Loukeris T G, Arcà B, Livadaras I, Dialektaki G, Savakis C
Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology Hellas, Heraklion, Greece.
Proc Natl Acad Sci U S A. 1995 Oct 10;92(21):9485-9. doi: 10.1073/pnas.92.21.9485.
A transposon based on the transposable element Minos from Drosophila hydei was introduced into the genome of Drosophila melanogaster using transformation mediated by the Minos transposase. The transposon carries a wild-type version of the white gene (w) of Drosophila inserted into the second exon of Minos. Transformation was obtained by injecting the transposon into preblastoderm embryos that were expressing transposase either from a Hsp70-Minos fusion inserted into the genome via P-element-mediated transformation or from a coinjected plasmid carrying the Hsp70-Minos fusion. Between 1% and 6% of the fertile injected individuals gave transformed progeny. Four of the insertions were cloned and the DNA sequences flanking the transposon ends were determined. The "empty" sites corresponding to three of the insertions were amplified from the recipient strain by PCR, cloned, and sequenced. In all cases, the transposon has inserted into a TA dinucleotide and has created the characteristic TA target site duplication. In the absence of transposase, the insertions were stable in the soma and the germ line. However, in the presence of the Hsp70-Minos gene the Minos-w transposon excises, resulting in mosaic eyes and germ-line reversion to the white phenotype. Minos could be utilized as an alternative to existing systems for transposon tagging and enhancer trapping in Drosophila; it might also be of use as a germ-line transformation vector for non-Drosophila insects.
一种基于果蝇海德氏果蝇可转座元件Minos的转座子,通过Minos转座酶介导的转化被引入黑腹果蝇的基因组中。该转座子携带果蝇白色基因(w)的野生型版本,插入到Minos的第二个外显子中。通过将转座子注射到前胚盘胚胎中实现转化,这些胚胎要么从通过P元件介导的转化插入基因组的Hsp70-Minos融合基因表达转座酶,要么从共注射携带Hsp70-Minos融合基因的质粒表达转座酶。1%至6%的可育注射个体产生了转化后代。克隆了四个插入位点,并确定了转座子末端两侧的DNA序列。通过PCR从受体菌株中扩增出与其中三个插入位点对应的“空”位点,进行克隆和测序。在所有情况下,转座子都插入到TA二核苷酸中,并产生了特征性的TA靶位点重复。在没有转座酶的情况下,插入在体细胞和生殖系中都是稳定的。然而,在存在Hsp70-Minos基因的情况下,Minos-w转座子会切除,导致眼睛出现嵌合体,生殖系恢复为白色表型。Minos可作为果蝇中转座子标签和增强子捕获现有系统的替代方法;它也可能用作非果蝇昆虫的生殖系转化载体。