The development of gene targeting approaches has had a tremendous impact on the functional analysis of the mouse genome. A specific application of this technique has been the adaptation of the bacteriophage P1 Cre/loxP site-specific recombinase system which allows for the precise recombination between two loxP sites, resulting in deletion or inversion of the intervening sequences. Because of the efficiency of this system, it can be applied to conditional deletions of relatively short coding sequences or regulatory elements but also to more extensive chromosomal rearrangement strategies. Both mechanistic and functional studies of genomic imprinting have benefited from the development of the Cre/loxP technology. Since imprinted genes within large chromosomal regions are regulated by the action of cis-acting sequences known as imprinting centers, chromosomal engineering approaches are particularly well suited to the elucidation of long-range mechanisms controlling the imprinting of autosomal genes. Here we review the applications of the Cre/loxP technology to the study of genomic imprinting, highlight important insights gained from these studies and discuss future directions in the field.

Some insight into the developmental basis for imprinting specific genes during the evolution of mammals can be gained from conventional gene ‘knockout’ studies. However, the consequences of full loss of function are often wide-ranging and may obscure the critical, dosage-related phenotype. This review focuses on transgenic techniques employed to alter the dosage of imprinted genes, including the application of bacterial artificial chromosome transgenic mice, in imprinting research. Advantages of dosage-based techniques over conventional knockout studies will be discussed, with examples. Important applications of transgenic mice in imprinting research, including studying gene expression patterns, the identification of imprinting centres and isolating the consequences of altered gene dosage, are reviewed with a particular focus on the imprinted domain on mouse distal chromosome 7.

In the mid-1980s, elegant studies on mouse embryos revealed that both parental genomes are required for normal development leading to the discovery of genomic imprinting. Imprinting is a parent-of-origin-dependent epigenetic mechanism whereby a subset of autosomal genes is expressed from only one of the parental alleles. Imprinting control involves both DNA- and histone-methylation, which differentially mark the parental alleles. More than a hundred imprinted genes have been identified so far, many of which play important roles in the regulation of growth and development. Nonetheless, the full extent of imprinting and its biological functions remain underestimated. In this review, we describe recently developed strategies to identify novel imprinted genes and highlight the potential of combining several high throughput approaches. By integrating databases obtained from epigenome- and transcriptome-wide analyses, we now have the unique opportunity to identify all the imprinted genes in the human/mouse genomes.

Genomic imprinting is the differential expression of genes according to their transmitting parent and is achieved by labelling of the two alleles with different epigenetic marks. The majority of described imprinted genes are present in clusters with coordinate regulation. Multiple mechanisms are known to regulate this differential expression, including repression of one allele by the action of cis-acting macro non-coding RNAs, insulator elements, allele specific histone modifications and DNA methylation. A hallmark of all imprinted regions described so far is the presence of one or more differentially methylated regions (DMRs). A DMR is a nucleotide sequence rich in CpG dinucleotides that is specifically methylated on one parental chromosome and unmethylated on the allele derived from the other parent. This parent-specific differential methylation may be imparted during spermatogenesis or oogenesis (as is the case for gametic DMRs) or may be acquired during embryogenesis (somatic DMRs). This review will describe the advantages and disadvantages of some of the techniques that can be used to compare epigenetic marks between parental chromosomes and to understand how these marks affect the 3D interactions and monoallelic expression at imprinted loci. Recent advances in sequencing technologies, in particular, provide exciting new opportunities to study imprinting. These analyses are likely to lead to the full characterization of the ‘imprintome’, which includes uncovering the totality of imprinted genes within a genome, their epigenetic landscape and unique features that render them resistant to epigenetic reprogramming in the early embryo.

Genetic events alone cannot explain the entire process of carcinogenesis. It is estimated that there are more epigenetic alterations in cancer than DNA mutations, and disiphering driver and secondary events is essential to understand early processes of tumorigenesis. Epigenetic modifications control gene activity, governing whether a gene is transcribed or silent. In cancer, global patterns of two epigenetic marks, histone modifications and DNA methylation, are known to be extensively deregulated. Tumour cells are also characterized by loss-of-imprinting, a key epigenetic developmental mechanism. Genomic imprinting is the parent-of-origin, monoallelic expression of genes and is controlled by differentially DNA-methylated regions and allelic-histone modifications. With specific emphasis on imprinted loci this review will discuss alterations in DNA methylation and histone modifications in cancer. The recent advances in technology that might facilitate the identification and characterization of the epigenetic profiles of cancer will also be described.

Studies of large imprinted clusters, such as the Gnas locus, have revealed much about the significance of DNA methylation, transcription and other factors in the establishment and maintenance of imprinted gene expression. However, the complexity of such loci can make manipulating them and interpreting the results challenging. We review here a distinct class of imprinted genes, which have arisen by retrotransposition, and which have the potential to be used as models for the dissection of the fundamental features and mechanisms required for imprinting. They are also of interest in their own right, generating diversity in the transcriptome and providing raw material upon which selection can act.

Recent data have revealed that the paradigmatic tumour suppressor gene RB1 on chromosome 13 is preferentially expressed from the maternal allele. Imprinted expression of RB1 is linked to a differentially methylated CpG island in intron 2 of this gene (CpG 85). On the paternal chromosome, CpG 85 is unmethylated and acts as a weak promoter of an alternative RB1 transcript. Paternal mRNA levels are probably reduced as the result of transcriptional interference of the regular promoter and the alternative promoter on this chromosome. CpG 85 is part of a truncated processed pseudogene (KIAA0649P) that integrated into the RB1 gene prior to the speciation of extant primates. It is plausible that differential penetrance and variation of age at diagnosis, which have been observed in patients with hereditary and non-hereditary retinoblastoma, respectively, are a consequence of imprinted expression of the RB1 gene. Interestingly, RB1 is imprinted in the same direction as CDKN1C, which operates upstream of RB1. The imprinting of two components of the same pathway indicates that there has been strong evolutionary selection for maternal inhibition of cell proliferation.