Li X, Lobo N, Bauser C A, Fraser M J
Department of Biological Sciences, and Center for Tropical Diseases Research and Training, University of Notre Dame, IN 46556, USA.
Mol Genet Genomics. 2001 Oct;266(2):190-8. doi: 10.1007/s004380100525.
The piggyBac element from Trichoplusia ni is recognized as a useful vector for transgenesis of a wide variety of species. This transposable element is 2472 bp in length, and has a complex repeat configuration consisting of an internal repeat (IR), spacer, and terminal repeat (TR) at both ends, and a single ORF encoding the transposase. Excision assays performed in microinjected T. ni embryos using plasmids deleted for progressively larger portions of the piggyBac internal sequence reveal that the 5' and 3' IR, spacer, and TR configuration is sufficient for precise excision of piggyBac when transposase is provided in trans. Interplasmid transposition assays using plasmids carrying varying lengths of intervening sequence between the piggyBac termini in T. ni demonstrate that a minimum of 55 bp of intervening sequence is required for optimal transposition, while lengths less than 40 bp result in a dramatic decrease in transposition frequency. These results suggest that the piggyBac transposase may bind both termini simultaneously before cleavage can occur, and/or that the formation of a transposition complex requires DNA bending between the two termini. Based on these results we constructed a 702-bp cartridge with minimal piggyBac 5' and 3' terminal regions separated by an intervening sequence of optimal length. Interplasmid transposition assays demonstrate that the minimal terminal configuration is sufficient to mediate transposition, and also verify that simply inserting this cartridge into an existing plasmid converts that plasmid into a non-autonomous piggyBac transposon. We also constructed a minimal piggyBac vector, pXL-Bac, that contains an internal multiple cloning site sequence between the minimal terminal regions. These vectors should greatly facilitate the utilization of the piggyBac transposon in a wide range of hosts.
来自粉纹夜蛾的piggyBac元件被认为是用于多种物种转基因的有用载体。这种转座元件长度为2472 bp，具有复杂的重复结构，两端由一个内部重复序列（IR）、间隔区和末端重复序列（TR）组成，还有一个编码转座酶的单一开放阅读框。在显微注射的粉纹夜蛾胚胎中使用删除了piggyBac内部序列越来越大部分的质粒进行的切除试验表明，当通过反式提供转座酶时，5'和3' IR、间隔区和TR结构足以实现piggyBac的精确切除。在粉纹夜蛾中使用携带piggyBac末端之间不同长度间隔序列的质粒进行的质粒间转座试验表明，最佳转座需要至少55 bp的间隔序列，而长度小于40 bp会导致转座频率急剧下降。这些结果表明，piggyBac转座酶可能在切割发生之前同时结合两个末端，和/或转座复合物的形成需要两个末端之间的DNA弯曲。基于这些结果，我们构建了一个702 bp的盒式结构，其具有最小的piggyBac 5'和3'末端区域，由最佳长度的间隔序列隔开。质粒间转座试验表明，最小末端结构足以介导转座，并且还验证了将此盒式结构简单插入现有质粒可将该质粒转化为非自主piggyBac转座子。我们还构建了一个最小的piggyBac载体pXL - Bac，其在最小末端区域之间包含一个内部多克隆位点序列。这些载体应极大地促进piggyBac转座子在广泛宿主中的应用。
