Lidholm D A, Lohe A R, Hartl D L
Department of Organismic and Evolutionary Biology, Harvard University, Cambridge, Massachusetts 02138.
Genetics. 1993 Jul;134(3):859-68. doi: 10.1093/genetics/134.3.859.
A vector for germline transformation in Drosophila melanogaster was constructed using the transposable element mariner. The vector, denoted pMlwB, contains a mariner element disrupted by an insertion containing the wild-type white gene from D. melanogaster, the beta-galactosidase gene from Escherichia coli and sequences that enable plasmid replication and selection in E. coli. The white gene is controlled by the promoter of the D. melanogaster gene for heat-shock protein 70, and the beta-galactosidase gene is flanked upstream by the promoter of the transposable element P as well as that of mariner. The MlwB element was introduced into the germline of D. melanogaster by co-injection into embryos with an active mariner element, Mos1, which codes for a functional transposase and serves as a helper. Two independent germline insertions were isolated and characterized. The results show that the MlwB element inserted into the genome in a mariner-dependent manner with the termini of the inverted repeats inserted at a TA dinucleotide. Both insertions exhibit an unexpected degree of germline and somatic stability, even in the presence of an active mariner element in the genetic background. These results demonstrate that the mariner transposable element, which is small (1286 bp) and relatively homogeneous in size among different copies, is nevertheless capable of promoting the insertion of the large (13.2 kb) MlwB element. Because of the widespread phylogenetic distribution of mariner among insects, these results suggest that mariner might provide a wide host-range transformation vector for insects.
利用转座元件水手座构建了一种用于黑腹果蝇种系转化的载体。该载体命名为pMlwB,包含一个水手座元件,该元件被一个插入片段破坏,该插入片段含有来自黑腹果蝇的野生型白眼基因、来自大肠杆菌的β-半乳糖苷酶基因以及能使质粒在大肠杆菌中复制和筛选的序列。白眼基因由黑腹果蝇热休克蛋白70基因的启动子控制,β-半乳糖苷酶基因的上游侧翼是转座元件P以及水手座的启动子。通过与编码功能性转座酶并作为辅助元件的活性水手座元件Mos1共同注射到胚胎中,将MlwB元件导入黑腹果蝇的种系。分离并鉴定了两个独立的种系插入。结果表明,MlwB元件以依赖水手座的方式插入基因组,反向重复序列的末端插入到TA二核苷酸处。即使在遗传背景中存在活性水手座元件的情况下,这两个插入都表现出意想不到的种系和体细胞稳定性。这些结果表明,尽管水手座转座元件很小(1286 bp)且不同拷贝之间大小相对均匀,但它仍能够促进大的(13.2 kb)MlwB元件的插入。由于水手座在昆虫中的系统发育分布广泛,这些结果表明水手座可能为昆虫提供一种广泛宿主范围的转化载体。