| Budget Amount *help |
¥4,000,000 (Direct Cost: ¥4,000,000)
Fiscal Year 1993: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1992: ¥3,000,000 (Direct Cost: ¥3,000,000)
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| Research Abstract |
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.
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