›› 2018, Vol. 61 ›› Issue (7): 795-800.doi: 10.16380/j.kcxb.2018.07.005

• RESEARCH PAPERS • Previous Articles     Next Articles

Cellular location of antisense oligonucleotide of snoRNA Bm-15 and its interference efficiency with Bm-15 in Spodoptera frugiperda Sf9 cells

LI Xin-Mei, QIU Wu-Jie, CUI Bin, YANG Zong-Lin, SHEN Ya-Wen, LU Yi-Ping, KAN Yun-Chao, LI Dan-Dan*   

  1.  (Henan Provincial Key Laboratory of Funiu Mountain Insect Biology, Nanyang Normal University, Nanyang, Henan 473061, China)
  • Online:2018-07-20 Published:2018-07-20

Abstract: 【Aim】 To study the interference efficiency of antisense oligonucleotide (ASO) with insect small nucleolar RNA (snoRNA) Bm-15, and to investigate the intracellular delivery of ASO in cells. 【Methods】 Spodoptera frugiperda Sf9 cells were transfected with Cy5-labeled, 2′-O ribosomal methylation and phosphorothioate-modified ASO of Bm-15 through liposome. Co-localization of Bm-15 ASO (labeled with Cy5) and lysosome as well as mitochondria was screened by immunofluorescence. The interference efficiency of ASO with snoRNA Bm-15 was analyzed by detecting the expression level of Bm-15 in Sf9 cells transfected with Bm-15 ASO using real-time PCR. 【Results】 The proportions of ASO fluorescence signals inundating the whole cell and at the cell edge were 34% and 66%, respectively, in the transfected Sf9 cells after 48 h transfection of Bm-15 ASO, suggesting that ASO might be delivered into different subcellular organelles. Further co-localization analysis showed that Bm-15 ASO was transported into lysosomes but not mitochondria. qPCR result showed that the expression level of Bm-15 decreased by 47% in Sf9 cells transfected with Bm-15 ASO. 【Conclusion】 ASO can not escape from the cellular endogenous degradation machinery even after various chemical modifications, and this effectively explains the low interference efficiency of ASO with target gene in some circumstances.

Key words: Insect, Spodoptera frugiperda, antisense oligonucleotide, snoRNA, Bm-15, lysosome