Analysis of Genomic Imprinting Genes in Mammals

父本基因组和母本基因组在哺乳动物发育过程中发挥着不同的作用。某些基因的不同表达似乎是基因组印记的机制。为了阐明基因组印迹的生物学意义，我们建立了一种新的消减方法并结合聚合酶链式反应来系统分离这些基因。受精胚胎同时含有父系和母系基因组，而孤雌生殖胚胎只含有母系基因组。因此，从父本基因组中减去后者的cDNA，就可以获得父本基因组中特异表达的基因。孤雌生殖胚胎最多可以发育到第9天(25-体节体期)。由于现阶段小鼠胚胎数量较小，很难获得足够数量的胚胎来构建文库并进行差减克隆。为了解决这个问题，我们用PCR扩增了小鼠的cDNAs，通过在cDNAs的两侧引入特定的连接子，并改进了几种反应条件，使含有0.510kb片段的cDNAs能够在没有明显偏倚的情况下被扩增出来。此外，用两种不同类型的连接子构建不同的文库，在两种不同的cDNA混合物之间只能扩增出一种类型的cdna，并在这些cDNA之间进行了一系列的消减。结果经过三次差减后，LGF-2(父系特异性基因)在本方法中得到了有效的浓缩，而β-肌动蛋白(非特异性基因)则几乎降到了背景水平。到目前为止，以消减后的cDNA为探针，获得了4个父系表达基因(包括2个新基因)。目前正在进行更广泛的筛查。我们希望通过对这些基因的分析，获得更多关于基因组印迹相关基因的数量和功能的信息，以阐明这种现象的生物学意义。

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.