Portet Anaïs, Galinier Richard, Lassalle Damien, Faille Alexandre, Gourbal Benjamin, Duval David
IHPE UMR 5244, CNRS, IFREMER, University of Montpellier, University of Perpignan, Perpignan, France.
Department of Medicine, Molecular Immunity Unit, University of Cambridge, Cambridge, United Kingdom.
PeerJ. 2021 Feb 23;9:e10895. doi: 10.7717/peerj.10895. eCollection 2021.
is one of the snail intermediate hosts of , the causative agent of intestinal schistosomiasis disease. Numerous molecular studies using comparative approaches between susceptible and resistant snails to infection have helped identify numerous snail key candidates supporting such susceptible/resistant status. The functional approach using RNA interference (RNAi) remains crucial to validate the function of such candidates. CRISPR-Cas systems are still under development in many laboratories, and RNA interference remains the best tool to study snail genetics. Herein, we describe the use of modified small interfering RNA (siRNA) molecules to enhance cell delivery, especially into hemocytes, the snail immune cells. Modification of siRNA with 5' Cholesteryl TriEthylene Glycol (Chol-TEG) promotes cellular uptake by hemocytes, nearly eightfold over that of unmodified siRNA. FACS analysis reveals that more than 50% of hemocytes have internalized Chol-TEG siRNA conjugated to Cy3 fluorophores, 2 hours only after injection into snails. Chol-TEG siRNA targeting BgTEP1 (ThioEster-containing Protein), a parasite binding protein, reduced BgTEP1 transcript expression by 70-80% compared to control. The level of BgTEP1 protein secreted in the hemolymph was also decreased. However, despite the BgTEP1 knock-down at both RNA and protein levels, snail compatibility with its sympatric parasite is not affected suggesting functional redundancy among the BgTEP genes family in snail-schistosoma interaction.
是肠道血吸虫病病原体的蜗牛中间宿主之一。许多使用易感和抗性蜗牛之间的比较方法对感染进行的分子研究，有助于识别众多支持这种易感/抗性状态的蜗牛关键候选基因。使用RNA干扰（RNAi）的功能方法对于验证此类候选基因的功能仍然至关重要。CRISPR-Cas系统在许多实验室仍在开发中，RNA干扰仍然是研究蜗牛遗传学的最佳工具。在此，我们描述了使用修饰的小干扰RNA（siRNA）分子来增强细胞递送，特别是递送至血细胞（蜗牛的免疫细胞）。用5'胆固醇三乙二醇（Chol-TEG）修饰siRNA可促进血细胞对其的摄取，比未修饰的siRNA高出近八倍。流式细胞仪分析显示，在注射到蜗牛体内仅2小时后，超过50%的血细胞已内化与Cy3荧光团偶联的Chol-TEG siRNA。靶向BgTEP1（含硫酯蛋白）（一种寄生虫结合蛋白）的Chol-TEG siRNA与对照相比，使BgTEP1转录本表达降低了70-80%。血淋巴中分泌的BgTEP1蛋白水平也降低了。然而，尽管在RNA和蛋白质水平上都敲低了BgTEP1，但蜗牛与其同域寄生虫的相容性并未受到影响，这表明在蜗牛-血吸虫相互作用中BgTEP基因家族存在功能冗余。
