Yan Ying, Hosseini Bashir, Scheld Annemarie, Pasham Srilakshmi, Rehling Tanja, Schetelig Marc F
Department of Insect Biotechnology in Plant Protection, Institute for Insect Biotechnology, Justus-Liebig-University Giessen, Giessen, Germany.
Liebig Centre for Agroecology and Climate Impact Research, Justus-Liebig-University Giessen, Giessen, Germany.
Front Bioeng Biotechnol. 2023 Feb 14;11:876492. doi: 10.3389/fbioe.2023.876492. eCollection 2023.
Genetic control strategies such as the Release of Insects Carrying a Dominant Lethal (RIDL) gene and Transgenic Embryonic Sexing System (TESS) have been demonstrated in the laboratory and/or deployed in the field. These strategies are based on tetracycline-off (Tet-off) systems which are regulated by antibiotics such as Tet and doxycycline (Dox). Here, we generated several Tet-off constructs carrying a reporter gene cassette mediated by a 2A peptide. Different concentrations (0.1, 10, 100, 500, and 1,000 μg/mL) and types (Tet or Dox) of antibiotics were used to evaluate their effects on the expression of the Tet-off constructs in the S2 cells. One or both of the two concentrations, 100 and 250 μg/mL, of Tet or Dox were used to check the influence on the performances of a wild-type strain and female-killing (FK) strains employing TESS. Specifically, the Tet-off construct for these FK strains contains a promoter to regulate the gene and a sex-specifically spliced pro-apoptotic gene to eliminate females. The results suggested that the expression of the Tet-off constructs was controlled by antibiotics in a dose-dependent manner. ELISA experiments were carried out identifying Tet at 34.8 ng/g in adult females that fed on food supplemented with Tet at 100 μg/mL. However, such method did not detect Tet in the eggs produced by antibiotic-treated flies. Additionally, feeding Tet to the parents showed negative impact on the fly development but not the survival in the next generation. Importantly, we demonstrated that under certain antibiotic treatments females could survive in the FK strains with different transgene activities. For the strain V229_M4f1 which showed moderate transgene activity, feeding Dox to fathers or mothers suppressed the female lethality in the next generation and feeding Tet or Dox to mothers generated long-lived female survivors. For the strain V229_M8f2 which showed weak transgene activity, feeding Tet to mothers delayed the female lethality for one generation. Therefore, for genetic control strategies employing the Tet-off system, the parental and transgenerational effects of antibiotics on the engineered lethality and insect fitness must be carefully evaluated for a safe and efficient control program.
诸如携带显性致死基因的昆虫释放（RIDL）和转基因胚胎性别鉴定系统（TESS）等遗传控制策略已在实验室得到验证和/或在田间得到应用。这些策略基于四环素调控（Tet-off）系统，该系统由四环素（Tet）和强力霉素（Dox）等抗生素调控。在此，我们构建了几个携带由2A肽介导的报告基因盒的Tet-off构建体。使用不同浓度（0.1、10、100、500和1000μg/mL）和类型（Tet或Dox）的抗生素来评估它们对S2细胞中Tet-off构建体表达的影响。使用100和250μg/mL这两种浓度中的一种或两种Tet或Dox来检查对采用TESS的野生型菌株和杀雌（FK）菌株性能的影响。具体而言，这些FK菌株的Tet-off构建体包含一个调控基因的启动子和一个性别特异性剪接的促凋亡基因以消除雌性。结果表明，Tet-off构建体的表达受抗生素剂量依赖性控制。酶联免疫吸附测定（ELISA）实验显示，以添加100μg/mL Tet的食物为食的成年雌性体内Tet含量为34.8 ng/g。然而，这种方法未在经抗生素处理的果蝇所产的卵中检测到Tet。此外，给亲代喂食Tet对果蝇发育有负面影响，但对下一代的存活没有影响。重要的是，我们证明在某些抗生素处理下，雌性能够在具有不同转基因活性的FK菌株中存活。对于转基因活性中等的V229_M4f1菌株，给父本或母本喂食Dox可抑制下一代的雌性致死率，给母本喂食Tet或Dox可产生长寿的雌性幸存者。对于转基因活性较弱的V229_M8f2菌株，给母本喂食Tet可使雌性致死率延迟一代。因此，对于采用Tet-off系统的遗传控制策略，为了制定安全有效的控制方案，必须仔细评估抗生素对工程致死性和昆虫适应性的亲代和跨代影响。
