Negative selection using yeast cytosine deaminase/uracil phosphoribosyl transferase in Plasmodium falciparum for targeted gene deletion by double crossover recombination
Negative selection using yeast cytosine deaminase/uracil phosphoribosyl transferase in Plasmodium falciparum for targeted gene deletion by double crossover recombination
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DOI：
10.1016/j.molbiopara.2006.06.014
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发表时间：
2006-11-01
影响因子：
1.5
通讯作者：
Cowman, Alan F.
中科院分区：
文献类型：
--
作者：
Maier, Alexander G.;Braks, Joanna A. M.;Cowman, Alan F.
恶性疟原虫的基因操纵能力已经建立了许多年，它是利用基因破坏[1]、等位基因交换[2]和转基因表达[3]进行寄生虫基因功能分析的重要工具。这是使用阳性选择来维持携带药物选择标记的环状质粒而建立的，所述药物选择标记例如分别赋予对乙胺嘧啶或WR 99210的抗性的刚地弓形虫二氢叶酸还原酶（TgDHFR）[4，5]或人dhfr基因[6]，尽管最近已经使用了其他选择系统[7]。携带通过同源重组整合到基因组中的这些质粒的拷贝的转染的恶性疟原虫寄生虫通常通过药物选择和停药选择的生长循环随时间选择。在没有药物选择的情况下，附加型质粒迅速丢失，因为它们在子代中分配不均匀，而整合到基因组中的质粒通常与每条染色体分离[8]。该方法确保在随后的开/关药物循环后，获得的所有转染的恶性疟原虫寄生虫都通过单交换同源重组整合了质粒。虽然这被证明是一种非常有用的基因破坏方法，但由于环状附加体形式的持续存在，需要长时间的培养来获得具有质粒整合拷贝的寄生虫，因此这是有限的。另一个缺点是，在单次交叉的情况下：CD，胞嘧啶脱氨酶; DHFR，二氢叶酸还原酶; 5-FC，5-氟胞嘧啶; FCU，酿酒酵母CD和URPT的嵌合基因; TK，胸苷激酶; URPT，尿嘧啶磷酸核糖基转移酶C对应作者：沃尔特和伊丽莎霍尔医学研究所，1G皇家游行，墨尔本3050，澳大利亚.电话：+传真：+ 61 3 93470852。
The ability to genetically manipulatePlasmodium falciparum has been established for a number of years and it is an important tool for functional analysis of parasite genes using gene disruption [1], allelic exchange [2] and transgene expression [3]. This was established using positive selection for maintenance of circular plasmids carrying a drug selectable marker such as Toxoplasma gondii dihydrofolate reductase (TgDHFR)[4, 5], or the human dhfr gene [6] conferring resistance to pyrimethamine or WR99210 respectively although more recently other selection systems have been used [7]. Transfected P. falciparum parasites carrying copies of these plasmids integrated into the genome by homologous recombination were generally selected over time by cycles of growth on and off drug selection. The episomal plasmids are lost rapidly in the absence of drug selection as they are partitioned unevenly amongst the daughter progeny whereas the plasmids integrated into the genome are segregated normally with each chromosome [8]. This method ensured that after subsequent on/off drug cycles all transfected P. falciparum parasites obtained had integrated the plasmid by single crossover homologous recombination. Whilst this proved to be an extremely useful method for gene disruption it was limited because of the long periods of culture required to derive parasites with integrated copies of the plasmid due to persistence of the circular episomal forms. Another drawback is that with a single cross over theAbbreviations: CD, cytosine deaminase; DHFR, dihydrofolate reductase; 5-FC, 5-fluorocytosine; FCU, chimeric gene of Saccharomyces cerevisiae CD and URPT; TK, thymidine kinase; URPT, uracil phosphoribosyl transferase∗ Corresponding author at: The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Melbourne 3050, Australia. Tel.:+ 61 3 93452446; fax:+ 61 3 93470852.