Transfection of Babesia bovis by Double Selection with WR99210 and Blasticidin-S and Its Application for Functional Analysis of Thioredoxin Peroxidase-1.
Transfection of Babesia bovis by Double Selection with WR99210 and Blasticidin-S and Its Application for Functional Analysis of Thioredoxin Peroxidase-1.
复制标题
用WR99210和Blasticidin-S进行双重选择，并应用其用于硫氧还蛋白过氧化物酶-1的功能分析，将Babesia Bovis转染。
DOI：
10.1371/journal.pone.0125993
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发表时间：
2015
期刊：
影响因子：
3.7
通讯作者：
Kawazu S
中科院分区：
文献类型：
--
作者：
Asada M;Yahata K;Hakimi H;Yokoyama N;Igarashi I;Kaneko O;Suarez CE;Kawazu S
遗传操作是分析基因功能的必要技术，然而，有限的方法可用于牛巴贝斯虫，全球重要的牛疾病，牛巴贝斯虫病的病原体。迄今为止，已经开发了两种用于B的稳定转染系统。牛，使用选择性标记杀稻瘟菌素-S脱氨酶（bsd）或人二氢叶酸还原酶（hdhfr）。在这项工作中，我们联合收割机这两个选择标记在一个连续的转染系统。具体地，亲本转基因B.附加型表达绿色荧光蛋白（GFP）和人二氢叶酸还原酶（hDHFR）的牛系，用编码由红色荧光蛋白（RFP）和杀稻瘟菌素-S脱氨酶（BSD）组成的融合蛋白的质粒转染。用WR 99210和杀稻瘟菌素-S选择导致出现GFP和RFP双阳性的寄生虫。然后，我们应用这种方法来补充基因功能的寄生虫线，其中硫氧还蛋白过氧化物酶-1（Bbtpx-1）基因敲除使用hDHFR作为选择标记。构建携带RFP-BSD和Bbtpx-1表达盒的质粒，并转染到Bbtpx-1敲除（KO）寄生虫中。通过两种转染方法，附加型转染和基因组整合独立地获得转染子。Bbtpx-1的互补导致对亚硝化应激的抗性通过一氧化氮供体硝普钠完全恢复，这在Bbtpx-1 KO寄生虫中受损。综上所述，我们建立了B细胞的序贯转染方法。牛，并随后将该技术应用于基因互补研究。这种方法将使更广泛的基因操纵巴氏疟原虫，以提高我们对这种寄生虫生物学的理解。
Genetic manipulation is an essential technique to analyze gene function; however, limited methods are available for Babesia bovis, a causative pathogen of the globally important cattle disease, bovine babesiosis. To date, two stable transfection systems have been developed for B. bovis, using selectable markers blasticidin-S deaminase (bsd) or human dihydrofolate reductase (hdhfr). In this work, we combine these two selectable markers in a sequential transfection system. Specifically, a parent transgenic B. bovis line which episomally expresses green fluorescent protein (GFP) and human dihydrofolate reductase (hDHFR), was transfected with a plasmid encoding a fusion protein consisting of red fluorescent protein (RFP) and blasticidin-S deaminase (BSD). Selection with WR99210 and blasticidin-S resulted in the emergence of parasites double positive for GFP and RFP. We then applied this method to complement gene function in a parasite line in which thioredoxin peroxidase-1 (Bbtpx-1) gene was knocked out using hDHFR as a selectable marker. A plasmid was constructed harboring both RFP-BSD and Bbtpx-1 expression cassettes, and transfected into a Bbtpx-1 knockout (KO) parasite. Transfectants were independently obtained by two transfection methods, episomal transfection and genome integration. Complementation of Bbtpx-1 resulted in full recovery of resistance to nitrosative stress, via the nitric oxide donor sodium nitroprusside, which was impaired in the Bbtpx-1 KO parasites. In conclusion, we developed a sequential transfection method in B. bovis and subsequently applied this technique in a gene complementation study. This method will enable broader genetic manipulation of Babesia toward enhancing our understanding of the biology of this parasite.