Airs Paul M, Kudrna Katherine E, Lubinski Bailey, Phanse Yashdeep, Bartholomay Lyric C
Department of Pathobiological Sciences, University of Wisconsin-Madison, Madison, WI 53706, USA.
Midwest Center of Excellence for Vector-Borne Diseases, University of Wisconsin-Madison, Madison, WI 53706, USA.
Insects. 2023 Jun 15;14(6):556. doi: 10.3390/insects14060556.
In mosquitoes, the utilization of RNAi for functional genetics is widespread, usually mediated through introduced double-stranded RNAs (dsRNAs) with sequence identity to a gene of interest. However, RNAi in mosquitoes is often hampered by inconsistencies in target gene knockdown between experimental setups. While the core RNAi pathway is known to function in most mosquito strains, the uptake and biodistribution of dsRNAs across different mosquito species and life stages have yet to be extensively explored as a source of variation in RNAi experiments. To better understand mosquito-RNAi dynamics, the biodistribution of a dsRNA to a heterologous gene, LacZ (iLacZ), was tracked following various routes of exposure in the larval and adult stages of , , and . iLacZ was largely limited to the gut lumen when exposed per os, or to the cuticle when topically applied, but spread through the hemocoel when injected. Uptake of dsRNA was noted in a subset of cells including: hemocytes, pericardial cells of the dorsal vessel, ovarian follicles, and ganglia of the ventral nerve cord. These cell types are all known to undergo phagocytosis, pinocytosis, or both, and as such may actively take up RNAi triggers. In , iLacZ was detected for up to one week post exposure by Northern blotting, but uptake and degradation drastically differed across tissues. The results presented here reveal that the uptake of RNAi triggers is distinct and specific to the cell type in vivo.
在蚊子中，RNA干扰（RNAi）在功能遗传学中的应用十分广泛，通常是通过引入与目标基因具有序列同一性的双链RNA（dsRNA）来介导的。然而，蚊子中的RNAi常常受到不同实验设置中目标基因敲低不一致的阻碍。虽然已知核心RNAi途径在大多数蚊子品系中发挥作用，但dsRNA在不同蚊子物种和生命阶段的摄取和生物分布，作为RNAi实验中变异的一个来源，尚未得到广泛研究。为了更好地理解蚊子RNAi的动态过程，在埃及伊蚊、白纹伊蚊和致倦库蚊的幼虫和成虫阶段，通过各种暴露途径追踪了一种针对异源基因LacZ的dsRNA（iLacZ）的生物分布。经口暴露时，iLacZ主要局限于肠腔，局部应用时局限于表皮，但注射时则会扩散到血腔。在包括血细胞、背血管的心包细胞、卵巢卵泡和腹神经索神经节在内的一部分细胞中发现了dsRNA的摄取。已知这些细胞类型都会进行吞噬作用、胞饮作用或两者兼而有之，因此可能会主动摄取RNAi触发物。在埃及伊蚊中，通过Northern印迹法在暴露后长达一周的时间内都检测到了iLacZ，但不同组织中的摄取和降解情况差异很大。此处给出的结果表明，RNAi触发物的摄取在体内对细胞类型具有独特性和特异性。
