Isolation of embryonic cDNA subtracted by parthenogenic driver cDNA

Abstract

Genomic imprinting by which, depending on their parental origin, certain genes are depressed, was first described in the studies of fertilized mouse embryos which had been reconstituted using nuclear transplantation technique. This was also supported by the studies of chromosomal translocations. Recent progress on the identifications of several imprinted genes made trials to explain the biological teleonomy and epigenetic mechanism of genomic imprinting. It is believed that DNA methylation may be responsible for discriminating parental genomes. However, the number of identified genes is too small to understand the biology of genomic imprinting. In this study, it was attempted to screen the imprinted genes. Because the parthenogenetic embryos carry only maternal genomes, maternally imprinted genes are believed to be depressed in these embryos. Therefore, it was designed to isolate the genes that are expressed in normal embryos while silent in parthenogenons. Total RNAs were isolated from the parthenogenetic and normal embryos. RNA was then served as template for cDNA synthesis. cDNAs were amplified by PCR. Parthenogenetic cDNA was used as driver DNAs which eliminate cDNAs common in both embryos from normal embryonic cDNA pool. Subtracted DNA was then rescreened by differential hybridization method. Seven clones were selected, and two of them were sequenced. These two sequences were absent in public databases. Instead, canl sequence showed partial homologies with an EST sequence which was sequenced from cDNA library of human leg tissues.