Allele-specific detection of nascent transcripts by fluorescence in situ hybridization reveals temporal and culture-induced changes in Igf2 imprinting during pre-implantation mouse development

Abstract

Background: Genomic imprinting causes parental-origin-specific monoallelic transcription of a subset of mammalian genes in the embryo and adult. There is conflicting evidence, however, for the monoallelic transcription of some imprinted genes, such as Igf2, in pre-implantation embryos. Results: We have developed an allele-specific fluorescence in situ hybridization method which involves a pair of oligonucleotide probes designed to detect an intronic polymorphism. The method, called ASO-RNA-FISH, enabled us to distinguish allelic nascent Igf2 transcripts in the cell nuclei of early mouse embryos, avoiding signals from the stored oocyte-specific transcripts. Igf2 transcription was first detectable in two-cell embryos, and biallelic transcription was predominant up to the morula stage. Then, the maternal allele became silenced during the blastocyst stage. When embryos were cultured in vitro, however, a strong bias to maternal transcription was observed up to the morula stage. Conclusion: ASO-RNA-FISH revealed that a transition of Igf2 from biallelic to monoallelic transcription occurs in the blastocyst stage. This developmental regulation was modified temporarily by in vitro culture, suggesting a possible link between altered imprinting and abnormalities of the foetuses experienced in vitro culture. ASO-RNA-FISH is therefore a powerful technique for the study of allele-specific gene expression.