Construction of transgenic Drosophila by using the site-specific integrase from phage φC31
Construction of transgenic Drosophila by using the site-specific integrase from phage φC31
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DOI：
10.1534/genetics.166.4.1775
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发表时间：
2004-04-01
期刊：
影响因子：
3.3
通讯作者：
Calos, MP
中科院分区：
文献类型：
--
作者：
Groth, AC;Fish, M;Calos, MP
phiC31整合酶在体外和大肠杆菌、酵母和哺乳动物细胞中有效地发挥作用，介导其attB和attP识别位点之间的单向位点特异性重组。在这里，我们表明，这种位点特异性的整合系统也有效地在培养的细胞和胚胎中的果蝇。携带attB和attP识别位点的转染质粒DNA在S2细胞中发生分子内重组的频率为47%。此外，在果蝇基因组中鉴定了几个内源性假attP位点，这些位点被整合酶识别并用作S2细胞中整合的底物。通过将attP位点与P元件整合到基因组中来创建果蝇的两个品系。将phiC31整合酶作为mRNA注射到胚胎中，其功能是促进含有attB的质粒整合到attP位点中，导致55%的可育成年人产生转基因后代。总共100%的这些后代在基因组attP位点携带精确整合事件。这些实验证明了用phiC31整合酶系统对果蝇基因组进行精确基因工程的潜力，并可能有利于果蝇和其他昆虫的研究。
The phiC31 integrase functions efficiently in vitro and in Escherichia coli, yeast, and mammalian cells, mediating unidirectional site-specific recombination between its attB and attP recognition sites. Here we show that this site-specific integration system also functions efficiently in Drosophila melanogaster in cultured cells and in embryos. Intramolecular recombination in S2 cells on transfected plasmid DNA carrying the attB and attP recognition sites occurred at a frequency of 47%. In addition, several endogenous pseudo attP sites were identified in the fly genome that were recognized by the integrase and used as substrates for integration in S2 cells. Two lines of Drosophila were created by integrating an attP site into the genome with a P element. phiC31 integrase injected into embryos as mRNA functioned to promote integration of an attB-containing plasmid into the attP site, resulting in tip to 55% of fertile adults producing transgenic offspring. A total of 100% of these progeny carried a precise integration event at the genomic attP site. These experiments demonstrate the potential for precise genetic engineering of the Drosophila genome with the phiC31 integrase system and will likely benefit research in Drosciphila and other insects.