Purified mariner (Mos1) transposase catalyzes the integration of marked elements into the germ-line of the yellow fever mosquito, Aedes aegypti
Purified mariner (Mos1) transposase catalyzes the integration of marked elements into the germ-line of the yellow fever mosquito, Aedes aegypti
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DOI：
10.1016/s0965-1748(00)00110-7
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发表时间：
2000-11-01
影响因子：
3.8
通讯作者：
James, AA
中科院分区：
文献类型：
--
作者：
Coates, CJ;Jasinskiene, N;James, AA
来自毛果蝇的Mariner转座元件Mos1的衍生物可以整合到黄热病蚊子的生殖系中。以前，动员Mos1所需的转座酶是由一个表达该酶的辅助质粒反式提供的，该酶是在拟暗乳杆菌热休克蛋白82启动子的控制下表达的。在此，我们测试了纯化的重组Mos1转座酶是否能提高Ae的回收率。埃及伊蚊变形剂。将Mos1转座酶注入白眼Kh(W)/Kh(W)，Ae。埃及人胚胎中含有一份含有朱砂基因野生型等位基因的Mos1供体质粒。在G(1)动物中，通过部分恢复眼睛的颜色来识别转化的蚊子，并通过基因组DNA的Southern分析证实，当Mos1转座酶浓度接近100 nM时，在G(O)动物中由独立插入产生的种系转化率比使用辅助质粒实验中看到的提高了2倍。此外，总G(1)转化子的回收率比共注射辅助质粒的频率高7.5倍。Southern杂交分析和基因扩增实验证实转座子已整合到蚊子基因组中，尽管并不是所有的整合都是预期的剪贴式转座。利用纯化的转座酶获得胚系整合的频率的提高将促进Mos1转基因蚊子的产生，并将转基因方法应用于这一重要的多种病原体载体的生物学。(C)2000爱思唯尔科学有限公司。保留所有权利。
Derivatives of the mariner transposable element, Mos1, from Drosophila mauritiana, can integrate into the germ-line of the yellow fever mosquito, Aedes aegypti. Previously, the transposase required to mobilize Mos1 was provided in trans by a helper plasmid expressing the enzyme under the control of the D. psuedoobscura heat-shock protein 82 promoter. Here we tested whether purified recombinant Mos1 transposase could increase the recovery of Ae. aegypti transformants. Mos1 transposase was injected into white-eyed, kh(w)/kh(w), Ae. aegypti embryos with a Mos1 donor plasmid containing a copy of the wild-type allele of the D. melanogaster cinnabar gene. Transformed mosquitoes were recognized by partial restoration of eye color in the G(1) animals and confirmed by Southern analyses of genomic DNA, At Mos1 transposase concentrations approaching 100 nM, the rate of germ-line transformants arising from independent insertions in G(o) animals was elevated 2-fold compared to that seen in experiments with helper plasmids. Furthermore, the recovery of total G(1) transformants was increased 7.5-fold over the frequency seen with co-injected helper plasmid. Southern blot analyses and gene amplification experiments confirmed the integration of the transposons into the mosquito genome, although not all integrations were of the expected cut-and-paste type transposition. The increased frequency of germ-line integrations obtained with purified transposase will facilitate the generation of Mos1 transgenic mosquitoes and the application of transgenic approaches to the biology of this important vector of multiple pathogens. (C) 2000 Elsevier Science Ltd. All rights reserved.