Germline transformation of the malaria vector, Anopheles gambiae, with the piggyBac transposable element
Germline transformation of the malaria vector, Anopheles gambiae, with the piggyBac transposable element
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DOI：
10.1046/j.0962-1075.2001.00299.x
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发表时间：
2001-12-01
影响因子：
2.6
通讯作者：
Benedict, MQ
中科院分区：
文献类型：
--
作者：
Grossman, GL;Rafferty, CS;Benedict, MQ
将带有增强绿色荧光蛋白（EGFP）标记的piggyBac转座元件注射到蚊子胚胎中，实现了非洲主要疟疾媒介冈比亚按蚊的种系转化。在筛选的34143只幼蚊中，筛选出2只表达EGFP的G(1)代雄蚊。基因组Southern数据和piggyBac插入边界的测序表明，这两个雄性在注射的G(0)胚胎中由一个piggyBac插入事件产生。遗传交叉数据表明，该元素的插入位点要么导致了隐性致死，要么与隐性致死密切相关。这表现在自交系中表达egfp的后代数量不足，但外交与未转化个体的比例预期不足，以及未能建立纯种系。插入位点与2号染色体上的无项圈位点弱连锁，原位杂交显示插入位点位于该染色体的28D区。在唾液腺和大多数个体的前胃中均观察到特别高的表达水平。在研究结束时，注射技术的改进导致G(1)后代中Go的孵化率，瞬时表达率和egfp的表达率增加。
Germline transformation of the major African malaria vector, Anopheles gambiae, was achieved using the piggyBac transposable element marked with the enhanced green fluorescent protein (EGFP) Injected Into mosquito embryos. Two G(1) generation male mosquitoes expressing EGFP were identified among 34 143 larvae screened. Genomic Southern data and sequencing of the piggyBac insertion boundaries showed that these two males arose from one piggyBac insertion event In the Injected G(0) embryos. Genetic cross data suggest that the Insertion site of the element either resulted in, or Is tightly linked to, a recessive lethal. This was demonstrated by a deficiency in the number of EGFP-expressing offspring from inbred crosses but expected ratios In outcrosses to nontransformed individuals and failure to establish a pure-breeding line. The Insertion was weakly linked to the collarless locus on chromosome 2 and was shown by in situ hybridization to be located in division 28D of that chromosome. Particularly high levels of expression were observed uniformly in salivary glands and, in most individuals, in the anterior stomach. An improvement In the Injection technique at the end of the studies resulted in Increased Go hatching, transient expression and EGFP-expression rates among G(1) progeny.