Duvillié B, Bucchini D, Tang T, Jami J, Pàldi A
Institut Cochin de Génétique Moléculaire, INSERM U257, Paris, France.
Genomics. 1998 Jan 1;47(1):52-7. doi: 10.1006/geno.1997.5070.
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 the Ins2 paternal allele in the visceral yolk sac during the last part of gestation indicates that Ins2 also is imprinted. However, in other tissues in which Ins2 is expressed, both alleles are active at all developmental stages. Taking advantage of two mouse strains carrying different null mutations introduced at the Ins2 locus via homologous recombination in ES cells, we examined whether genes inserted at the Ins2 locus become imprinted and have the same restricted pattern of monoallelic expression. In the first null allele, Ins2 was replaced by LacZ, under the control of the endogenous Ins2 promoter, and a Neo cassette with its own promoter was inserted 3' to LacZ (Zneo allele). In the second null allele, Ins2 and 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 x wildtype) F1 embryos, the pattern of LacZ expression was similar to that of Ins2; i.e., LacZ is 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, Neo was transcribed only when paternally inherited, in the yolk sac as well as in the embryo. Unexpectedly, we found that LacZ transcription 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 inherited LacZ is 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-range cis-acting effects. In addition, our data suggest that the two parental alleles may "communicate" with each other and influence the transcription at the locus.
编码胰岛素原2（Ins2）的小鼠基因位于7号染色体远端几百千碱基的区域内，该区域包含几个印记基因。在妊娠后期，Ins2父本等位基因在内脏卵黄囊中特异性表达，这表明Ins2也是印记基因。然而，在Ins2表达的其他组织中，两个等位基因在所有发育阶段均有活性。利用通过胚胎干细胞中的同源重组在Ins2基因座引入不同无效突变的两种小鼠品系，我们研究了插入Ins2基因座的基因是否会被印记并具有相同的单等位基因表达受限模式。在第一个无效等位基因中，Ins2被LacZ取代，由内源性Ins2启动子控制，并且带有自身启动子的Neo盒被插入到LacZ的3'端（Zneo等位基因）。在第二个无效等位基因中，Ins2及其启动子被相同的Neo盒取代（Neo等位基因）。通过对涉及两个突变体和野生型等位基因的各种正反交进行RT-PCR来监测母本和父本遗传基因的表达。在（Zneo×野生型）F1胚胎中，LacZ的表达模式与Ins2相似；即，LacZ仅在父本遗传时在卵黄囊中表达，而其在胚胎本身中的表达与其父本或母本来源无关。对于这两个突变等位基因，Neo仅在父本遗传时才转录，在卵黄囊和胚胎中均如此。出乎意料的是，我们发现母本染色体上的LacZ转录取决于父本染色体上等位基因的性质。当父本染色体携带野生型等位基因时，母本遗传的LacZ在胚胎中完全表达，而当父本等位基因为Neo等位基因时，母本遗传的LacZ则被抑制。我们结果的主要结论是，引入印记染色体区域的单个基因可以被印记，这表明存在长程顺式作用效应的影响。此外，我们的数据表明，两个亲本等位基因可能相互“交流”并影响该基因座的转录。
