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.
In the case of a new viral disease outbreak, an immediate development of virus detection kits and vaccines is required. For COVID-19, we established a rapid production procedure for SARS-CoV-2 spike protein (S protein) by using the baculovirus-silkworm expression system. The baculovirus vector-derived S proteins were successfully secreted to silkworm serum, whereas those formed insoluble structure in the larval fat body and the pupal cells. The ectodomain of S protein with the native sequence was cleaved by the host furin-protease, resulting in less recombinant protein production. The S protein modified in furin protease-target site was efficiently secreted to silkworm serum and was purified as oligomers, which showed immunoreactivity for anti-SARS-CoV-2 S2 antibody. By using the direct transfection of recombinant bacmid to silkworms, we achieved the efficient production of SARS-CoV-2 S protein as fetal bovine serum (FBS)-free system. The resultant purified S protein would be useful tools for the development of immunodetection kits, antigen for immunization for immunoglobulin production, and vaccines.
Heterochromatin protein 1s (HP1s) are nonhistone chromosomal proteins that play a direct role in the formation and maintenance of heterochromatin structure. Similarly to Caenorhabditis elegans, silkworms possess holocentric chromosomes, in which diffused kinetochores extend along the length of each chromosome. We have isolated two silkworm HP1 homologues, BmHP1a and BmHP1b. Cytological analysis showed a unique localization of BmHP1s during cell division, in which these proteins first appear to dissociate from the chromosomes, but then return to enclose the chromosomes during metaphase. BmHP1s formed homo- and hetero-dimers and interacted with BmSu(var)3-9, which is a methyltransferase for histone H3 lysine 9 (H3K9). We further showed, using a silkworm cell-based reporter system, that BmHP1b had higher transcriptional repression activity than BmHP1a, whereas BmHP1a interacted more strongly with BmSu(var)3-9 than did BmHP1b. These results suggest that silkworm HP1a and HP1b may play different roles in heterochromatin formation in holocentric silkworm chromosomes.
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