Imprinted genes occur in clusters, several of which are regulated by a macro long non-protein-coding (lnc) RNA. Inner genes within the cluster can be overlapped by the macro lncRNA and show multi-lineage (ML) imprinted expression in the embryo and extra-embryonic tissues, while outer genes are not overlapped by the macro lncRNA and show extra-embryonic-lineage (EXEL) specific imprinted expression. Extra-embryonic tissues are a mixture of ML and EXEL cell types, so imprinted expression may be

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... ked by biallelic expression in ML tissues if a whole organ is examined. We have developed an efficient method to isolate a pure population of visceral endoderm (VE), an EXEL cell type, providing a system to discover new imprinted genes as EXEL tissues show imprinted expression of both, ML and EXEL genes, and masking is avoided. Using this system I have taken advantage of the T-hairpin uniparental deletion to systematically check for the limits of the well-characterised Igf2r cluster. Imprinted genes can be definitively shown to be part of these Igf2r cluster by determining if they are regulated by the Airn macro lncRNA. This is done by comparing wildtype embryos with those containing a truncated and non-functional Airn. Using this system I have tested 15 genes and identified three genes showing biased imprinted expression in VE and shown that they are part of the Igf2r cluster. In addition, I have preliminary data confirming a recent report that Pde10a shows imprinted expression in placenta, and have shown that imprinted silencing is regulated by Airn. Taken together these results extend the known region showing imprinted expression in the Igf2r cluster from 440Kb up to 4Mb in extra-embryonic tissues.