To product the blue fluorescent protein (AmCyan) expressed cocoon, we were fused AmCyan cDNA to the heavy chain gene and injected the gene into a silkworm. AmCyan was one of the existing violet fluorochromes and originally derived from the fluorescent protein amFP486. AmFP486 was cloned from the sea anemone Anemonia majano (GenBank accession number AF168421), and belongs to the family of fluorescent proteins (FPs) isolated from coral reef organisms. The AmCyan fusion protein, each with N- and C- terminal sequences or the fibroin H-chain, were designed to be secreted into the lumen of the posterior silk glands. The expression of the AmCyan/H-chain fusion gene was regulated by the fibroin H-chain promoter. The use of the 3xP3 EGFP as a marker allowed us to rapidly distinguish transgenic silkworm. Mixtures of the donor and helper vectors were micro-injected into 300 eggs of bivoltine silkworms (Baegokjam). EGFP fluorescence was observed in 3 broods of transgenic silkworms under a florescence stereomicroscope. The cocoon was displayed strong blue fluorescence, proving that the fusion protein was present in the cocoon. Accordingly, we suggest that the AmCyan gene expressed cocoon will be enable the production of the novel biomaterials based on the transgenic silk.

BmCecB1 are antimicrobial peptides from Bombyx mori and belongs to cecropin family. Antimicrobial peptides are important components of the innate immune systems in all living organism. This peptide has antibacterial activity against several Gram-positive and Gram-negative bacteria. To produce the BmCecB1 antimicrobial peptide, we constructed transgenic silkworm that expressed BmCecB1 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 600 eggs of bivoltin silkworms, Baegokjam. In total, 49 larvae (G0) were hatched and allowed to develop into moths. The resulting G1 generation consisted of 22 broods, and we selected 2 broods containing at least 1 EGFP-positive embryo. The rate of successful transgenesis for the G1 broods was 11%. We identified 9 EGFP-positive G1 moths and these were backcrossed with wild-type moths. With the aim of identifying a BmCecB1 as antimicrobial peptide, we investigated the Radical diffusion Assay (RDA) and then demonstrated that BmCecB1 possesses high antibacterial activities against Gram-negative bacteria.

Immune-inducible antimicrobial peptides were produced using transgenic silkworms that expressed Rel family transcription factor, truncated BmRelish1 (BmRelish1t) genes under the control of the BmA3 promoter using the piggyBac vector. BmRelish1t gene contains all domains of Bmrelish: a Rel homolog domain (RHD), nuclear localization signal (NLS), acidic and hydrophobic amino acids (AHAA) rich region except the Ankyrin repeat domain (ANK) and the death domain (DD). (1:1) Mixtures of the donor vector (pG-3xP3EGFP-BmA3BmRelish1t) and helper vector were micro-injected into 1,800 eggs of bivoltin silkworms, Baegokjam and EGFP-induced fluorescence was observed for 25 broods of transgenic silkworms under a florescence stereomicroscope. Analysis by real-time PCR indicated that transgenic silkworms expressing BmRelish1t recombinant proteins displayed higher mRNA expression levels of the Bombyx mori antimicrobial peptides such as lebocin, moricin, and nuecin than the normal silkworms. Moreover, transgenic silkworms expressing BmRelish1t showed antibacterial activity against Escherichia coli. We suggest that transgenic expression of BmRelish1t may find useful applications for the production of various antimicrobial peptides at the same time in transgenic silkworms.

Silkworm transgenesis is now a routine method leading to a satisfactory yield of transformed animals and the reliable expression of transgenes during multiple successive generations. However, the screening of G1 transgenic individuals from numerous progeny has proved to be difficult and time-consuming work. Previously, we characterized the promoter of heat shock protein 70 from Bombyx mori (bHsp70), which is ubiquitously expressed in all tissues and developmental stages. To investigate the utilization of the bHsp70 promoter to screen transgenic individuals, the EGFP marker gene was inserted into the piggyBac vector under the control of the bHsp70 promoter. Mixtures of the donor and helper vectors were micro-injected into 3,060 eggs of bivoltine silkworms (Keomokjam). EGFP fluorescence was observed in 17 broods of transgenic silkworms under a florescence stereomicroscope. Interestingly, this fluorescent marker protein was detected not only in parts of the embryo segments on the seventh day of the G1 embryonic developmental stage but it was also detected in a part of the body of G1 hatched larvae, in the middle silk gland of G1 fifth instar larvae, and in the wings of seven-day-old G1 pupae and G1 moths. Therefore, we suggest that the bHsp70 promoter can be used for the rapid and simple screening of transgenic silkworms.

본 연구는 두 개의 편광판 사이에서 홀로그램과 같은 독특한 외관을 갖는 라멜라 액정상 메이크업 클렌징 제형에 관한 것이다. 라멜라 액정상을 갖는 메이크업 클렌징 제형 연구는 오래전부터 많이 시도되어 왔지만, 탁 도 증가 및 점도 상승 등의 문제로 실제 상용화에는 한계가 있었다. 본 연구에서는 이러한 문제를 해결하기 위해 계면활성제의 알킬 체인에 의한 액정상의 유동성 증가와 음이온 계면활성제 도입에 따른 정전기적 반발력 증가 를 이용하였다. 이를 통해 액정상의 결정화를 억제하여 안정한 액정상 제형을 확보할 수 있었다. 또한, 본 연구에 서는 객관적이고 정량적인 클렌징력 평가를 위해 형광물질과 in vivo imaging system (IVIS) 장비를 이용하여 in vitro 클렌징력 평가법을 새롭게 도입하였다. 이렇게 개발된 클렌징력 평가법을 이용하여 라멜라 액정상 메이 크업 클렌징 제형의 우수한 메이크업 클렌징력을 확인할 수 있었다. 한편, 본 연구에서 개발된 라멜라 액정상 메이크업 클렌징 제형을 직교 배열된 두 편광판 사이에 두면 홀로그램과 유사한 특이적인 무늬를 관측할 수 있 다. 이러한 현상은 액정상 제형을 통과하는 가시광선과 라멜라 구조 사이의 간섭현상에 의한 것으로 추정된다. 클렌징 제형에서의 라멜라 구조는 소각 X선 산란분석(SAXS)을 이용하여 Bragg spacing ratio가 1 : 2의 정수 비로 관찰되는 것을 통해 확인하였다. 본 연구를 통해 개발된 라멜라 액정상 메이크업 클렌징 제형은 우수한 경시 안정성과 클렌징력, 그리고 편광판 사이에서 관찰되는 홀로그램과 유사한 독특한 외관으로 인해 향후 새로 운 메이크업 클렌징 제품 개발에 도움이 될 것으로 기대된다.

마이셀을 이용한 가용화 제형은 화장품 산업에서 스킨 로션, 토너, 미스트 등 다양한 제형으로 이용되고 있다. 마이셀은 입자 자체가 매우 작기 때문에 효능 물질의 담지체 역할보다는 향을 가용화시키는 정도로 활용 되고 있다. 본 연구에서는 효능 물질인 β-sitosterol을 담지 할 수 있는 새로운 마이셀을 개발하기 위하여 투 명한 외관을 갖는 swollen micelle을 고려하였다. 특히, 효능 성분과의 용해도 계수를 고려하여 swollen micelle을 제조함으로써 난용성 효능 성분이 마이셀 내부에 안정하게 존재할 수 있는 새로운 방법을 개발하였다. 이렇게 만들어진 swollen micelle의 안정도는 동적광산란장치(dynamic light scattering, DLS)를 이용하여 확인하였고, 투명한 입자의 외관과 모양은 육안 관찰 및 cryo-TEM을 통해 확인하였다. 또한, DSC를 이용한 열분석을 통해 난용성 효능 성분인 β-sitosterol이 swollen micelle 내에서 안정하게 존재함을 확인하였다. 본 연구를 통해 용해도 계수를 고려한 swollen micelle은 난용성 효능 성분의 새로운 담지체로서 이용할 수 있 음을 확인하였다.