Meyers G, Helftenbein E
Abteilung Zellbiologie, Universität Tübingen, F.R.G.
Gene. 1988;63(1):31-40. doi: 10.1016/0378-1119(88)90543-4.
We present a new protocol for the transfection of Stylonychia lemnae with linear DNA vectors containing the neomycin-resistance gene from Escherichia coli transposon Tn5. The taking up of heterologous DNA is achieved by damaging the cells' protein coat with urea prior to transfection according to the calcium phosphate co-precipitation procedure. After transfection, transformed cells can be enriched by selection with the antibiotic drug G418. Hybridization experiments show that macronuclear DNA of these G418-selected cells contains molecules homologous to the transfected vector DNA, which are altered by some recombination process. Transfected cells, which have grown for more than 100 cell cycles in antibiotic-free medium, still contain vector-homologous DNA, but recombination continued during this time. We are able to transfect Stylonychia early after conjugation, a process which is followed by complex genome rearrangements and amplification. In these experiments we observed considerable amplification of vector-homologous DNA molecules as compared to transfected vegetative cells. Again these molecules are altered by recombination in respect to the original vector DNA. As soon as suitable vectors are available, the transfection protocol presented here can be a basic tool for the study of DNA replication, transcription and macronuclear development in Stylonychia.
我们提出了一种新的转染方案，用于将含有来自大肠杆菌转座子Tn5的新霉素抗性基因的线性DNA载体转染到纤细半眉虫（Stylonychia lemnae）中。根据磷酸钙共沉淀程序，在转染前用尿素破坏细胞的蛋白质外壳，从而实现异源DNA的摄取。转染后，可通过用抗生素药物G418进行筛选来富集转化细胞。杂交实验表明，这些经G418筛选的细胞的大核DNA含有与转染载体DNA同源的分子，这些分子通过某种重组过程发生了改变。在无抗生素培养基中生长超过100个细胞周期的转染细胞，仍然含有与载体同源的DNA，但在此期间重组仍在继续。我们能够在接合后早期转染纤细半眉虫，这一过程之后会发生复杂的基因组重排和扩增。在这些实验中，与转染的营养细胞相比，我们观察到与载体同源的DNA分子有相当程度的扩增。同样，这些分子相对于原始载体DNA通过重组发生了改变。一旦有合适的载体，本文提出的转染方案可成为研究纤细半眉虫DNA复制、转录和大核发育的基本工具。
