Tagging of Endogenous Genes in a Toxoplasma gondii Strain Lacking Ku80
Tagging of Endogenous Genes in a Toxoplasma gondii Strain Lacking Ku80
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DOI：
10.1128/ec.00358-08
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发表时间：
2009-04-01
期刊：
影响因子：
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通讯作者：
Carruthers, Vern B.
中科院分区：
文献类型：
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作者：
Huynh, My-Hang;Carruthers, Vern B.
与其他具有完整基因组序列的生物一样，对弓形虫进行大规模研究（如表达和定位）的机会现在更加可行。我们提出了一个系统标记内源性基因与黄色荧光蛋白（YFP）在德尔塔ku 80株。Ku 80参与DNA链修复和非同源DNA末端连接;先前在其他生物体中的研究表明，在其缺失的情况下，随机整合被消除，允许将具有同源序列的构建体插入适当的基因座。我们产生了由YFP和二氢叶酸还原酶-胸苷酸合酶选择标记组成的载体。YFP之前是连接非依赖性克隆（LIC）盒，其允许插入含有互补LIC序列的PCR产物。我们证明了Delta ku 80菌株在将YFP标记的构建体整合到正确的基因座中比野生型菌株RH更有效和高效。然后，我们选择了几个假设的蛋白质，通过蛋白质组学筛选的排泄分泌抗原，并显示类似于已知的微线蛋白的微阵列表达谱，认为这些可能是新的蛋白质在细胞入侵中的作用。我们通过YFP荧光定位这些假设的蛋白质，并通过免疫印迹显示其表达。我们的研究结果表明，Delta ku 80菌株和pYFP的组合。LIC构建体减少了确定新的感兴趣基因的定位所需的时间和成本。这将为进行更大规模的新T。弓形虫基因
As with other organisms with a completed genome sequence, opportunities for performing large-scale studies, such as expression and localization, on Toxoplasma gondii are now much more feasible. We present a system for tagging genes endogenously with yellow fluorescent protein (YFP) in a Delta ku80 strain. Ku80 is involved in DNA strand repair and nonhomologous DNA end joining; previous studies in other organisms have shown that in its absence, random integration is eliminated, allowing the insertion of constructs with homologous sequences into the proper loci. We generated a vector consisting of YFP and a dihydrofolate reductase-thymidylate synthase selectable marker. The YFP is preceded by a ligation-independent cloning (LIC) cassette, which allows the insertion of PCR products containing complementary LIC sequences. We demonstrated that the Delta ku80 strain is more effective and efficient in integrating the YFP-tagged constructs into the correct locus than wild-type strain RH. We then selected several hypothetical proteins that were identified by a proteomic screen of excreted-secreted antigens and that displayed microarray expression profiles similar to known micronemal proteins, with the thought that these could potentially be new proteins with roles in cell invasion. We localized these hypothetical proteins by YFP fluorescence and showed expression by immunoblotting. Our findings demonstrate that the combination of the Delta ku80 strain and the pYFP. LIC constructs reduces both the time and cost required to determine localization of a new gene of interest. This should allow the opportunity for performing larger-scale studies of novel T. gondii genes.