Ren Shu-Yue, Angrand Pierre-Olivier, Rijli Filippo M
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, Illkirch Cedex, CU de Strasbourg, France.
Dev Dyn. 2002 Nov;225(3):305-15. doi: 10.1002/dvdy.10171.
Recent studies indicated that retention of selectable marker cassettes in targeted Hox loci may cause unexpected phenotypes in mutant mice, due to neighborhood effects. However, the molecular mechanisms have been poorly investigated. Here, we analysed the effects of the targeted insertion of a PGK-neo cassette in the 3' untranslated region of Hoxa2. Even at this 3' position, the insertion resulted in homozygous mutants that unexpectedly did not survive beyond 3 weeks of age. Molecular analysis of the targeted allele revealed a selective "knockdown" of Hoxa2 expression in rhombomere 2 and associated patterning abnormalities. Moreover, Hoxa1 was ectopically expressed in the hindbrain and branchial arches of mutant embryos. Of interest, we demonstrated that the ectopic expression was due to the generation of neo-Hoxa1 fusion transcripts, resulting from aberrant alternative splicing. These defects could be rescued after removal of the PGK-neo cassette by Flp-mediated recombination. These results underscore the complexity of transcriptional regulation at Hox loci and provide insights into the in vivo regulation of Hoxa2 segmental expression. They also provide a molecular basis for the interpretation of unexpected Hox knockout phenotypes in which the targeted selectable marker is retained in the locus.
最近的研究表明，由于邻域效应，靶向Hox基因座中选择标记盒的保留可能会在突变小鼠中导致意外的表型。然而，其分子机制尚未得到充分研究。在这里，我们分析了在Hoxa2的3'非翻译区靶向插入PGK-neo盒的影响。即使在这个3'位置，插入也导致纯合突变体意外地在3周龄后无法存活。对靶向等位基因的分子分析揭示了Hoxa2在菱脑节2中的选择性“敲低”以及相关的模式异常。此外，Hoxa1在突变胚胎的后脑和鳃弓中异位表达。有趣的是，我们证明这种异位表达是由于异常的可变剪接产生了neo-Hoxa1融合转录本。通过Flp介导的重组去除PGK-neo盒后，这些缺陷可以得到挽救。这些结果强调了Hox基因座转录调控的复杂性，并为Hoxa2节段性表达的体内调控提供了见解。它们还为解释在基因座中保留靶向选择标记的意外Hox基因敲除表型提供了分子基础。
