Targeted insertion results in a rhombomere 2-specific Hoxa2 knockdown and ectopic activation of Hoxa1 expression
Targeted insertion results in a rhombomere 2-specific Hoxa2 knockdown and ectopic activation of Hoxa1 expression
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DOI：
10.1002/dvdy.10171
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发表时间：
2002-11-01
影响因子：
2.5
通讯作者：
Rijli, FM
中科院分区：
文献类型：
--
作者：
Ren, SY;Angrand, PO;Rijli, FM
最近的研究表明，由于邻近效应，在靶向Hox基因座中保留选择性标记盒可能会导致突变小鼠出现意想不到的表型。然而，其分子机制研究甚少。在这里，我们分析了在Hoxa 2的3'非翻译区中靶向插入PGK-neo盒的效果。即使在该3'位置，插入也导致纯合突变体意外地不能存活超过3周龄。靶向等位基因的分子分析揭示了选择性的“敲低”Hoxa 2在菱形体2中的表达和相关的模式异常。此外，Hoxa 1在突变胚胎的后脑和鳃弓中异位表达。有趣的是，我们证明了异位表达是由于产生新的HSP 1融合转录，导致异常的选择性剪接。这些缺陷可以通过Flp介导的重组去除PGK-neo盒后被挽救。这些结果强调了在Hox基因座转录调控的复杂性，并提供了深入了解体内调控Hoxa 2节段性表达。它们还提供了用于解释意外的Hox敲除表型的分子基础，其中靶向选择标记保留在基因座中。(C)2002 Wiley-Liss，Inc.
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-Hoxal1 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. (C) 2002 Wiley-Liss, Inc.