Imprinting at the MouseIns2Locus: Evidence forcis- andtrans-Allelic Interactions

The mouse gene encoding preproinsulin 2 (Ins2) is located on the distal end of chromosome 7 in a region of several hundred kilobases that contains several imprinted genes. The exclusive expression of theIns2paternal allele in the visceral yolk sac during the last part of gestation indicates thatIns2also is imprinted. However, in other tissues in whichIns2is expressed, both alleles are active at all developmental stages. Taking advantage of two mouse strains carrying different null mutations introduced at theIns2locus via homologous recombination in ES cells, we examined whether genes inserted at theIns2locus become imprinted and have the same restricted pattern of monoallelic expression. In the first null allele,Ins2was replaced byLacZ,under the control of the endogenousIns2promoter, and a Neo cassette with its own promoter was inserted 3′ toLacZ(Zneo allele). In the second null allele,Ins2and its promoter were replaced by the same Neo cassette (Neo allele). Expression of the maternally and paternally inherited genes was monitored by RT-PCR performed on various reciprocal crosses involving the two mutants and the wildtype alleles. In (Zneo × wildtype) F1 embryos, the pattern ofLacZexpression was similar to that ofIns2;i.e.,LacZis expressed in the yolk sac only when paternally inherited, while its expression in the embryo proper is independent of its paternal or maternal origin. For both of the mutant alleles,Neowas transcribed only when paternally inherited, in the yolk sac as well as in the embryo. Unexpectedly, we found thatLacZtranscription on the maternal chromosome varied depending on the nature of the allele on the paternal chromosome. While fully expressed in the embryo when the paternal chromosome carries the wildtype allele, the maternally inheritedLacZis extinguished when the paternal allele is the Neo allele. The major conclusion from our results is that individual genes introduced into an imprinted chromosomal domain can become imprinted, indicating the influence of long-rangecis-acting effects. In addition, our data suggest that the two parental alleles may “communicate” with each other and influence the transcription at the locus.