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김성완 ( Sung Wan Kim ),윤은영 ( Eun Young Yun ),최광호 ( Kwang Ho Choi ),김성렬 ( Seong Ryul Kim ),박승원 ( Seung Won Park ),강석우 ( Seok Woo Kang ),권오유 ( O Yu Kwon ),구태원 ( Tae Won Goo ) 한국잠사학회 2012 한국잠사곤충학회지 Vol.50 No.2
We constructed the fibroin H-chain expression system to produce Discosoma sp. red fluorescent protein variant2 (DsRed2) in transgenic silkworm cocoon. Fluorescent cocoon could be made by fusing DsRed2 cDNA to the heavy chain gene and injecting it into a silkworm. The DsRed2 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 DsRed2/ 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 red fluorescence, proving that the fusion protein was present in the cocoon. Accordingly, we suggest that the DsRed2 fluorescence silk will enable the production of novel biomaterial based on the transgenic silk.
김성완 ( Sung Wan Kim ),강민옥 ( Min Uk Kang ),강석우 ( Seok Woo Kang ),윤은영 ( Eun Young Yun ),최광호 ( Kwang Ho Choi ),김성렬 ( Seong Ryul Kim ),박승원 ( Seung Won Park ),노시갑 ( Si Kab Nho ),구태원 ( Tae Won Goo ) 한국잠사학회 2013 한국잠사곤충학회지 Vol.51 No.1
Silkworm transgenesis scientists have done some genetic modification work on multivoltine silkworms, but that type of silkworms is less commercial feasible. They are easy to manipulate, because they breed all year round. But the commercial silkworm variety must undergo hydrochloric acid treatment at a high temperature to be artificially hatched. Hydrochloric acid penetrates through the holes in the silkworm eggs, fatally damaging their reproduction. So it had been thought that altering the properties of the commercial silkworm variety would be very difficult. So we have tried to make from diapause to non-diapause eggs using diapauses varieties, ``Backokjam`` and ``Jam 124``. At present, our group has establishing the conditions for non-diapause eggs. Oviposited eggs after 40 ~ 60 hours were incubated for 24 hours at 15 ~ 20oC with dark condition. Non-diapause eggs were completely induced. The hatching rate, molting rate and pupation rate of non-diapause ``Jam 124`` and ``Backokjam`` eggs showed no differences compared to diapause eggs. When transgenic silkworm using the non-diapause eggs, the hatching rate showed that non-diapause eggs induced from diapause were 40 ~ 70%, diapause eggs treated with artificial incubation were 10 ~ 30%, and polyvoltine strains, HM eggs were 30 ~ 50%. Therefore, we suggest that modification techniques of the commercial silkworm eggs adequate for silkworm transgenesis can be used to develop transgenic silkworms more easily.
Construction of Transgenic Silkworms Expressing Human Stem Cell Factor (hSCF)
Sung Wan Kim(김성완),Eun Young Yun(윤은영),Seong Ryul Kim(김성렬),Seung Won Park(박승원),Seok Woo Kang(강석우),O-Yu Kwon(권오유),Tae Won Goo(구태원) 한국생명과학회 2011 생명과학회지 Vol.21 No.12
본 연구의 목적은 누에형질전환체를 이용하여 재조합단백질 대량생산 시스템을 개발하는 것으로서, 본 실험에서는 hSCF유전자를 이용하여 누에에서 재조합단백질을 생산하였다. 실험에 사용된 piggyBac 전이벡터는 hSCF 유전자의 발현 조절을 위해 초파리 유래의 dHsp70 promoter를 사용하였고, EGFP marker유전자는 3xP3 promoter로 발현을 조절하였다. 총 1,020 개의 누에알에 microinjection 하여 G1 세대에서 22 bloods의 형질전환체를 선발하였고, 선발된 누에형질전환체는 초기배 단계의 눈과 신경조직, 유충과 번데기 그리고 성충의 눈에서 GFP 형광을 관찰 할 수 있었다. hSCF 재조합단백질의 발현은 Western blot 분석으로 확인 할 수 있었고, inverse PCR 분석을 통해서 누에 게놈에 전이벡터가 삽입된 것을 확인할 수 있었다. 지금까지의 실험 결과에서 hSCF 재조합 단백질이 누에에서 생산될 수 있음을 확인 할 수 있었다. 비록 누에에서 생산된 hSCF 재조합단백질의 생리활성에 대한 실험이 추후에 요구되지만, 이러한 실험결과는 piggyBac 전이벡터와 microinjection 법으로 누에에서 고부가가치의 재조합단백질을 대량생산 할 수 있음을 보여 주었다고 할 수 있겠다. 따라서 누에를 유용물질 생산을 위한 생체반응기로서 활용할 수 있을 것으로 기대된다. Human Stem Cell Factor (hSCF) is a cytokine that binds to the c-Kit receptor and plays an important role in hematopoiesis, spermatogenesis, and melanogenesis. To produce the human Stem Cell Factor (hSCF) recombinant protein, we constructed a germline transgenic silkworm using the piggyback vector. The expression of the hSCF gene was driven by the Drosophila heat shock protein 70 (dHsp70) promoter. 3XP3 promotor-driven EGFP was used as a marker which allowed us to rapidly distinguish the transgenic silkworm. A mixture of the donor and helper vector was micro-injected into 1,020 eggs of bivoltin silkworms, Keomokjam. We obtained approximately 22 G1 broods that were EGFP-positive. The expression of the hSCF gene in the transgenic silkworm was analyzed by SDS-PAGE and immunoblotting. Also, analysis of insertion sites into the silkworm genome using inverse PCR showed that exogenous DNA was inserted into the transgenic silkworm genome. These results show that successfully constructed transgenic silkworm expresses the hSCF recombinant protein.
김주년(Kim, Joo-Nyun),이상래(Lee, Sang-Rae),김성완(Kim, Sung-Wan),윤원주(Yun,Won-Ju),김석권(Kim, Seok-Kwon),노성민(Noh, Sung-Min),마근수(Ma, Keun-su) 한국항공우주연구원 2014 항공우주산업기술동향 Vol.12 No.1
항공우주분야의 원격측정시스템은 비행체의 내부 상태 및 비행정보를 지상으로 전송하는 역할을 하는 탑재부분과 지상에서 비행 데이터를 수신하여 처리하는 지상시스템으로 구성된다. 원격측정시스템을 통해 취득된 데이터는 모든 비행시험 및 임무 수행의 성공여부를 결정하는데 매우 중요한 역할을 한다. 본 논문에서는 항공우주분야 원격측정 기술의 기술동향에 관하여 기술하였는데 고전적 1세대 기술에서부터 최근의 3세대 기술에 이르기까지 기술경향을 기술하였다. 특히 최근 무인항공기 분야를 비롯한 항공분야 원격측정 데이터 전송율의 요구가 급격히 증대됨에 따라 고속 네트워크를 활용한 4세대 항공우주 원격측정기술의 표준화가 진행되고 있으며 본 논문에서는 이에 관한 기술 동향도 함께 기술하였다. Telemetry system aerospace industry consists of the on board section which gathers and transmits status of the aerospace vehicles and the ground section which receives and processes the telemetry data. Telemetry data acquired through the flight tests of all the mission can be used to determine the success or failure of the mission. In this paper, aerospace telemetry technology trend is described from the conventional first generation telemetry technology to the latest technology trends, the third generation technology. Since the aeronautical telemetry industry including the unmanned aircraft telemetry data rate increases rapidly, new telemetry system, the fourth generation telemetry technology, is underway to cope with the future telemetry mission using high speed Ethernet network. The trend of the fourth generation telemetry system is also described in this paper.
Artificial Mutation for Silkworm Molecular Breeding Using Gene Scissors
Jeong Won Hong(홍정원),Chan Young Jeong(정찬영),Jeong Hee Yu(유정희),Su-Bae Kim(김수배),Sang Kuk Kang(강상국),Seong-Wan Kim(김성완),Nam-Suk Kim(김남숙),Kee Young Kim(김기영),Jong Woo Park(박종우) 한국생명과학회 2020 생명과학회지 Vol.30 No.8
Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated protein (Cas)9을 이용하는 유전자 가위 기술은 미래 육종 기술로서 주목받고 있다. 본 연구에서는 3세대 유전자 가위 CRISPR/Cas9을 이용한 누에 kynurenine 3-monooxygenase (KMO) 유전자 편집을 통한 돌연변이 유발 및 선택 교배를 통하여 유전자의 세대간 전달을 분석하고자 하였다. 유전자 편집을 위하여 누에의 KMO 유전자에 대한 3종의 가이드 RNA를 제작하고, 제작된 gRNA는 Cas9 단백질과 복합체를 형성시켜 누에 세포주(BM-N)에 도입 후 T7 endonucleaseI 분석을 수행하여 최적의 gRNA를 선발하였다. 선발된 K1N gRNA는 누에 유전자를 편집하기 위하여 Cas9 단백질과 복합체를 형성시킨 후 누에 초가 배아에 미세주사하고 사육하였다. 미세주사 후 부화율은 18% 가량으로 낮게 나타났으나 생존한 개체 중 돌연변이 발생율은 60% 이상으로 비교적 높게 나타났다. 돌연변이가 발생된 G0세대의 KMO 유전자는 이형접합자 형태로 나타났으며, 표현형의 변화는 관찰되지 않았다. 하지만 형접합자들 사이의 근친 교배에 의해 탄생한 G1세대 돌연변이에서는 일부에서 알과 눈의 색 변화가 확인되었으며, 변이가 확인된 개체들사이의 근친교배를 통해 생산된 G2세대에서는 모든 개체에서 표현형의 변화가 나타났다. 이러한 결과에 비추어 볼 때, 유전자 가위를 이용한 돌연변이 육종에는 한계가 있으나 전통 교배 육종과 융합을 통하여 육종 기간을 비약적으로 단축시킬 수 있는 곤충 육종 기술로 발전가능성이 높다고 판단된다. Gene editing technology using the clustered regularly interspaced short palindromic repeat (CRISPR) and the CRISPR associated protein (Cas)9 has been highly anticipated in developing breeding techniques. In this study, we discuss gene scissors as a tool for silkworm molecular breeding through analysis of Bombyx mori Kynurenine 3-Monooxygenase (BmKMO) gene editing using the CRISPR/Cas9 system and analysis of generational transmission through mutagenesis and selective crossing. The nucleotide sequence of the BmKMO gene was analyzed, and three guide RNAs (gRNAs) were prepared. Each synthesized gRNA was combined with Cas9 protein and then analyzed by T7 endonuclease I after introduction into the BM-N silkworm cell line. To edit the silkworm gene, K1P gRNA and Cas9 complexes were subsequently microinjected into the silkworm embryos; the hatching rate was 18% and the incidence of mutation was 60%. The gene mutation was verified in the heterozygous G0 generation, but no phenotypic change was observed. In homozygotes generated by self-crossing, a mutant phenotype was observed. These results suggest that silkworm molecular breeding using the CRISPR/ Cas9 system is possible and could be an effective way of shortening the time required.