piggyBac internal sequences are necessary for efficient transformation of target genomes
piggyBac internal sequences are necessary for efficient transformation of target genomes
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DOI：
10.1111/j.1365-2583.2004.00525.x
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发表时间：
2005-01-01
影响因子：
2.6
通讯作者：
Fraser, MJ
中科院分区：
文献类型：
--
作者：
Li, X;Harrell, RA;Fraser, MJ
先前报道的piggyBac最小序列试剂盒能够在胚胎间质粒转座试验中有效地进行转座，但与全长或较少广泛的内部缺失构建体相比，在黑腹果蝇中未能以显著的频率产生转化子。我们使用PCR策略重新检查了这些内部结构域（ID）序列对种系转化的重要性，该策略有效地增加了每个末端的ID序列长度。我们比较了一系列含有3xP3-ECFP标记基因的piggyBac ID合成缺失质粒对黑腹龙种系转化的影响。我们的分析确定了一个最小的序列配置，足以将piggyBac载体序列从质粒转移到昆虫基因组中。南方杂交证实了piggyBac转座子序列的存在，插入位点分析证实了这些整合靶向TTAA位点。结果证实，毗邻5‘和3’末端重复结构域的ID序列对于piggyBac有效的种系转化至关重要，尽管它们不是切除或质粒转位所必需的。利用这些信息，我们重建了一个倒置重复盒ITR1.1k和一个最小的piggyBac转座子载体pXL-BacII-ECFP，除了之前描述的对迁移至关重要的末端重复配置外，每个转座子都包含这些鉴定的ID序列。我们在独立实验中证实，这些新的最小结构产生的转换频率与控制piggyBac载体相似。我们构建的序列分析证实了在我们的载体的3'内部重复序列中点突变的位置和来源，对转化效率没有明显影响。
A previously reported piggyBac minimal sequence cartridge, which is capable of efficient transposition in embryo interplasmid transposition assays, failed to produce transformants at a significant frequency in Drosophila melanogaster compared with full-length or less extensive internal deletion constructs. We have re-examined the importance of these internal domain (ID) sequences for germline transformation using a PCR strategy that effectively adds increasing lengths of ID sequences to each terminus. A series of these piggyBac ID synthetic deletion plasmids containing the 3xP3-ECFP marker gene are compared for germline transformation of D. melanogaster. Our analyses identify a minimal sequence configuration that is sufficient for movement of piggyBac vectored sequences from plasmids into the insect genome. Southern hybridizations confirm the presence of the piggyBac transposon sequences, and insertion site analyses confirm these integrations target TTAA sites. The results verify that ID sequences adjacent to the 5' and 3' terminal repeat domains are crucial for effective germline transformation with piggyBac even though they are not required for excision or interplasmid transposition. Using this information we reconstructed an inverted repeat cartridge, ITR1.1k, and a minimal piggyBac transposon vector, pXL-BacII-ECFP, each of which contains these identified ID sequences in addition to the terminal repeat configuration previously described as essential for mobility. We confirm in independent experiments that these new minimal constructs yield transformation frequencies similar to the control piggyBac vector. Sequencing analyses of our constructs verify the position and the source of a point mutation within the 3' internal repeat sequence of our vectors that has no apparent effect on transformation efficiency.