González-Estévez C, Momose T, Gehring W J, Saló E
Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain.
Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14046-51. doi: 10.1073/pnas.2335980100. Epub 2003 Nov 13.
To generate transgenic planarians we used a set of versatile vectors for animal transgenesis based on the promiscuous transposons, mariner, Hermes and piggyBac, and a universal enhanced GFP (EGFP) marker system with three Pax6 dimeric binding sites, the 3xP3-EGFP developed by Berghammer et al. [Berghammer, A. J., Klinger, M. & Wimmer, E. A. (1999) Nature 402, 370-371]. This marker is expressed specifically in the eyes of various arthropod taxa. Upon microinjection into the parenchyma of adult planarians and subsequent electroporation, these vectors transpose efficiently into the planarian genome. One of the cell types transformed are the totipotent "neoblast" stem cells present in the adults, representing 30% of total cells. The neoblast represents a unique cell type with the capacity to proliferate and to differentiate into all somatic cell types as well as into germ cells. All three transposon vectors have high transformation efficiency, but only Hermes and piggyBac show stable integration. The mariner vector is frequently lost presumably because of the presence of active mariner-type transposons in the genome of the Girardia tigrina. Transformed animals are mosaics containing both transformed and untransformed neoblasts. These differentiate to form EGFP-positive and -negative photoreceptor cells. Such mosaicism is maintained through several cycles of regeneration induced by decapitation or asexual reproduction. Transformed neoblasts also contribute to the germ line, and can give rise to pure transgenic planarian lines in which EGFP is expressed in all photoreceptor cells after sexual reproduction. The presence of the transgenes was confirmed by PCR, plasmid rescue assay, inverse PCR, and Southern blotting. Our results with the 3xP3-EGFP marker confirm the presence of Pax6 activity in the differentiated photoreceptor cells of planarian eyes. Transgenesis will be an important tool to dissect developmental molecular mechanisms in planarian regeneration, development and stem cell biology, and may also be an entry point to analyze the biology of parasitic Platyhelminthes.
为了生成转基因涡虫，我们使用了一组基于混杂转座子（水手座、赫耳墨斯座和猪尾巴座）的通用动物转基因载体，以及一个带有三个Pax6二聚体结合位点的通用增强型绿色荧光蛋白（EGFP）标记系统，即由伯格哈默等人开发的3xP3 - EGFP[伯格哈默，A. J.，克林格，M. & 维默，E. A.（1999年）《自然》402卷，370 - 371页]。该标记在各种节肢动物类群的眼睛中特异性表达。将这些载体显微注射到成年涡虫的实质组织中并随后进行电穿孔后，它们能高效地转座进入涡虫基因组。被转化的细胞类型之一是成年涡虫中存在的全能“新胚层”干细胞，占总细胞数的30%。新胚层代表一种独特的细胞类型，具有增殖能力，并能分化为所有体细胞类型以及生殖细胞。所有三种转座子载体都具有高转化效率，但只有赫耳墨斯座和猪尾巴座显示出稳定整合。水手座载体经常丢失，推测是因为在虎纹涡虫的基因组中存在活跃的水手座型转座子。转化后的动物是嵌合体，包含转化和未转化的新胚层细胞。这些细胞分化形成EGFP阳性和阴性的光感受器细胞。这种嵌合现象通过断头或无性繁殖诱导的几个再生周期得以维持。转化后的新胚层细胞也对生殖系有贡献，并且在有性繁殖后能产生纯转基因涡虫品系，其中所有光感受器细胞都表达EGFP。通过聚合酶链反应（PCR）、质粒拯救试验、反向PCR和Southern印迹法证实了转基因的存在。我们使用3xP3 - EGFP标记的结果证实了涡虫眼睛分化的光感受器细胞中存在Pax6活性。转基因技术将成为剖析涡虫再生、发育和干细胞生物学中发育分子机制的重要工具，也可能是分析寄生扁形动物生物学的切入点。
