Stable RNA Interference in Spodoptera frugiperda Cells by a DNA Vector-based Method

Lin, Chih-Chien; Hsu, Ju Wei; Huang, Kuo Cheng; Tang, Hung-Kuan; Lai, Yiu‐Kay

doi:10.1007/s10529-005-5530-3

Cited by 13 publications

(9 citation statements)

References 17 publications

(20 reference statements)

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“…This is the first indication that gene-specific RNAi is effective for suppressing endogenous gene expression in cells of S. frugiperda. Two other reports have demonstrated RNAi-mediated downregulation of gene expression via baculovirus in S. frugiperda cells (Agrawal et al, 2004;Lin et al, 2006).…”

Section: Rnai In Cultured Cell Lines-host Gene Suppressionmentioning

confidence: 98%

Metabolic engineering of the baculovirus‐expression system via inverse “shotgun” genomic analysis and RNA interference (dsRNA) increases product yield and cell longevity

Kim

Kramer

Hebert

et al. 2007

Biotech & Bioengineering

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RNA interference (RNAi) is as powerful tool for characterizing gene function in eukaryotic organisms and cultured cell lines. Its use in metabolic engineering has been limited and few reports have targeted protein expression systems to increase yield. In this work, we examine the use of in vitro synthesized double stranded RNA (dsRNA) in the baculovirus expression vector system (BEVS), using commercially relevant cultured cells (Spodoptera frugiperda, Sf-9) and larvae (Trichoplusia ni) as hosts. First, we employed an inverse "shotgun" genomic analysis to "find" an array of 16 putative insect gene targets. We then synthesized dsRNA in vitro targeting these genes and investigated the effects of injected dsRNA on larval growth, development, and product yield. Growth and development was at times stunted and in several cases, the effects were lethal. However, dsRNA targeting an acidic juvenile hormone-suppressible protein (AJHSP1), and translational elongation factor 2 (Ef-2) resulted in significantly increased yield of model product, GFP. Next, we targeted known genes, v-cath and apoptosis inducer, sf-caspase 1, in cultured Sf-9 cells. We confirm RNAi-mediated sf-caspase 1 suppression in Sf-9 cells, but not in baculovirus-infected cells, likely due to the overriding effects of inhibitor of apoptosis protein, p35. We also demonstrate suppression of v-cath in infected cells, which leads to a approximately 3-fold increase in product yield. Overall, our results support the application of RNAi in metabolic engineering, specifically for enhancing protein productivity in the baculovirus expression vector system.

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Section: Rnai In Cultured Cell Lines-host Gene Suppressionmentioning

confidence: 98%

Metabolic engineering of the baculovirus‐expression system via inverse “shotgun” genomic analysis and RNA interference (dsRNA) increases product yield and cell longevity

Kim

Kramer

Hebert

et al. 2007

Biotech & Bioengineering

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“…Baculoviral vectors contain multiple sites for foreign DNA insertion and are therefore able to encapsidate large amounts of foreign DNA [171] whilst maintaining stable and prolonged silencing of targeted genes [108]. However, cell lysis-associated proteolysis (if the protein of interest is a secreted protein, proteinases from lysed cells could severely compromise the yield of protein production the restricted host range [97].…”

Section: Non-integrating Viral Vectorsmentioning

confidence: 99%

“…There are three main non-viral vector systems that involve the treatment of cells by chemical (cationic liposomes and polymer complexes) or physical means (direct injection of naked plasmid DNA) [64,89,108,110,164]. The use of chemical and physical methods of gene transfer is relatively simple and does not provoke specific immune responses of the host.…”

Section: Non-viral Vectorsmentioning

confidence: 99%

RNA Interference with Special Reference to Combating Viruses of Crustacea

Fauce

Owens

2012

Indian J. Virol.

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RNA interference has evolved from being a nuisance biological phenomenon to a valuable research tool to determine gene function and as a therapeutic agent. Since pioneering observations regarding RNA interference were first reported in the 1990s from the nematode worm, plants and Drosophila, the RNAi phenomenon has since been reported in all eukaryotic organisms investigated from protozoans, plants, arthropods, fish and mammals. The design of RNAi therapeutics has progressed rapidly to designing dsRNA that can specifically and effectively silence disease related genes. Such technology has demonstrated the effective use of short interfering as therapeutics. In the absence of a B cell lineage in arthropods, and hence no long term vaccination strategy being available, the introduction of using RNA interference in crustacea may serve as an effective control and preventative measure for viral diseases for application in aquaculture.

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“…Currently, chemically synthetic siRNAs are being evaluated for their use as highly-specific gene-silencing therapeutics, as well as their traditional role as an extremely powerful instrument for functional genomic analyses (25). However, there are several disadvantages associated with the use of synthesized siRNAs: i) The transduction of siRNA into cells usually leads to only transient silencing effects; ii) the transfection efficiency of siRNA may influence the silencing effects in target cells; and iii) transfected siRNA is expensive, as they must be chemically or enzymatically synthesized (26). To overcome these shortcomings, a stable RNAi DNA vector-based method has been developed (27–29).…”

Section: Discussionmentioning

confidence: 99%

“…However, there are several disadvantages associated with the use of synthesized siRNAs: i) The transduction of siRNA into cells usually leads to only transient silencing effects; ii) the transfection efficiency of siRNA may influence the silencing effects in target cells; and iii) transfected siRNA is expensive, as they must be chemically or enzymatically synthesized (26). To overcome these shortcomings, a stable RNAi DNA vector-based method has been developed (27–29). Approximately 70% of the vectors used in gene therapy clinical trials are represented by viral-based delivery systems (30).…”

Section: Discussionmentioning

confidence: 99%

Effects of RNA silencing of matrix metalloproteinase-2 on the growth of esophageal carcinoma cells in vivo

Shen

Feng

et al. 2016

Oncology Letters

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Esophageal carcinoma is one of the most common malignancies in China. Previous studies reported that matrix metalloproteinases (MMPs) have important roles in the progression and invasion of numerous types of solid tumors. Among the MMPs, MMP-2 has been closely associated with tumor growth and invasion. In the present study, a short hairpin RNA (shRNA) lentiviral expression vector targeting the MMP-2 gene was constructed in order to observe the inhibitory effect of MMP-2 gene silencing on the growth of the KYSE150 esophageal carcinoma cell line in vivo. Three small hairpin RNA sequences targeting MMP-2 were designed and cloned into lentiviral vectors. Following transfection of the lentiviral vectors into KTSE150 cells, MMP-2 mRNA and protein expression levels were examined by reverse transcription-quantitative polymerase chain reaction and western blotting, and the growth rate of cells was analyzed by MTT assays. Subsequently, tumor growth was assessed in nude mice. Lentivirus-mediated RNA interference effectively inhibited the expression of MMP-2 mRNA and protein in KYSE150 esophageal carcinoma cells, and suppressed the growth of esophageal carcinoma cells in vivo. The results of the present study suggested that lentivirus-mediated gene therapy targeting MMP-2 may be an attractive strategy for the treatment of esophageal carcinoma and justifies the performance of further studies on the application of lentivirus vectors to cancer gene therapy.

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Stable RNA Interference in Spodoptera frugiperda Cells by a DNA Vector-based Method

Cited by 13 publications

References 17 publications

Metabolic engineering of the baculovirus‐expression system via inverse “shotgun” genomic analysis and RNA interference (dsRNA) increases product yield and cell longevity

Metabolic engineering of the baculovirus‐expression system via inverse “shotgun” genomic analysis and RNA interference (dsRNA) increases product yield and cell longevity

RNA Interference with Special Reference to Combating Viruses of Crustacea

Effects of RNA silencing of matrix metalloproteinase-2 on the growth of esophageal carcinoma cells in vivo

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