BACs as tools for the study of genomic imprinting

Abstract

Genomic imprinting in mammals results in the expression of genes from only one parental allele. Imprinting occurs as a consequence of epigenetic marks set down either in the father's or the mother's germ line and affects a very specific category of mammalian gene. A greater understanding of this distinctive phenomenon can be gained from studies using large genomic clones, called bacterial artificial chromosomes (BACs). Here, we review the important applications of BACs to imprinting research, covering physical mapping studies and the use of BACs as transgenes in mice to study gene expression patterns, to identify imprinting centres, and to isolate the consequences of altered gene dosage. We also highlight the significant and unique advantages that rapid BAC engineering brings to genomic imprinting research.

Figure 1

During mammalian gametogenesis, the diploid primordial germ cell undergoes meiosis to produce haploid male and female gametes. As meiosis progresses, specific DNA sequences acquire a DNA methylation imprint (black lollypops) in one parental germline but not the other. After fertilization, this imprint is recognized within the somatic cells establishing an imprinted domain. Maternal inheritance is indicated in red and paternal inheritance is indicated in blue.

Figure 2

Mouse distal chromosome 7 contains two complex imprinted loci. (a) A diagrammatic representation of mouse distal chromosome 7 with maternal inheritance indicated in red and paternal inheritance indicated in blue. Genes are represented by the black boxes with the parent-of-origin expression indicated by either a red (maternal) or blue (paternal) arrow. Biallelic expression is indicated by a black arrow. Noncoding RNAs are represented by dashed arrows. DNA methylation is represented by the black lollypops. Green boxes indicate position of ICs whilst green doubleheaded arrows indicate the region influenced by the IC. (b) BAC clones that are used to examine expression, imprinting, and function. Blue boxes indicate the position of lacZ insertion. (c) Images of transgenic embryos inheriting the modified BACs through either the maternal or paternal germline as indicated by gender symbols below image. Expression of Cdkn1c from the BAC is revealed by lacZ reporter expression.

Figure 3

Mouse distal chromosome 2 contains a minimalistic imprinted locus. (a) A diagrammatic representation of mouse distal chromosome 2 imprinted region with maternal inheritance indicated in red and paternal inheritance indicated in blue. Genes are represented by the boxes with the parent-of-origin expression indicated by either a red (maternal) or blue (paternal) arrow. DNA methylation is represented by the black lollypops. Green box indicates position of proposed IC whilst green doubleheaded arrow indicates the known region influenced by this IC. (b) BAC clones and subclone used to examine expression, imprinting, and function. Blue boxes indicate the position of lacZ reporter. (c) Images of transgenic embryos inheriting the modified BACs through either the maternal or paternal germline as indicated by gender symbols below image. Expression of Neuronatin from the transgenic insertion is revealed by lacZ reporter expression.