A fibroin secretion-deficient silkworm mutant, , provides an efficient system for producing recombinant proteins

Inoue, Satoshi; Kanda, Toshio; Imamura, Morikazu; Quan, Guoxing; Kojima, Katsura; Tanaka, Hiromitsu; Tomita, Masahiro; Hino, Rika; Yoshizato, Katsutoshi; Mizuno, Seitoku

doi:10.1016/j.ibmb.2004.10.002

Cited by 86 publications

(90 citation statements)

References 25 publications

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“…23) Accumulation of defective L-chain protein and H-chain in the PSG of Nd-s D has been reported to cause failure of PSG development. 15,24) The production of foreign protein in the PSG and the lack of effective secretion of protein into the lumen can lead to abnormal PSG development. The PSG of the KH25 strain appeared to develop normally (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…The resulting plasmid was designated pHC-EGFP. Finally, the Asc I-Fse I fragment of pHC-EGFP was cloned into Asc I-Fse I-digested pBac[3Xp3-DsRed2afm], 15) and the resulting plasmid was designated pBac[3Xp3-DsRed2afm] HC-EGFP.…”

Section: Methodsmentioning

confidence: 99%

“…The transgenic technique has been used in the functional analysis of genes 10,11) and as a tool in foreign gene production. [12][13][14] There have been several reports of modification of the L-chain 12,14,15) and the P25 protein, 13) but there have been no reports of modification of the H-chain in silkworms by this transgenic technique. Furthermore, these modifications of the L-chain and P25 have not demonstrated changes in the physical properties of fibroin fiber.…”

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confidence: 99%

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A New Method for the Modification of Fibroin Heavy Chain Protein in the Transgenic Silkworm

Kojima¹,

Kuwana²,

Sezutsu³

et al. 2007

Bioscience, Biotechnology, and Biochemistry

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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A New Method for the Modification of Fibroin Heavy Chain Protein in the Transgenic Silkworm

Kojima¹,

Kuwana²,

Sezutsu³

et al. 2007

Bioscience, Biotechnology, and Biochemistry

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“…Electroporation-mediated somatic transgenesis for rapid functional analysis in insects Toshiya Ando and Haruhiko Fujiwara* pPIG-A3GR was obtained by cloning the DsRed2 ORF cassette from pBac3xP3DsRed2 (Inoue et al, 2005) into BamHI/NotI-digested pPIG-A3GG.…”

Section: Abstract: In Vivo Electroporation Piggybac Non-model Orgamentioning

confidence: 99%

Electroporation-mediated somatic transgenesis for rapid functional analysis in insects

Ando

Fujiwara

2013

Development

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Transgenesis is a powerful technique for determining gene function; however, it is time-consuming. It is virtually impossible to carry out in non-model insects in which egg manipulation and screening are difficult. We have established a rapid genetic functional analysis system for non-model insects using a low-cost electroporator (costing under US$200) designed for somatic transformation with the piggyBac transposon. Using this system, we successfully generated somatic transgenic cell clones in various target tissues (e.g. olfactory neurons, wing epidermis, larval epidermis, muscle, fat body and trachea) of the silkworm Bombyx mori during development. We also induced stable and transient RNA interference (RNAi) using short hairpin RNA (shRNA)-mediating DNA vectors and direct transfer of small interfering RNAs (siRNAs), respectively. We found that these electroporation-mediated approaches could also be applied to the swallowtail butterfly Papilio xuthus and the red flour beetle Tribolium castaneum. Thus, this method could be a powerful genetic tool for elucidating various developmental phenomena in non-model insects.

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“…Tomita et al (2003) reported on the expression of human type-IIIcollagen into the cocoon as a fibroin L-chain fusion protein using the piggyBac transposon vector. To secrete the collagen into the cocoon, the gene encoding collagen was connected with the cDNA C-terminal of fibroin L-chain with the L-chain promoter (Inoue et al, 2005). In this study, we demonstrated the fibroin H-chain expression system using the piggyBac transposonderived vector to produce the enhanced cyan fluorescent protein (ECFP) in the cocoon.…”

Section: Plasmid Dna Constructionmentioning

confidence: 99%

Expression of the cyan fluorescent protein in fibroin H-chain of transgenic silkworm

Goo¹,

Choi²,

Kim³

et al. 2017

International Journal of Industrial Entomology

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We constructed the fibroin H-chain expression system to produce enhanced cyan fluorescent proteins (ECFP) in transgenic silkworm cocoon. Fluorescent cocoon could be made by fusing ECFP cDNA to the heavy chain gene and injecting it into a silkworm. The ECFP fusion protein, each with N-and C-terminal sequences of the fibroin H-chain, was designed to be secreted into the lumen of the posterior silk glands. The expression of the ECFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworms. The EGFP fluorescence became visible in the ocelli and in the central and peripheral nervous system on the seventh day of embryonic development. A mixture of the donor and helper vector was micro-injected into 1,020 Kumokjam, bivoltin silkworm eggs. We obtained 6 broods. The cocoon was displayed strong blue fluorescence, proving that the fusion protein was present in the cocoon. Accordingly, we suggest that the ECFP fluorescence silk will enable the production of novel biomaterial based on the transgenic silk.

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A fibroin secretion-deficient silkworm mutant, , provides an efficient system for producing recombinant proteins

Cited by 86 publications

References 25 publications

A New Method for the Modification of Fibroin Heavy Chain Protein in the Transgenic Silkworm

A New Method for the Modification of Fibroin Heavy Chain Protein in the Transgenic Silkworm

Electroporation-mediated somatic transgenesis for rapid functional analysis in insects

Expression of the cyan fluorescent protein in fibroin H-chain of transgenic silkworm

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