“…The fragment comprised 24 bp of exon1, 713 bp of intron1, 8 bp of partial sequence of exon2 (9 bp distance from the initiation codon), and 374 bp of 5′-flanking region upstream of transcription initiation site. Besides the core promoter sequences including the TATA-box and CAAT-box, several conserved motifs reported previously were detected including Spl-binding and Pbx-1 consensus sequences, an octamer-like sequence, a Bm1 element and a common sequence found in storage protein genes (Mori et al 1991a, b; Sakurai et al 1988a, b; Willott et al 1989; Matsumoto et al 1986; Delaney et al 1986; Fujiwara and Yamashita 1992; Ogawa et al 2005) (Fig. 1a).Fig.…”
The fat body plays multiple, crucial roles in the life of silkworms. Targeted expression of transgenes in the fat body of the silkworm, Bombyx mori, is important not only for clarifying the function of endogenous genes expressed in this tissue, but also for producing valuable recombinant proteins. However, fat body-specific gene expression remains difficult due to a lack of suitable tissue-specific promoters. Here we report the isolation of the fat body-specific promoter of Bmlp3, a member of the 30K protein family of silkworms. The 1.1 kb fragment from −374 to +738 of Bmlp3 displayed strong promoter activity in the cell lines BmE and Spli-221. In transgenic silkworms, a DsRed reporter gene controlled by the 1.1 kb Bmlp3 promoter fragment was expressed specifically in the fat body in a stage-specific pattern that was nearly identical to the endogenous Bmlp3 gene. We conclude that the 1.1 kb Bmlp3 promoter fragment is sufficient to direct tissue- and stage-specific expression of transgenes in the fat body of silkworms, highlighting the potential use of this promoter for both functional genomics research and biotechnology applications.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-013-9705-8) contains supplementary material, which is available to authorized users.
“…The fragment comprised 24 bp of exon1, 713 bp of intron1, 8 bp of partial sequence of exon2 (9 bp distance from the initiation codon), and 374 bp of 5′-flanking region upstream of transcription initiation site. Besides the core promoter sequences including the TATA-box and CAAT-box, several conserved motifs reported previously were detected including Spl-binding and Pbx-1 consensus sequences, an octamer-like sequence, a Bm1 element and a common sequence found in storage protein genes (Mori et al 1991a, b; Sakurai et al 1988a, b; Willott et al 1989; Matsumoto et al 1986; Delaney et al 1986; Fujiwara and Yamashita 1992; Ogawa et al 2005) (Fig. 1a).Fig.…”
The fat body plays multiple, crucial roles in the life of silkworms. Targeted expression of transgenes in the fat body of the silkworm, Bombyx mori, is important not only for clarifying the function of endogenous genes expressed in this tissue, but also for producing valuable recombinant proteins. However, fat body-specific gene expression remains difficult due to a lack of suitable tissue-specific promoters. Here we report the isolation of the fat body-specific promoter of Bmlp3, a member of the 30K protein family of silkworms. The 1.1 kb fragment from −374 to +738 of Bmlp3 displayed strong promoter activity in the cell lines BmE and Spli-221. In transgenic silkworms, a DsRed reporter gene controlled by the 1.1 kb Bmlp3 promoter fragment was expressed specifically in the fat body in a stage-specific pattern that was nearly identical to the endogenous Bmlp3 gene. We conclude that the 1.1 kb Bmlp3 promoter fragment is sufficient to direct tissue- and stage-specific expression of transgenes in the fat body of silkworms, highlighting the potential use of this promoter for both functional genomics research and biotechnology applications.Electronic supplementary materialThe online version of this article (doi:10.1007/s11248-013-9705-8) contains supplementary material, which is available to authorized users.
“…These proteins have molecular weights of around 30 kDa, and 30Kc19 protein is the most abundant among 30K proteins (30Kc6, 30Kc12, 30Kc19, 30Kc21 and 30Kc23) in the hemolymph [37]. During the 5th instar larva to early pupa stage, these 30K proteins are synthesized in fat body cells and accumulate in the hemolymph [38,39]. They are then transferred from the hemolymph to fat body cells during metamorphosis from larva to pupa, and are deposited there until later use [40,41].…”
Recently, the recombinant 30Kc19 protein, originating from silkworm hemolymph of Bombyx mori has attracted attention due to its cell-penetrating property and potential application as a protein delivery system. However, this observation of penetration across cell membrane has raised questions concerning the interaction of the protein-lipid bilayer. Here, we report a dimerization propensity of the 30Kc19 protein in the presence of amphiphilic moieties; sodium dodecyl sulfate (SDS) or phospholipid. Native PAGE showed that the 30Kc19 monomer formed a dimer when SDS or phospholipid was present. In the glutathione-S-transferase (GST) pull-down assay, supplementation of the 30Kc19 protein to mammalian cell culture medium showed dimerization and penetration; due to phospholipids at the cell membrane, the main components of the lipid bilayer. Mutagenesis was performed, and penetration was observed by 30Kc19 C76A and not 30Kc19 C57A, which meant that the presence of cysteine at position 57 (Cys-57) is involved in dimerization of the 30Kc19 at the cell membrane during penetration. We anticipate application of the native 30Kc19 protein with high cell-penetrating efficiency for delivery of cargos to various cell types. The intracellular cargo delivery using the 30Kc19 protein is a non-virus derived (e.g. TAT) delivery method, which can open up new approaches for the delivery of therapeutics in bioindustries, such as pharma- and cosmeceuticals.
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