Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator-like effector nucleases
Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator-like effector nucleases
复制标题
DOI：
10.1111/dgd.12105
复制
发表时间：
2014-01-01
影响因子：
2.5
通讯作者：
Suzuki, Ken-ichi T.
中科院分区：
文献类型：
--
作者：
Sakane, Yuto;Sakuma, Tetsushi;Suzuki, Ken-ichi T.
转录激活剂样效应核酸酶（TALEN）已广泛用于各种生物体的基因组编辑。然而，在某些情况下，由于非洲爪蟾的多倍体性，很难使用一对 TALEN 有效破坏非洲爪蟾的两个旁系同源基因。在这里，我们报告了使用两对 TALEN 在 X.laevis 中进行多个和旁系同源基因的定向诱变。首先，我们通过在携带egfp的转基因胚胎中注射两对TALENs，展示了酪氨酸酶旁系同源物（tyra和tyrb）和增强型绿色荧光蛋白（egfp）这三个基因的同时定向诱变。 tyr旁系同源物和egfp的移码突变率达到40-80%，这与白化病和GFP荧光丧失这两种严重表型性状的高频率一致。接下来，我们展示了在胚胎发生过程中早期引入 TALEN 介导的这些靶基因座的诱变。最后，我们还证明两对不同的 TALEN 可以同时高效地向编码组蛋白伴侣的两个旁系同源物引入突变。我们的结果表明，使用 TALEN 对多个基因进行定向诱变可用于分析非洲爪蟾中具有冗余性的旁系同源基因的功能。
Transcription activator-like effector nucleases (TALENs) have been extensively used in genome editing in various organisms. In some cases, however, it is difficult to efficiently disrupt both paralogous genes using a single pair of TALENs in Xenopus laevis because of its polyploidy. Here, we report targeted mutagenesis of multiple and paralogous genes using two pairs of TALENs in X.laevis. First, we show simultaneous targeted mutagenesis of three genes, tyrosinase paralogues (tyra and tyrb) and enhanced green fluorescent protein (egfp) by injection of two TALENs pairs in transgenic embryos carrying egfp. Consistent with the high frequency of both severe phenotypic traits, albinism and loss of GFP fluorescence, frameshift mutation rates of tyr paralogues and egfp reached 40-80%. Next, we show early introduction of TALEN-mediated mutagenesis of these target loci during embryogenesis. Finally, we also demonstrate that two different pairs of TALENs can simultaneously introduce mutations to both paralogues encoding histone chaperone with high efficiency. Our results suggest that targeted mutagenesis of multiple genes using TALENs can be applied to analyze the functions of paralogous genes with redundancy in X.laevis.