Analysis of Genomic Imprinting Genes in Mammals

哺乳动物基因组印记基因分析

• 批准号: 04455013
• 负责人: ISHINO Fumitoshi
• 金额: $ 2.56万
• 依托单位: Tokyo Institute of Technology
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04455013/
• 关键词: genomic imprinting; subtractive cloning; parthenogenesis; PCR; molecular biology; development; early embryos; cDNAライブラリー; サブトラクション; ゲノミック・インプリンテング; 遺伝子増幅法

Paternal and maternal genomes play different role in mammalian development. Different expression of some kind of genes seems to be the mechanism of genomic imprinting. In order to elucidate the biological importance of the genomic imprinting, we developed new subtraction method combined with PCR to isolate such genes systematically. Fertilized embryos contain both paternal and maternal genomes, on the other hand, parthenogenetic embryos contain only maternal genomes. Therefore, we could obtain genes specifically expressed from paternal genome by subtraction the latter cDNA from the former cDNA.Parthenogenetic embryos could develop at most day 9 (25-somite stage). It is very difficult to obtain enough amout of embryols to make cDNA library and to carry out subtractive cloning, because the mouse embryos at this stage are very small. In order to solve this problem, we amplified the mouse cDNAs with PCR.By introducing specific linkers at both side of cDNA,and improving several reaction conditions of PCR,cDNAs which are consisted with 0.5 10kb fragments were able to amplified without significant bias. Moreover, using two types of linkers to make different cDNA library, we can amplify only one type of cDNA between the mixture of two different cDNA.A series of subtraction were carried out between these cDNAs. Results after third cycle of subtraction showed that lgf-2 (paternal specific gene) was effectively concentrated in this method, while beta-actin (non-specific gene) was decreased almost to the background level. So far, four paternally expressed genes (including 2 new genes) were obtained by using the subtracted cDNA as probes. More extensive screening are now being carried out. We hope we get more information about the number and the function of genes concerning genomic imprinting to elucidate the biological importance of this phenomenon by analyzing these genes.

在哺乳动物的发育过程中，父源基因组和母源基因组起着不同的作用。某些基因的差异表达可能是基因组印记的机制。为了阐明基因组印记的生物学意义，我们发展了一种新的消减法结合PCR技术系统地分离基因组印记基因。受精胚胎含有父本和母本基因组，而孤雌生殖胚胎只含有母本基因组。因此，我们可以通过从前一个cDNA中减去后一个cDNA来获得在父本基因组中特异表达的基因。孤雌胚胎最多可发育到第9天（25体节期）。由于这一阶段的小鼠胚胎体积很小，很难获得足够数量的胚胎用于制作cDNA文库和进行差减克隆。为了解决这一问题，我们采用PCR方法扩增小鼠cDNA，通过在cDNA两端引入特异性连接子，并对PCR反应条件进行了改进，使扩增出的cDNA片段大小为0.5 ~ 10 kb，无明显偏倚。此外，利用两种接头构建不同的cDNA文库，在两种不同cDNA的混合物中只能扩增出一种cDNA，并对这两种cDNA进行了一系列的差减。第三次消减后的结果表明，该方法有效地浓缩了lgf-2（父本特异性基因），而β-actin（非特异性基因）几乎降至背景水平。迄今为止，利用扣除的cDNA作为探针，获得了4个父本表达基因（包括2个新基因）。目前正在进行更广泛的筛查。我们希望通过对基因组印记相关基因的分析，获得更多关于基因组印记相关基因的数量和功能的信息，从而阐明这一现象的生物学意义。

项目成果

T.Kneko-Ishino et al: "Genes differentially expressed between fertilized and parthenogenetic embryos in the mouse" (in preparation). (1995)

T.Kneko-Ishino 等人：“小鼠受精胚胎和孤雌胚胎之间差异表达的基因”（准备中）。

T.KANEKO-ISHINO el al: "Genes differentially expressed between fertilized and parthenogenetic embryos in the mouse." (in preparation). (1995)

T.KANEKO-ISHINO 等人：“小鼠受精胚胎和孤雌胚胎之间表达差异的基因。”

F.Ishino et al: "Screening of imprinting genes in the mouse" Abstracts in The Eighth International Conference of the International Society of Differentiation Hiroshima. 186 (1994)

F.Ishino 等人：《小鼠印记基因的筛选》摘要，刊登于广岛国际分化学会第八届国际会议。

F.ISHINO et al: "Screening of imprinting in the mouse." Abstracts in The Eighth International Conference of the International Society of Differentiation.186- (1994)

F.ISHINO 等人：“小鼠印记的筛选。”