Analysis of Genomic Imprinting Genes in Mammals

父本和母本基因组在哺乳动物发育过程中发挥着不同的作用。一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，我们开发了新的减法与PCR相结合的方法来系统地分离这些基因。受精胚胎包含父亲和母亲的基因组，另一方面，孤雌生殖胚胎只包含母亲的基因组。因此，我们可以通过从父本基因组中减去后者的cDNA，获得特异性表达的基因。孤雌胚胎最多可在第9天发育（25-somite期）。由于这一阶段的小鼠胚胎非常小，很难获得足够数量的胚胎来制作cDNA文库并进行减法克隆。为了解决这个问题，我们用PCR扩增了小鼠的cdna。通过在cDNA两侧引入特异性连接体，并改进PCR的若干反应条件，可以扩增出0.5 10kb片段组成的cDNA，且无明显偏倚。此外，利用两种类型的连接体构建不同的cDNA文库，我们可以在两种不同cDNA的混合物中只扩增出一种类型的cDNA。在这些cdna之间进行了一系列的减法。第三次减法后的结果表明，该方法有效地浓缩了父本特异性基因lgf-2，而非特异性基因β -actin几乎降低到本底水平。目前为止，用减去的cDNA作为探针获得了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检查。我们希望通过对基因组印迹相关基因的分析，进一步了解基因组印迹相关基因的数量和功能，阐明这一现象的生物学意义。

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.