Simple, fast, tissue-specific bacterial artificial chromosome transgenesis in Xenopus.
Simple, fast, tissue-specific bacterial artificial chromosome transgenesis in Xenopus.
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DOI：
10.1002/dvg.20819
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发表时间：
2012-03
期刊：
影响因子：
1.5
通讯作者：
Grainger, Robert M.
中科院分区：
文献类型：
--
作者：
Fish, Margaret B.;Nakayama, Takuya;Grainger, Robert M.
我们已经开发了一种方法，细菌人工染色体（BAC）DNA注入非洲爪蟾胚胎，是简单而有效的，并在一致的和组织特异性表达的转基因克隆到BAC载体的结果。当研究大的或复杂的基因时，与大的DNA片段一起工作是必要的，因为可以由BAC容纳，并且有助于研究用于控制发育限制基因表达的长程调控元件的功能。我们将荧光报告基因重组到三个非洲爪蟾BAC克隆中，靶向三个不同的基因，并报告高达60%的注射胚胎以与内源性表达一致的方式表达报告基因。这些BAC在注射后复制的行为与较小质粒的行为形成对比，较小质粒在作为环状分子注射时降解相对较快，并且在未整合到非洲爪蟾基因组中时通常不能重现内源性表达。
We have developed a method of injecting Bacterial Artificial Chromosome (BAC) DNA into Xenopus embryos that is simple and efficient, and results in consistent and tissue-specific expression of transgenes cloned into BAC vectors. Working with large pieces of DNA, as can be accommodated by BACs, is necessary when studying large or complex genes and conducive to studying the function of long-range regulatory elements that act to control developmentally restricted gene expression. We recombineered fluorescent reporters into three Xenopus tropicalis BAC clones targeting three different genes and report that up to 60% of injected embryos express the reporter in a manner consistent with endogenous expression. The behavior of these BACs, which are replicated after injection, contrasts with that of smaller plasmids, which degrade relatively quickly when injected as circular molecules and generally fail to recapitulate endogenous expression when not integrated into the Xenopus genome.
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