Expansion of experimental genetics approaches for Plasmodium berghei with versatile transfection vectors
Expansion of experimental genetics approaches for Plasmodium berghei with versatile transfection vectors
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DOI：
10.1016/j.molbiopara.2012.06.001
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发表时间：
2012-09-01
影响因子：
1.5
通讯作者：
Matuschewski, Kai
中科院分区：
文献类型：
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作者：
Kooij, Taco W. A.;Rauch, Manuel M.;Matuschewski, Kai
实验性反向遗传学方法已被证明在疟原虫生物学研究中非常有效。鼠疟疾模型寄生虫伯氏疟原虫是遗传上最容易修正的疟原虫物种，并且可以完全进入体内的整个生命周期。在这里，我们描述了下一代高度通用的转染载体集，有助于将实验遗传策略推向全基因组规模。通过 36 个连续克隆和 17 个亚克隆步骤，从标准转染质粒生成了优化的载体集。这些靶向载体统称为 Berghei 适应性转染 (pBAT) 质粒，包含允许药物选择盒回收、重组寄生虫的强大绿色荧光标记、具有红色荧光表位标签融合的靶蛋白羧基末端标记以及异源基因表达的关键元件。这些载体针对小尺寸、多功能限制性内切酶识别位点和单个载体元件的潜在交换进行了进一步优化。我们表明，稳定整合到转基因表达位点（伯氏疟原虫第 6 号染色体同线性断点处的基因间位点）中是表型沉默的，并产生用于成像应用的亮绿色荧光寄生虫系。我们提供一个例子。 P. 伯氏肌动蛋白 2，用于靶向基因删除，并说明正选择标记可以回收，从而允许多轮遗传操作。我们提出，本文描述的载体将极大地促进通过多种实验遗传学方法对预测的和孤儿疟原虫基因模型进行功能分配。 (C) 2012 Elsevier B.V. 保留所有权利。
Experimental reverse genetic approaches have proven powerful in the study of the biology of the malaria parasite. The murine malaria model parasite Plasmodium berghei is the genetically most amendable Plasmodium species and allows full access to the entire life cycle in vivo. Here, we describe a next-generation, highly versatile transfection vector set that facilitates advancing experimental genetic strategies towards a genome-wide scale. Through 36 consecutive cloning and 17 subcloning steps an optimized vector set was generated from the standard transfection plasmid. These targeting vectors, collectively referred to as the Berghei Adaptable Transfection (pBAT) plasmids, contain key elements that permit recycling of the drug-selectable cassette, robust green fluorescent labelling of recombinant parasites, carboxy-terminal tagging of target proteins with a red fluorescent-epitope tag fusion, and expression of heterologous genes. The vectors were further optimized for small size, versatile restriction endonuclease recognition sites and potential exchange of individual vector elements. We show that stable integration into a transgene expression site, an intergenic locus at a synteny breakpoint on P. berghei chromosome 6, is phenotypically silent and generated a bright green fluorescent parasite line for imaging applications. We provide an example. P. berghei actin 2, for targeted gene deletion and illustrate that the positive selection marker can be recycled, thereby permitting multiple rounds of genetic manipulations. We propose that the vectors described herein will greatly facilitate functional assignment to predicted and orphan Plasmodium gene models by multiple experimental genetics approaches. (C) 2012 Elsevier B.V. All rights reserved.