Germline transmission and efficient DNA recombination in mouse embryonic stem cells mediated by adenoviral-Cre transduction.
Germline transmission and efficient DNA recombination in mouse embryonic stem cells mediated by adenoviral-Cre transduction.
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腺病毒-Cre 转导介导的小鼠胚胎干细胞中的种系传递和有效 DNA 重组。
DOI：
10.1002/gene.20044
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Tan,Tse-Hua
中科院分区：
文献类型：
--
作者：
Shui,Jr-Wen;Tan,Tse-Hua
基因靶向后，aloxP-neo-loxPcassette 被引入 ES 细胞中。目标等位基因中存在选择性标记（例如 neo）可能会导致 flox 小鼠的基因干扰，或由于直接敲除小鼠的遗传模糊性而导致意外的表型。通常，neo 盒通过 Cre 重组酶在目标 ES 细胞中的瞬时表达而被选择性去除。然而，这种方法涉及选择、扩增和筛选 ES 细胞克隆的繁琐过程，这可能会损害种系能力。在这里，我们描述了一种将腺病毒-Cre介导的基因重组与ES基因靶向相结合的新方法，以促进ES细胞中有效的loxP-neo-loxPremoval。我们证明，腺病毒-Cre 感染的 ES 细胞可以保留其种系能力。这里描述的程序有助于对 ES 细胞进行快速遗传操作，以获得无新基因敲除动物、多基因靶向、适合体外表征的纯合突变 ES 细胞或 Rag 缺陷囊胚互补。创世纪 39:217–223, 2004。© 2004 Wiley‐Liss, Inc.
Following gene targeting, aloxP‐neo‐loxPcassette was introduced into ES cells. The presence of a selectable marker such as neo in the targeted allele may result in gene interference in flox mice or unexpected phenotypes due to genetic ambiguity in direct knockout mice. Typically, the neo cassette is selectively removed by transient expression of the Cre recombinase in targeted ES cell. However, this method involves a tedious process of selecting, expanding, and screening ES cell clones which may compromise germline competency. Here, we describe a novel method of combining adenovirus‐Cre mediated gene recombination with ES gene targeting to facilitate efficientloxP‐neo‐loxPremoval in ES cells. We demonstrate that adenovirus‐Cre infected ES cells can retain their germline competency. The procedures described here facilitate a rapid genetic manipulation of ES cells to obtain neo‐free knockout animals, multiple gene targeting, homozygous mutant ES cells ideal forin vitrocharacterization, orRag‐deficient blastocyst complementation. genesis 39:217–223, 2004. © 2004 Wiley‐Liss, Inc.