A method for the generation of conditional gene repair mutations in mice
A method for the generation of conditional gene repair mutations in mice
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DOI：
10.1093/nar/29.3.e10
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发表时间：
2001-02-01
影响因子：
14.9
通讯作者：
Zeitlin, S
中科院分区：
文献类型：
--
作者：
Dragatsis, I;Zeitlin, S
小鼠的条件性基因修复突变可以帮助细胞谱系分析，并为条件性基因失活策略提供有价值的补充。我们提出了一种产生条件性基因修复突变的方法，该方法使用位于目标基因第一内含子内的IoxP侧翼(漂浮式)可选择标记和转录/翻译停止盒(NeStop)。在没有Cre重组酶的情况下，目标等位基因的表达被抑制，产生零等位基因，而在Cre存在的情况下，新STOP的切除将使表达恢复到野生型水平。为了测试这一策略，我们生成了小鼠亨廷顿病基因同源(HDH)的条件基因修复等位基因。在hdh内含子1插入新的STOP产生了一个空等位基因，该等位基因纯合的小鼠类似于无效的hdh突变，并在胚胎8.5天后死亡。在发育早期普遍表达的OREE转基因的存在下，切除新的STOP恢复了HDH的表达，挽救了早期的胚胎死亡。讨论了这一策略的简单修改，允许使用一个靶向构建体产生常规基因敲除、条件性基因敲除和条件性基因修复等位基因。
Conditional gene repair mutations in the mouse can assist in cell lineage analyses and provide a valuable complement to conditional gene inactivation strategies. We present a method for the generation of conditional gene repair mutations that employs a IoxP-flanked (floxed) selectable marker and tran-scriptional/translational stop cassette (neostop) located within the first intron of a target gene. In the absence of Cre recombinase, expression of the targeted allele is suppressed generating a null allele, while in the presence of Cre, excision of neostop restores expression to wild-type levels. To test this strategy, we have generated a conditional gene repair allele of the mouse Huntington's disease gene homolog (Hdh). Insertion of neostop within the Hdh intron 1 generated a null allele and mice homozygous for this allele resembled nullizygous Hdh mutants and died after embryonic day 8.5. In the presence of a ore transgene expressed ubiquitously early in development, excision of neostop restored Hdh expression and rescued the early embryonic lethality. A simple modification of this strategy that permits the generation of conventional gene knockout, conditional gene knockout and conditional gene repair alleles using one targeting construct is discussed.