shRNA Expression Plasmids Generated by a Novel Method Efficiently Induce Gene-Specific Knockdown in a Silkworm Cell Line

Abstract

RNAi knockdown by using shRNA expression plasmids is widely used to determine the function of individual genes in mammals. Here we developed a simple method to create an IR DNA in a U6 small nuclear RNA promoter-based parent vector using a single-stranded IR DNA with short hairpin structure and Bst DNA polymerase. Furthermore, we demonstrated that the shRNA expression plasmids constructed by our method effectively induced target-specific RNAi in the silkworm cell line. We also found that sequence preference in the silkworm cell line was much lower than in mammalian cells and shRNA-induced RNAi was influenced by the length of the stem region.

Construction of shRNA Expression Plasmids for Silkworm Cell Lines Using Single-Stranded DNA and Bst DNA Polymerase

“…In general, RNAi ef fi ciency in the cells is known to depend on the sequence of the stem region, and only approximately 30% of random siRNAs have been reported to show highly effective RNAi in cultured mammalian cells ( 15 ) . However, in our experiment, six of eight shRNA expression plasmids-each having randomly designed nucleotide sequences at the stem region-suppressed the expression of the reporter gene by more than 95% in silkworm cells ( 11 ) . Another two constructs also showed 75-80% reductions.…”

“…shRNA with 19 or 17 nt stem regions were inef fi cient in silkworm cell lines ( 11 ) . Therefore, the length of the stem region should be at least 21 nt.…”

“…The second method is a polymerase chain reaction (PCR)-based strategy in which the promoter sequence serves as the template ( 10 ) . We developed another simple method to create IR DNA in the parent vector using a single-stranded DNA possessing a short hairpin structure and Bst DNA polymerase, which has strand displacement activity ( 11 ) . This method comprises the following steps: (a) linearization of the plasmid with the 5 ¢ end of one terminus dephosphorylated by treating stepwise with one restriction enzyme, alkaline phosphatase, and then a second restriction enzyme; (b) ligation of a hairpin oligonucleotide to one end of the linear plasmid; (c) execution of the strand displacement reaction by Bst DNA polymerase; and (d) self-ligation of the linear plasmid ( Fig.…”

BmEts upregulates promoter activity of lebocin in Bombyx mori

“…25 to 1194 of BmEts cDNA was amplified by PCR and subcloned into the NcoI and XhoI sites of pIEx-4 (Novagen). For short hairpin RNA expression plasmids targeting BmRelishes (GenBank accession numbers AB298441 and AB298442 for BmRelish1 and BmRelish2, respectively) (pSH-BmRelishes) and BmRels (GenBank accession numbers AB096087 and AB096088 for BmRelA and BmRelB, respectively) (pSH-BmRels), inverted repeated DNAs 5 0 -AGGAT-GAATGCCTCATGTTGTGTGTGCTGTCCACAACATGAGGCATTCATCCT-3 0 and 5 0 -GTACACTTCCAGCTGAAGAGGGTGTGCTGTCCCCTCTTCAGCTG GAAGTGTAC-3 0 were inserted between the NcoI and StuI sites of pIEx-4-BmU6M (Tanaka et al, 2009a), respectively, according to the method of Tanaka et al (2009a). pSH-EGFP corresponds to "EGFP-L1" described by Tanaka et al (2009a).…”

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

“…We assumed that the expression of clathrin would be suppressed only in the cells transfected with clathrin RNAi expression plasmid, and not in non-transfected cells. The result that the expression of the reporter gene was suppressed nearly 100% in NIAS-Bm-oyanagi2 cells, and that sequence preference of RNAi in NIAS-Bm-oyanagi2 cells was much lower than in mammalian cells, 42) lead us to infer that clathrin transcription is significantly suppressed in cells transfected with clathrin RNAi expression plasmid. Thus the cells used in experiment in Fig.…”