New and highly efficient method for silkworm transgenesis using Autographa californica nucleopolyhedrovirus and piggyBac transposable elements
New and highly efficient method for silkworm transgenesis using Autographa californica nucleopolyhedrovirus and piggyBac transposable elements
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DOI：
10.1002/bit.20296
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发表时间：
2004-12-30
影响因子：
3.8
通讯作者：
Mori, H
中科院分区：
文献类型：
--
作者：
Yamamoto, M;Yamao, M;Mori, H
我们开发了一种新的家蚕转基因方法。该方法将重组杆状病毒的使用与piggyBac转座因子的使用相结合。一种称为辅助病毒的重组AcNPV被设计成在果蝇hsp70启动子的控制下表达piggyBac转座酶。另一种重组AcNPV编码的基因将被整合到家蚕基因组中，在这种情况下，是绿色荧光蛋白（GFP）基因，受B控制。mori actin A3启动子，并由piggyBac反向末端重复序列标记。用涂有这两种重组杆状病毒混合物的细针接种前胚盘卵。大多数接种的幼虫孵化，高比例的新孵化的GO幼虫表达GFP标记。通过G1昆虫的Southern印迹分析、对通过反向PCR分离的插入位点接头进行测序以及以孟德尔方式分离的标记物（如G2昆虫中出现绿色荧光所证明）来证实转基因。因此，利用这种新的方法可以容易而有效地获得转基因家蚕。(C)2004 Wiley Periodicals，Inc.
We have developed a new method for the transgenesis of the silkworm, Bombyx mori. This method couples the use of recombinant baculoviruses with the use of the piggyBac transposable element. One recombinant AcNPV, designated the helper virus, is designed to express the piggyBac transposase under the control of the Drosophila hsp70 promoter. Another recombinant AcNPV encoded the gene to be incorporated into the silkworm genome, in this case a green fluorescent protein (GFP) gene, under the control of B. mori actin A3 promoter and franked by the piggyBac inverted terminal repeats. Preblastoderm eggs were inoculated with a fine needle coated with a mixture of these two recombinant baculoviruses. Most of the inoculated larvae hatched and a high proportion of the newly hatched GO larvae expressed the GFP marker. Transgenesis was confirmed by Southern blot analysis of G1 insects, sequencing the insertion site junctions isolated by inverse PCR, and the marker segregated in Mendelian fashion, as evidenced by the appearance of green fluorescence in G2 insects. Thus, transgenic silkworms were easily and efficiently obtained using this new method. (C) 2004 Wiley Periodicals, Inc.