FLP Recombinase-Mediated Induction of Cu/Zn-Superoxide Dismutase Transgene Expression Can Extend the Life Span of Adult Drosophila melanogaster Flies

Yeast FLP recombinase was used in a binary transgenic system (“FLP-OUT”) to allow induced overexpression of catalase and/or Cu/Zn-superoxide dismutase (Cu/ZnSOD) in adult Drosophila melanogaster. Expression of FLP recombinase was driven by the heat-inducible hsp70 promoter. Once expressed, FLP catalyzed the rearrangement and activation of a target construct in which expression of catalase or Cu/ZnSOD cDNAs was driven by the constitutive actin5C promoter. In this way a brief heat pulse (120 or 180 min, total) of young adult flies activated transgene expression for the rest of the life span. FLP-OUT allows the effects of induced transgene expression to be analyzed in control (no heat pulse) and experimental (heat pulse) populations with identical genetic backgrounds. Under the conditions used, the heat pulse itself always had neutral or slightly negative effects on the life span. Catalase overexpression significantly increased resistance to hydrogen peroxide but had neutral or slightly negative effects on the mean life span. Cu/ZnSOD overexpression extended the mean life span up to 48%. Simultaneous overexpression of catalase with Cu/ZnSOD had no added benefit, presumably due to a preexisting excess of catalase. The data suggest that oxidative damage is one rate-limiting factor for the life span of adult Drosophila. Finally, experimental manipulation of the genetic background demonstrated that the life span is affected by epistatic interactions between the transgene and allele(s) at other loci.

本研究采用酵母FLP重组酶（FLP recombinase）构建双元转基因系统"FLP-OUT"，以实现在成年黑腹果蝇（Drosophila melanogaster）中诱导性过表达过氧化氢酶（catalase）和/或铜锌超氧化物歧化酶（Cu/Zn-superoxide dismutase，Cu/ZnSOD）。FLP重组酶的表达由热诱导型hsp70启动子驱动。FLP重组酶表达后，可催化靶标载体的重排与激活：该靶标载体中，过氧化氢酶或Cu/ZnSOD的cDNA表达由组成型actin5C启动子驱动。通过对年轻成年果蝇进行短时热激处理（总时长120或180分钟），即可在其剩余寿命周期内激活转基因表达。FLP-OUT系统可在遗传背景完全一致的对照（无热激处理）与实验（热激处理）种群中，分析诱导型转基因表达的生物学效应。在本实验所用条件下，热激处理本身对果蝇寿命仅存在中性或轻微负面作用。过氧化氢酶过表达可显著提升果蝇对过氧化氢的抗性，但对平均寿命仅表现为中性或轻微负面影响。铜锌超氧化物歧化酶过表达可将果蝇平均寿命延长至多48%。同时过表达过氧化氢酶与铜锌超氧化物歧化酶未产生额外增益，推测原因为细胞内过氧化氢酶本底水平已过量。实验数据表明，氧化损伤是成年黑腹果蝇寿命的限速因素之一。最后，对遗传背景的实验操作证实，转基因与其他位点的等位基因之间存在上位性互作（epistatic interactions），该互作可影响果蝇寿命。