result, demonstrating the existence of a common default state of genomic imprinting to male and female germlines. When we produced clone embryos from day 11.5 PGCs, their development was significantly improved, allowing them to survive until at least the day 11.5 embryonic stage. Interestingly, several intermediate states of genomic imprinting between somatic cell states and the default states were seen in these embryos. Loss of the monoallelic expression of imprinted genes proceeded in a step-wise manner coordinated specifically for each imprinted gene. DNA demethylation of the DMRs of the imprinted genes in exact accordance with the loss of their imprinted monoallelic expression was also observed. Analysis of DNA methylation in day 10.5 to day 12.5 PGCs demonstrated that PGC clones represented the DNA methylation status of donor PGCs well. These findings provide strong evidence that the erasure process of genomic imprinting memory proceeds in the day 10.5 to day 11.5 PGCs, with the timing precisely controlled for each imprinted gene. The nuclear transfer technique enabled us to analyze the imprinting status of each PGC and clearly demonstrated a close relationship between expression and DNA methylation patterns and the ability of imprinted genes to support development.

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