Wang W, Swevers L, Iatrou K
Department of Biochemistry, University of Calgary, Canada.
Insect Mol Biol. 2000 Apr;9(2):145-55. doi: 10.1046/j.1365-2583.2000.00172.x.
Widespread occurrence in insects and the capacity to transpose in the absence of host-derived factors means that mariner-like elements are considered to be attractive candidates for the development of a universal insect genetic transformation system. Here we show that the Mos1 mariner element of Drosophila mauritiana is capable of mediating excision and transposition events in a silkmoth (Bombyx mori) derived tissue culture cell line (Bm5 cells). Plasmid rescue assays, in combination with Southern hybridization and polymerase chain reaction (PCR) analyses, confirm that the Mos1 transposase can mediate excision of DNA sequences, inserted between terminally repeated sequences recognized by the transposase, and integration into the chromosomal DNA of the Bm5 cells. In addition to chromosomal integration events, inter- and intraplasmid transposition and target element excision events were also detected. Approximately 50% of the plasmids recovered from plasmid rescue assays were found to contain the 'signature' of Mos1-specific excision and/or integration events, indicating that the mariner transposase functions efficiently in the Bombyx cells. Because mariner-induced excision and integration events are strictly dependent on the presence of a co-transfected Mos1 transposase expression vector, it is clear that the multiple copies of endogenous mariner-like elements (Bmmar1) that exist in the Bombyx genome are neither functional nor do they interfere with the efficiency of the transposition process. Thus, the Mos1 element and, probably, mariner elements, in general, hold great promise for the development of genetic transformation systems for lepidopteran insects.
在昆虫中广泛存在且能够在没有宿主衍生因子的情况下进行转座,这意味着类水手元件被认为是开发通用昆虫遗传转化系统的有吸引力的候选者。在这里,我们表明果蝇毛里求斯种的Mos1水手元件能够在家蚕(Bombyx mori)来源的组织培养细胞系(Bm5细胞)中介导切除和转座事件。质粒拯救分析,结合Southern杂交和聚合酶链反应(PCR)分析,证实Mos1转座酶能够介导插入在转座酶识别的末端重复序列之间的DNA序列的切除,并整合到家蚕Bm5细胞的染色体DNA中。除了染色体整合事件外,还检测到了质粒间和质粒内的转座以及靶元件切除事件。从质粒拯救分析中回收的大约50%的质粒被发现含有Mos1特异性切除和/或整合事件的“特征”,这表明水手转座酶在家蚕细胞中高效发挥作用。由于水手诱导的切除和整合事件严格依赖于共转染的Mos1转座酶表达载体的存在,很明显家蚕基因组中存在的内源性类水手元件(Bmmar1)的多个拷贝既无功能,也不干扰转座过程的效率。因此,Mos1元件以及一般来说的水手元件,对于鳞翅目昆虫遗传转化系统的开发具有很大的前景。