Somatic mutagenesis with a Sleeping Beauty transposon system leads to solid tumor formation in zebrafish.
Somatic mutagenesis with a Sleeping Beauty transposon system leads to solid tumor formation in zebrafish.
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DOI：
10.1371/journal.pone.0018826
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发表时间：
2011-04-21
期刊：
影响因子：
3.7
通讯作者：
Essner JJ
中科院分区：
文献类型：
--
作者：
McGrail M;Hatler JM;Kuang X;Liao HK;Nannapaneni K;Watt KE;Uhl JD;Largaespada DA;Vollbrecht E;Scheetz TE;Dupuy AJ;Hostetter JM;Essner JJ
对人类癌症基因组和小鼠转座子诱导的肿瘤进行的大规模测序发现，在不同的癌症中存在大量基因突变。这一领域的突出挑战之一是确定哪些基因在突变时有助于细胞转化和肿瘤进展。为了确定驱动肿瘤发生的新的和保守的基因，我们在远亲脊椎动物物种斑马鱼Danio rerio中开发了一种新的癌症模型。采用睡美人T2/ONC转座子系统对斑马鱼进行体细胞诱变。将鲤鱼肌动蛋白启动子克隆到T2/ONC中，构建成T2/OncZ。通过将线性DNA注入斑马鱼胚胎中，获得了两个含有T2/OncZ大串联基因的转基因斑马鱼品系。通过在1-细胞阶段注射SB11转座酶RNA，T2/OncZ转座子在转基因阵列中的斑马鱼基因组中被动员起来。或者，将T2/OncZ斑马鱼与一个组成性表达SB11转座酶的转基因品系杂交。利用连接介导的聚合酶链式反应克隆了T2/OncZ转座子整合位点，并在Genome Analyzer II上进行了测序。斑马鱼基因组中有700-6800个独特的整合事件被定位。数据表明，通过转基因表达或RNA注射引入转座酶导致转座子重新整合事件在斑马鱼基因组中均匀分布。在∼10月龄时，注射SB11mRNA可导致10%的成鱼发生肿瘤。T2/OncZ诱导的斑马鱼肿瘤包含许多与人类和小鼠癌症基因相同的突变基因。这些分析验证了我们的突变方法，并为这些基因在人类癌症中的参与提供了额外的支持。斑马鱼T2/OncZ癌症模型将有助于识别人类癌症的新的和保守的遗传驱动因素。
Large-scale sequencing of human cancer genomes and mouse transposon-induced tumors has identified a vast number of genes mutated in different cancers. One of the outstanding challenges in this field is to determine which genes, when mutated, contribute to cellular transformation and tumor progression. To identify new and conserved genes that drive tumorigenesis we have developed a novel cancer model in a distantly related vertebrate species, the zebrafish, Danio rerio. The Sleeping Beauty (SB) T2/Onc transposon system was adapted for somatic mutagenesis in zebrafish. The carp ß-actin promoter was cloned into T2/Onc to create T2/OncZ. Two transgenic zebrafish lines that contain large concatemers of T2/OncZ were isolated by injection of linear DNA into the zebrafish embryo. The T2/OncZ transposons were mobilized throughout the zebrafish genome from the transgene array by injecting SB11 transposase RNA at the 1-cell stage. Alternatively, the T2/OncZ zebrafish were crossed to a transgenic line that constitutively expresses SB11 transposase. T2/OncZ transposon integration sites were cloned by ligation-mediated PCR and sequenced on a Genome Analyzer II. Between 700–6800 unique integration events in individual fish were mapped to the zebrafish genome. The data show that introduction of transposase by transgene expression or RNA injection results in an even distribution of transposon re-integration events across the zebrafish genome. SB11 mRNA injection resulted in neoplasms in 10% of adult fish at ∼10 months of age. T2/OncZ-induced zebrafish tumors contain many mutated genes in common with human and mouse cancer genes. These analyses validate our mutagenesis approach and provide additional support for the involvement of these genes in human cancers. The zebrafish T2/OncZ cancer model will be useful for identifying novel and conserved genetic drivers of human cancers.
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影响因子：
64.8
作者：
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影响因子：
11.4
作者：
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