Hersberger M, Kirby K, Phillips J P, Würgler F E, Koller T, Widmer R M
Institute of Cell Biology, ETH-Hönggerberg, Zürich, Switzerland.
Mutat Res. 1996 Dec 12;361(2-3):165-72. doi: 10.1016/s0165-1161(96)90251-4.
We present a plasmid rescue from transgenic Drosophila to study spontaneous and mutagen-induced mutations in vivo. Transgenic Drosophila lines were established by transformation with a shuttle vector containing the bacterial lacZ gene as a target for mutagenesis. The target gene can be recovered into bacteria by restriction endonuclease treatment of total genomic DNA, followed by ligation of the recircularized shuttle vectors. The resulting circular plasmids are then transformed back into E. coli lacZ- mutants, where the activity of the lacZ genes is scored on the induction substrate X-Gal. The number of inactivated versus intact lacZ genes directly indicates the mutation frequency. By the described target gene rescue procedure up to 5000 lacZ gene copies can be rescued from one fly routinely. Spontaneous background mutation rates using this system are 2.6 +/- 0.6 x 10(-4). Treatment of larvae with ethylnitrosourea (ENU) resulted in a dose-dependent increase of the mutation frequency to 4.8 +/- 0.6 x 10(-4) for 0.5 mM and 6.9 +/- 1.2 x 10(-4) for 1 mM ENU, respectively.
我们展示了一种从转基因果蝇中进行质粒拯救的方法，用于研究体内自发突变和诱变剂诱导的突变。通过用含有细菌lacZ基因作为诱变靶点的穿梭载体进行转化，建立了转基因果蝇品系。通过对总基因组DNA进行限制性内切酶处理，然后将重新环化的穿梭载体连接，可将目标基因回收至细菌中。然后将得到的环状质粒再转化回大肠杆菌lacZ - 突变体中，在诱导底物X - Gal上对lacZ基因的活性进行评分。失活的lacZ基因与完整的lacZ基因数量直接表明突变频率。通过所描述的目标基因拯救程序，通常可从一只果蝇中拯救多达5000个lacZ基因拷贝。使用该系统的自发背景突变率为2.6 +/- 0.6 x 10(-4)。用乙基亚硝基脲（ENU）处理幼虫导致突变频率呈剂量依赖性增加，0.5 mM ENU时为4.8 +/- 0.6 x 10(-4)，1 mM ENU时为6.9 +/- 1.2 x 10(-4)。
