Gene targeting of ErbB3 using a Cre-mediated unidirectional DNA inversion strategy
Gene targeting of ErbB3 using a Cre-mediated unidirectional DNA inversion strategy
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DOI：
10.1002/dvg.20243
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发表时间：
2006-10-01
期刊：
影响因子：
1.5
通讯作者：
Arteaga, Carlos L.
中科院分区：
文献类型：
--
作者：
Qu, Shimian;Rinehart, Cammie;Arteaga, Carlos L.
DNA酶介导的单向DNA倒位和转录阻滞是一种有前途的小鼠高通量条件突变策略。具有可由Cre重组酶激活的确定的转录沉默插入的小鼠胚胎干细胞库将利用现有的转基因Cre系来快速产生数百个谱系特异性和时间控制的每个基因的敲除小鼠，从而引入功能基因注释的显著平行性。然而，这种策略在多大程度上导致有效的基因敲除尚未建立。为了测试这种策略的可行性，我们使用这种策略靶向ErbB 3，ErbB酪氨酸激酶受体家族的成员。插入一个反向的“flipflox”载体组成的基因失活盒（GI）和内部核糖体进入位点（IRES）-GFP报告到ErbB 3的内含子1是转录沉默，并没有，不影响ErbB 3的表达。与普遍存在的和谱系特异性Cre重组酶表达系的杂交永久地反转插入的GI盒并阻断ErbB 3表达。通过体内重组实现DNA的单向倒位是一种有效的靶向或普遍存在的基因敲除策略。
Recombinase-mediated unidirectional DNA inversion and transcriptional arrest is a promising strategy for high throughput conditional mutagenesis in the mouse. Banks of mouse embryonic stem cells with defined, transcriptionally silent insertions that can be activated by Cre recombinase would take advantage of existing transgenic Cre lines to rapidly produce hundreds of lineage specific and temporally controlled knockout mice for each gene, thereby introducing significant parallelism to functional gene annotation. However, the extent to which this strategy results in effective gene knockout has not been established. To test the feasibility of this strategy we targeted ErbB3, a member of the ErbB family of tyrosine kinase receptors, using this strategy. Insertion of a reversed "flipflox" vector consisting of a gene inactivation cassette (GI) and an internal ribosome entry site (IRES)-GFP reporter into intron 1 of ErbB3 was transcriptionally silent and did, not affect ErbB3 expression. Crosses with ubiquitous and lineage specific Cre recombinase expressing lines permanently inverted the inserted GI cassette and blocked ErbB3 expression. Unidirectional DNA inversion by in vivo recombination is an effective strategy for targeted or ubiquitous gene knockout.