ABSTRACT

Transgenic technology for mosquitoes is now more than two decades old, and a wide array of control sequences have been described for regulating gene expression in various life stages or specific tissues. Despite this, comparatively little attention has been paid to the development and validation of other transgene-regulating elements, especially 3ʹ-untranslated regions (3ʹUTRs). As a consequence, the same regulatory sequences are often used multiple times in a single transgene array, potentially leading to instability of transgenic effector genes. To increase the repertoire of characterized 3ʹUTRs available for genetics-based mosquito control, we generated fifteen synthetic sequences based on the base composition of the widely used SV40 3ʹUTR sequence, and tested their ability to contribute to the expression of reporter genes EGFP or luciferase. Transient transfection in mosquito cells identified nine candidate 3ʹUTRs that conferred moderate to strong gene expression. Two of these were engineered into the mosquito genome through CRISPR/Cas9-mediated site-specific insertion and compared to the original SV40 3ʹUTR. Both synthetic 3ʹUTRs were shown to successfully promote transgene expression in all mosquito life stages (larva, pupa and adults), similar to the SV40 3ʹUTR, albeit with differences in intensity. Thus, the synthetic 3ʹUTR elements described here are suitable for regulating transgene expression in Ae. aegypti, and provide valuable alternatives in the design of multi-gene cassettes. Additionally, the synthetic-scramble approach we validate here could be used to generate additional functional 3ʹUTR elements in this or other organisms.

摘要

蚊子的转基因技术现在已有二十多年的历史，并且已经描述了多种控制序列，用于调节不同生命阶段或特定组织中的基因表达。尽管如此，对其他转基因调节元件的开发和验证，尤其是 3'-非翻译区 (3'UTR) 的关注相对较少。因此，相同的调控序列经常在单个转基因阵列中多次使用，可能导致转基因效应基因的不稳定。为了增加可用于基于遗传学的蚊子控制的特征 3ʹUTR 库，我们根据广泛使用的 SV40 3ʹUTR 序列的碱基组成生成了 15 个合成序列，并测试了它们对报告基因表达做出贡献的能力EGFP或荧光素酶。蚊子细胞中的瞬时转染鉴定了九个候选 3'UTR，它们赋予中度至强基因表达。其中两个通过 CRISPR/Cas9 介导的位点特异性插入被设计到蚊子基因组中，并与原始的 SV40 3'UTR 进行比较。两种合成的 3'UTR 均显示成功促进所有蚊子生命阶段（幼虫、蛹和成虫）的转基因表达，类似于 SV40 3'UTR，尽管强度不同。因此，这里描述的合成 3'UTR 元件适用于调节Ae 中的转基因表达。埃及人，并在多基因盒的设计中提供有价值的替代方案。此外，我们在此验证的合成加扰方法可用于在该生物体或其他生物体中生成额外的功能性 3'UTR 元素。