DNA Vector-Based RNA Interference in Cell Lines Derived from<i>Bombyx mori</i>

Fujita, Kosuke; Sagisaka, Aki; Tomimoto, Kazuya; Ishibashi, Jun Ichiro; Imanishi, Shigeo; Yamakawa, Minoru; Tanaka, Hiromitsu

doi:10.1271/bbb.90223

Cited by 18 publications

(9 citation statements)

References 31 publications

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“…Unlike the Drosophila S2 cells, the cultured silkworm BmN4 cells do not uptake dsRNA spontaneously, 11,12 but RNAi could be induced in these cells by transfection of dsRNA or exogenouly expressed hairpin RNA. 2,[4][5][6] To achieve the ectopic expression of CeSID-1 in BmN4 cells, we transfected the CeSID-1 expression vector, pBac CeSID1_GFPzeo, into BmN4 with a piggyBac helper plasmid, followed by selection with the antibiotic Zeocin (Fig. 1A).…”

Section: Resultsmentioning

confidence: 99%

“…1 RNAi has been used as a characterization tool in silkworm and cultured silkworm cell lines, as well as in other insects. [2][3][4][5][6] In cultured silkworm cells, RNAi-knockdown experiments have been successfully performed by dsRNA transfection or the expression of short or long hairpin RNA. In contrast to vertebrate cells, insect cells, including silkworm cells, have no interferon response to relatively long dsRNAs.…”

Section: Introductionmentioning

confidence: 99%

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Effective RNA interference in cultured silkworm cells mediated by overexpression ofCaenorhabditis elegansSID-1

et al. 2012

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RNA interference (RNAi) is a conserved mechanism that catalyzes sequence-specific gene silencing and has been used for loss-of-function genetic screens in many organisms. Here, we demonstrated that the expression of Caenorhabditis elegans SID-1 (CeSID-1) could trigger effective gene silencing in the cultured silkworm cell line, BmN4 (BmN4-SID1). Soaking the BmN4-SID1 in dsRNA corresponding to endogenous target genes induced a significant decrease of the amount of mRNA or protein. A small amount of dsRNA was enough to silence the target gene in a few days. Overexpression of CeSID-1 did not affect the cell viability. Our results suggest that BmN4-SID1 can be used in many applications in silkworm cells and will become a valuable resource for gene analysis.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Effective RNA interference in cultured silkworm cells mediated by overexpression ofCaenorhabditis elegansSID-1

et al. 2012

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“…Previously, we knocked down luciferase genes in silkworm cell lines by transfection of lhRNA expression plasmids via two-step cloning [28]. These constructs suppressed the expression of the luciferase gene by more than 98% [28]. The RNAi effect induced by lhRNA expression plasmids generated by these novel methods was similar to that generated by the two-step method.…”

Section: Discussionmentioning

confidence: 89%

“…The lhRNA expression plasmids comprising 200 and 300 bp of stem regions showed 93 and 95% of RNAi activity, respectively. Previously, we knocked down luciferase genes in silkworm cell lines by transfection of lhRNA expression plasmids via two-step cloning [28]. These constructs suppressed the expression of the luciferase gene by more than 98% [28].…”

Section: Discussionmentioning

confidence: 99%

A Novel Method to Convert a DNA Fragment Inserted into a Plasmid to an Inverted Repeat Structure

et al. 2011

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Transfection of an expression plasmid possessing inverted repeat (IR) DNA into cultured cells leads to the overexpression of hairpin RNA and efficient suppression of target gene expression. Such DNA vector-based RNA interference (RNAi) is widely used for characterizing genes of interest in cultured cell lines. In this study, we developed a new method to convert an inserted DNA fragment (IDF) in specially designed plasmid vectors into an IR structure by using nicking endonucleases and BcaBEST DNA polymerase. This method consists of the following steps: (1) linearization of the plasmid with a nick by using a restriction enzyme and a nicking endonuclease, (2) formation of the hairpin-loop DNA at the end near the IDF of the linearized plasmid, (3) insertion of a nick at the other end of the IDF by a nicking endonuclease, (4) execution of the strand displacement reaction from the nick to synthesize IR DNA, and (5) self-ligation of the linear double-stranded DNA. The IR DNA containing expression plasmids constructed by this method effectively induced target-specific RNAi in a silkworm cell line. We further established a method to purify expression plasmids containing IR DNA. Our new methods provide techniques for the construction of long hairpin RNA (lhRNA) expression plasmids for silencing specific genes in silkworms and other organisms, and offer a fundamental methodology for constructing an lhRNA expression library from a cDNA plasmid library.

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“…The majority of previous antiviral studies have reported the use of knockdown of baculovirus DNA replication factor immediate early‐1 (IE‐1) gene through RNAi technology, which significantly inhibited the virus replication in vivo and in vitro (Zhang, Wang et al , ; Zhou et al , ). Moreover, ie‐1 , lef‐1 , lef‐2 , lef‐3 , lef‐11 , oral infection factor p74 and gp64 have also significantly inhibited virus multiplication and replication in transgenic cells and silkworm (Fujita et al , ; Jiang et al , ; Xu et al , ; Zhang, He et al , ; Zhou et al , ; Cao et al , ). However, baculovirus early transcription activator and DNA replication factors, ie‐0 and ie‐2 , have not yet been studied as target genes.…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9‐mediated disruption of the immediate early‐0 and 2 as a therapeutic approach to Bombyx mori nucleopolyhedrovirus in transgenic silkworm

Dong

et al. 2018

Insect Molecular Biology

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The CRISPR/Cas9 system is a powerful tool for the treatment of infectious diseases. In our previous study, we knocked out the Bombyx mori nucleopolyhedrovirus (BmNPV) key genes and BmNPV-dependent host factor to generate transgenic antiviral strains. To further expand the range of target genes for BmNPV and more effectively prevent and control pathogenic infections, we performed gene editing and antiviral analysis by constructing a target-directed baculovirus early transcriptional activator immediate early-0 (ie-0) and 2 (ie-2) transgenic silkworm line. We hybridized it with Cas9 transgenic line to produce a double-positive transgenic Cas9(+)/sgIE0-sgIE2(+) line that could activate the CRISPR gene editing system. We first demonstrated that the system is capable of efficiently editing target genes and resulting in fragment deletions in the BmNPV genome. Survival rate of the transgenic Cas9(+)/sgIE0-sgIE2(+) line reached 65% after inoculation with 1 × 10 occlusion bodies/larva. Molecular analysis showed that BmNPV DNA replication and viral gene expression level in the transgenic Cas9(+)/sgIE0-sgIE2(+) line were significantly inhibited compared with the control Cas9(-)/sgIE0-sgIE2(-) line. These results indicated that IE-0 and IE-2, as baculovirus early transcriptional activators, can be used as target sites for gene therapy and that multigene editing could expand the range of target sites for research to create silkworm resistance breeds.

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DNA Vector-Based RNA Interference in Cell Lines Derived fromBombyx mori

Cited by 18 publications

References 31 publications

Effective RNA interference in cultured silkworm cells mediated by overexpression ofCaenorhabditis elegansSID-1

Effective RNA interference in cultured silkworm cells mediated by overexpression ofCaenorhabditis elegansSID-1

A Novel Method to Convert a DNA Fragment Inserted into a Plasmid to an Inverted Repeat Structure

CRISPR/Cas9‐mediated disruption of the immediate early‐0 and 2 as a therapeutic approach to Bombyx mori nucleopolyhedrovirus in transgenic silkworm

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