{"created":"2023-06-20T13:20:55.083211+00:00","id":988,"links":{},"metadata":{"_buckets":{"deposit":"68048433-ee35-4cc0-bd9b-60122dd9f422"},"_deposit":{"created_by":1,"id":"988","owners":[1],"pid":{"revision_id":0,"type":"depid","value":"988"},"status":"published"},"_oai":{"id":"oai:ir.soken.ac.jp:00000988","sets":["2:430:20"]},"author_link":["298","299","297"],"item_1_creator_2":{"attribute_name":"著者名","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"金田, 正弘"}],"nameIdentifiers":[{}]}]},"item_1_creator_3":{"attribute_name":"フリガナ","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"カネダ, マサヒロ"}],"nameIdentifiers":[{}]}]},"item_1_date_granted_11":{"attribute_name":"学位授与年月日","attribute_value_mlt":[{"subitem_dategranted":"2003-09-30"}]},"item_1_degree_grantor_5":{"attribute_name":"学位授与機関","attribute_value_mlt":[{"subitem_degreegrantor":[{"subitem_degreegrantor_name":"総合研究大学院大学"}]}]},"item_1_degree_name_6":{"attribute_name":"学位名","attribute_value_mlt":[{"subitem_degreename":"博士（理学）"}]},"item_1_description_12":{"attribute_name":"要旨","attribute_value_mlt":[{"subitem_description":"Genomic imprinting refers to the parental-origin-specific gene expression of a subset of autosomal genes in mammals.  Disruption of imprinting causes embryonic or postnatal lethality, growth retardation, abnormal behavior and many human diseases.  It has been proposed that DNA methylation marks the imprinted genes differently during male and female gametogenesis.  The epigenetic differences (imprints) between the two gametes lead to parental-origin-specific  gene expression in the offspring.  These imprints are maintained during development but erased in the fetal germ cells, and then re-established during gametogenesis in a sex-specific manner.  Mice deficient for Dnmt1, which is a maintenance methyltransferase, show embryonic lethality, genome-wide demethylation and disruption of genomic imprinting.  This indicates Dnmt1 is essential for the maintenance of imprints postfertilization.  On the other hand, the primary germline imprints are thought to be established during gametogenesis through the action of a de novo DNA methyltransferase(s).  However, targeted disruption of the de novo DNA methyltransferases (Dnmt3a and Dnmt3b) in mice results in embryonic or early postnatal lethality.  Therefore, the role of DNA methylation in the establishment of the germline imprints cannot be addressed in these mice.<br />  To circumvent this problem, I took advantage of the Cre-loxP system to inactivate the two de novo DNA methyltransferases in a germline-specific manner.  The floxed Dnmt3a and Dnmt3b alleles were disrupted in both male and female germlines by introducing Cre recombinase driven by the endogenous TNAP (tissue non-specific alkaline phosphatase) promoter.  These germline-specific Dnmt3a and Dnmt3b knockout mice should be viable and expected to grow up to adulthood because the deletion should occur only in germ cells.  Analysis of the embryos and gametes from these germline-specific Dnmt3a and Dnmt3b knockout mice will determine the function of each enzyme in the establishment of genomic imprints.<br />  The conditional Dnmt3a knockout mice that I generated were indeed viable and grew up to adulthood, though somatic tissues had various degrees (56%-80%) of the deletion.  Recombination efficiency was determined in fetal germ cells, oocyte and sperm, which suggested that the Dnmt3a alleles was mostly inactivated before the onset of methylation imprints.  Offsprings from the conditional Dnmt3a knockout females crossed with wild-type males died around embryonic day 9.5-10.5 (E9.5-10.5).  All embryos that I examined had recombined allele only, suggesting that the recombination efficiency was 100%.  Embryos appeared grossly normal at E9.5, but they showed growth retardation, defects in neural tube closure and lack of branchial arches by E10.5. At E11.5, only resorptions were seen.  The phenotype was similar to that of the embryos conceived by Dnmt3L knockout females, which are defective in establishing the maternal methylation imprints during oogenesis.  Indeed, the maternal methylation imprints and the allele-specific expression of several imprinted genes that I examined were lost in the embryos conceived by the conditional Dnmt3a knockout mice.  These results indicate that Dnmt3a is a key enzyme that establishes maternal methylation imprints during oogenesis.  Since Dnmt3L does not have any detectable DNA methyltransferase activity, it is conceivable that Dnmt3a and Dnmt3L cooperate in the process of maternal methylation imprinting.  The conditional Dnmt3a knockout males were also viable but showed impaired spermatogenesis, again resembling the Dnmt3L knockout males.  At postnatal day 11 (P11), the testes from these conditional Dnmt3a knockout males appeared normal, but, at 11 weeks of age, the size and weight of the testes were significantly reduced.  Virtually no spermatids or spermatozoa were observed in the seminiferous tubules of these testes.  These results showed that Dnmt3a, as well as Dnmt3L, is required for spermatogenesis.<br />  Both the conditional Dnmt3b knockout males and females were viable and grew up to adulthood, in contrast to the Dnmt3b-null mice, which die in late gestation.  No deletion was observed in most somatic tissues examined, except for a small proportion of skeletal muscle cells.  Most fetal germ cells had recombined Dnmt3b allele, suggesting that the floxed Dnmt3b allele was mostly deleted before the onset of methylation imprints.  A total of 88 pups were born from the conditional Dnmt3b knockout females and 87 pups had the recombined allele, suggesting a high rate of recombination by Cre.  These pups grew up to adulthood and were fertile.  No abnormalities were observed in the embryos.  The methylation levels of the imprinted genes and minor satellite DNA, which is the target sequence of Dnmt3b, were also normal.  These results suggest that Dnmt3b is not required for oogenesis or the establishment of maternal methylation imprints.  The pups and embryos derived from the conditional Dnmt3b knockout males were also normal and showed 100% recombination efficiency (102/102).  No abnormal phenotype, imprinting defect, or change in methylation level was observed.  Also, histological sections of the testes from these conditional Dnmt3b knockout males showed normal spermatogenesis.  These results suggest that Dnmt3b is not required for spermatogenesis or the establishment of paternal methylation imprints.  I also examined whether the Dnmt3b allele recombined by Cre was functionally null.  Embryos homozygous for the recombined Dnmt3b allele were embryonic lethal and the phenotype (growth retardation, rostral neural defects and demethylation of centromeric minor satellite repeats) was very similar to that of Dnmt3b-null embryos.  These observations confirmed that the floxed Dnmt3b allele was successfully inactivated by the Cre recombinase.<br />  This work suggests that Dnmt3a, but not Dnmt3b, is responsible for the establishment of the maternal imprints.  The results from my work also suggest that the primary imprints established in gametogenesis is DNA methylation.  Dnmt3a is also required for spermatogenesis, but whether Dnmt3a is responsible for the establishment of paternal methylation imprints is yet to be investigated.","subitem_description_type":"Other"}]},"item_1_description_18":{"attribute_name":"フォーマット","attribute_value_mlt":[{"subitem_description":"application/pdf","subitem_description_type":"Other"}]},"item_1_description_7":{"attribute_name":"学位記番号","attribute_value_mlt":[{"subitem_description":"総研大甲第722号","subitem_description_type":"Other"}]},"item_1_select_14":{"attribute_name":"所蔵","attribute_value_mlt":[{"subitem_select_item":"有"}]},"item_1_select_8":{"attribute_name":"研究科","attribute_value_mlt":[{"subitem_select_item":"生命科学研究科"}]},"item_1_select_9":{"attribute_name":"専攻","attribute_value_mlt":[{"subitem_select_item":"18 遺伝学専攻"}]},"item_1_text_10":{"attribute_name":"学位授与年度","attribute_value_mlt":[{"subitem_text_value":"2003"}]},"item_creator":{"attribute_name":"著者","attribute_type":"creator","attribute_value_mlt":[{"creatorNames":[{"creatorName":"KANEDA, Masahiro","creatorNameLang":"en"}],"nameIdentifiers":[{}]}]},"item_files":{"attribute_name":"ファイル情報","attribute_type":"file","attribute_value_mlt":[{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲722_要旨.pdf","filesize":[{"value":"326.5 kB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"要旨・審査要旨 / Abstract, Screening Result","url":"https://ir.soken.ac.jp/record/988/files/甲722_要旨.pdf"},"version_id":"84b0664d-d9e6-4fa4-84df-590634c051ff"},{"accessrole":"open_date","date":[{"dateType":"Available","dateValue":"2016-02-17"}],"displaytype":"simple","filename":"甲722_本文.pdf","filesize":[{"value":"3.5 MB"}],"format":"application/pdf","licensetype":"license_11","mimetype":"application/pdf","url":{"label":"本文","url":"https://ir.soken.ac.jp/record/988/files/甲722_本文.pdf"},"version_id":"4d210ecf-c9ab-4e64-85c9-87bea63f363c"}]},"item_language":{"attribute_name":"言語","attribute_value_mlt":[{"subitem_language":"jpn"}]},"item_resource_type":{"attribute_name":"資源タイプ","attribute_value_mlt":[{"resourcetype":"thesis","resourceuri":"http://purl.org/coar/resource_type/c_46ec"}]},"item_title":"Role of de novo DNA methyltransferases  Dnmt3a and Dnmt3b in the establishment of  genomic imprinting.","item_titles":{"attribute_name":"タイトル","attribute_value_mlt":[{"subitem_title":"Role of de novo DNA methyltransferases  Dnmt3a and Dnmt3b in the establishment of  genomic imprinting."},{"subitem_title":"Role of de novo DNA methyltransferases Dnmt3a and Dnmt3b in the establishment of genomic imprinting.","subitem_title_language":"en"}]},"item_type_id":"1","owner":"1","path":["20"],"pubdate":{"attribute_name":"公開日","attribute_value":"2010-02-22"},"publish_date":"2010-02-22","publish_status":"0","recid":"988","relation_version_is_last":true,"title":["Role of de novo DNA methyltransferases  Dnmt3a and Dnmt3b in the establishment of  genomic imprinting."],"weko_creator_id":"1","weko_shared_id":1},"updated":"2023-06-20T14:40:36.089586+00:00"}