The generation of germline transgenic silkworms for the production of biologically active recombinant fusion proteins of fibroin and human basic fibroblast growth factor

Hino, Rika; Tomita, Masahiro; Yoshizato, Katsutoshi

doi:10.1016/j.biomaterials.2006.07.028

Cited by 89 publications

(74 citation statements)

References 36 publications

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“…이 시스템은 벡터 및 헬퍼 플라스미드 DNA를 preblastodermal 누에알에 주입하여 형질전환누에를 제작 하는 방법으로서, 일본의 Tamura 연구팀이 나비목곤충인 Trichoplusia ni에서 유래한 piggyBac 전이벡터를 제작하 여 최초로 형질전환 누에 제작에 성공하였다 (Tamura et al 2000). 현재까지 piggyBac 전이벡터를 사용한 많은 누 에 형질전환 실험이 진행되었고, 이 발현 시스템을 사용 하여 basic fibroblast growth factor(bFGF), human serum albumin(HSA), feline interferon(FeIFN), insulin like growth factor-I(hIGF-I) 등이 성공적으로 발현된다고 보고되어 있 다 (Hino et al 2006, Kurihara et al 2007, Ogawa et al 2007, Tomita et al 2003, Zhao et al 2009). …”

Section: 서 론unclassified

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

Kim¹,

Yun²,

Choi³

et al. 2014

Journal of Sericultural and Entomological Science

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We produced the transgenic silkworm that expressed the enhanced blue fluorescent protein (EBFP) in the cocoon of silkworms. The EBFP 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 EBFP/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3 × P3-driven DsRed2 cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. A mixture of the donor and helper vector was micro-injected into 300 eggs of silkworms, Baegokjam. We obtained 5 broods. The cocoon displayed blue fluorescence, proving that the fusion protein was present in the cocoon. Also, the presence of fusion proteins in cocoons was demonstrated by SDS-PAGE and western blot analysis. Accordingly, we suggest that the EBFP fluorescence silk will enable the production of the silk-based biomaterials.

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Section: 서 론unclassified

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

Kim¹,

Yun²,

Choi³

et al. 2014

Journal of Sericultural and Entomological Science

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“…The cocoon is made mainly by silk fibroin, which is synthesized by the posterior silk gland (Hino et al, 2006;Ogawa et al, 2007). Silk fibroins consist of two subunits, fibroin H-and L-chains, which are linked by a disulfide-bond.…”

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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“…김성완·구태원·김성렬·박승원·최광호 이용하여 동물의 종양세포로 멜리틴을 운반하거나, HIV 를 파괴하는 데 효과가 있다고 보고되었다 (Hood et al 2013). 최초의 누에 형질전환은 1971년 일본의 Nawa에 의해 이루어진 실험으로 흑란계통의 게놈을 백란계통의 알에 주입하여 흑란계통의 누에를 얻는 실험이었다.…”

unclassified

“…그러나, 일본의 Tamura 등이 piggyBac유전자를 이용하여 누에 형질전환에 성공하였다 (Tamura et al 2000). 이후 이 방법을 사용하여 basic fibroblast growth factor(bFGF), human serum albumin (HSA), feline interferon(FeIFN), insulin like growth factor-I(hIGF-I) 등이 성공적으로 발현된다고 보고되어 있다 (Hino et al 2006, Kurihara et al 2007, Ogawa et al 2007, Tomita et al 2003, Zhao et al 2009). 최근에는 항균펩타 이드인 세크로핀을 발현시키는 실크가 제작되었다고 보고 되었다 (Li et al 2015 The Baegokjam strain was used as a host strain.…”

unclassified

Production of the melittin antimicrobial peptide in transgenic silkworm

Kim¹,

Goo²,

Kim³

et al. 2015

Journal of Sericultural and Entomological Science

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Melittin is the main component of Bee Venom and has antibacterial activity against several bacteria. To produce the melittin antimicrobial peptide, we constructed transgenic silkworm that expressed melittin gene under the control BmA3 promoter using piggyBac vector. The use of the 3xP3-driven EGFP cDNA as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor vector and helper vector were micro-injected into 300 eggs of bivoltin silkworms, Baegokjam. In total, 131 larvae (G0) were hatched and allowed to develop into moths. The resulting G1 generation consisted of 36 broods, and we selected 4 broods containing at least 1 EGFP-positive embryo. The rate of successful transgenesis for the G1 broods was 11%. We identified 12 EGFP-positive G1 moths and these were backcrossed with wild-type moths. With the aim of identifying a melittin as antimicrobial peptide, we investigated the Radical diffusion Assay (RDA) and then demonstrated that melittin possesses high antibacterial activities against gramnegative bacteria.

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The generation of germline transgenic silkworms for the production of biologically active recombinant fusion proteins of fibroin and human basic fibroblast growth factor

Cited by 89 publications

References 36 publications

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

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

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

Production of the melittin antimicrobial peptide in transgenic silkworm

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