Forward genetic screens in Xenopus using transposon-mediated insertional mutagenesis.
Forward genetic screens in Xenopus using transposon-mediated insertional mutagenesis.
复制标题
使用转座子介导的插入诱变对非洲爪蟾进行正向遗传筛选。
DOI：
10.1007/978-1-61779-992-1_6
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发表时间：
2012
期刊：
影响因子：
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通讯作者：
Mead,PaulE
中科院分区：
文献类型：
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作者：
Yergeau,DonaldA;Kelley,ClairM;Zhu,Haiqing;Kuliyev,Emin;Mead,PaulE
II类DNA“剪切粘贴”转座子已被用来有效地修改Xenopus基因组以用于转基因应用。一旦整合，转座子就是一种有效的底物，只需提供转座酶反式，就可以切除和重新整合(重新整合)到基因组的其他地方。我们已经使用了两种方法来重新动员驻留在青蛙基因组中的转座子：在单细胞期显微注射转座酶mRNA和通过转基因在种系中表达转座酶。同时携带底物转座子和转座酶转基因的双转基因青蛙(跳蛙)与野生型动物异交，并根据报告基因表达的变化对后代进行评分。虽然这两种方法都能有效地重新动员转座子，但育种介导的策略省去了耗时的微注射步骤；新的整合事件是通过简单地与漏斗蛙杂交产生的。由于非洲爪哇的每个异交通常产生2,000个或更多的后代，这种方法可以用来在这个高度易处理的发育模型系统中进行大规模的插入突变筛选。
The class II DNA “cut-and-paste” transposons have been used to efficiently modify theXenopusgenome for transgenesis applications. Once integrated, the transposon is an effective substrate for excision and re-integration (remobilization) elsewhere in the genome by simply supplying the transposase enzymein trans. We have used two methods to remobilize transposons resident in the frog genome: micro-injection of transposase mRNA at the one-cell stage and expression of the enzyme in the germline from a transgene. Double-transgenic frogs (hoppers) that harbor transgenes for both the substrate transposon and the transposase enzyme are outcrossed to wild-type animals and the progeny are scored for changes in reporter gene expression. Although both methods work effectively to remobilize transposons, the breeding-mediated strategy eliminates the time-consuming micro-injection step; novel integration events are produced by simply outcrossing the hopper frogs. As each outcross ofXenopus tropicalistypically produces 2,000, or more, progeny, this method can be used to perform large-scale insertional mutagenesis screens in this highly tractable developmental model system.