• KSBB Journal
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• 제19권5호
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• pp.341-347
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• 2004

Several gene delivery therapies are being developed for treatment of serum protein deficiency. EPO is one of the most promising therapeutic agent for this treatment which is currently being investigated in depth. This study has the ultimate purpose of improving the gene delivery system for an increase of red blood cell production. A plasmid DNA was constructed smaller than other plasmids for an increase in penetration into animal cells, and two genes were cloned into each vector as a co-delivery system to express erythropoietin, and interluekin-3 or thrombopoietin, which can act on erythroid cell, thus activating hematopoiesis synergically. This co-delivery system has an advantage of decreasing the labour required for industrial production of DNA vaccine. A new plasmid vector, pVAC, in size 2.9 kb, was constructed with the essential parts from PUC 19 and pSectagB, which is much smaller than other plasmid vector and is the size of 2.9 kb. Co-delivery system was constituted by cloning human erythropoietin with each of human interluekin-3 gene or human thrombopoietin gene into both pVAC and pSectagB. As a result, the transfection efficiency of pVAC was higer than that of pSectagB in vitro, and hematocrit level of the mice injected with pVAC is higher than that of other mice. And co-delivery system, made of several plasmid DNAs, was expressed in vitro.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.99-99
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• 2002

The hormone resistin is associated with typeII diabetes mellitus in rodent model. Resistin impairs glucose tolerance and insulin action. A new class of anti-diabetic drugs were called thiazolidinediones (TZDs) downregulates a resistin which is induced during adipocyte differentiation. But the connection between increased adiposity and resistin remains unknown. The objectives of this study was to clone a mouse resistin cDNA and to generate transgenic mice overexpressing mouse resistin gene. The 555 bp of mouse resistin was amplified from mob cDNAS by polymerase chain reaction (PCR) and cloned into pCR$\^$(R)/ 2.1 TOPO T-vector. Mouse resistin mRNA on the basis of Genbank sequence (acession no. AF323080). Then, the PCR product was cloned into pTargeT$\^$TM/ mammalian expression vector that has pCMV promoter and chimeric intron. Restriction enzyme analysis with BamH I and Not I was carried out to determine an orientation of the insert in the vector. The pCMV-mus/resistin gene was prepared from previous recombinant pTargeT$\^$TM/-mus/resistin by digestion of Bgl II, and has used for microinjection into pronuclei of one cell embryos. The microinjected embryos were transfered to pseudopregnant foster-mother. Mouse resistin expression was detected in transgenic F1 mice by Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR). Resistin gene expression mouse has heavier body weight which was measured higher level of plasma glucose than that of normal mouse. And in diet-induced experiments, the abdominal fat pads were isolated from each 24h starvation and re-feeding after fasting group mice that were assessed by RT-PCR analysis. In fasting group mice, resistin expression was higher than that of re-feeding group mice. This result suggests that the resistin gene overexpressing mice may be became to obesity and be useful as an animal disease model to be diabetes mellitus caused by insulin resistance of resistin.

• Reproductive and Developmental Biology
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• 제41권1호
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• pp.7-16
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• 2017

The knock-in efficiency in the fibroblast is very important to produce transgenic domestic animal using nuclear transfer. In this research, we constructed three kinds of different knock-in vectors to study the efficiency of knock-in depending on structure of knock-in vector with different size of homologous arm on the ${\beta}-casein$ gene locus in the somatic cells; DT-A_cEndo Knock-in vector, DT-A_tEndo Knock-in vector I, and DT-A_tEndo Knock-in vector II. The knock-in vector consists of 4.8 kb or 1.06 kb of 5' arm region and 1.8 kb or 0.64 kb of 3' arm region, and neomycin resistance gene(neor) as a positive selection marker gene. The cEndo Knock-in vector had 4.8 kb and 1.8 kb homologous arm. The tEndo Knock-in vector I had 1.06 kb and 0.64 kb homologous arm and tEndo Knock-in vector II had 1.06 kb and 1.8 kb homologous arm. To express endostatin gene as transgene, the F2A sequence was fused to the 5' terminal of endostatin gene and inserted into exon 7 of the ${\beta}-casein$ gene. The knock-in vector and TALEN were introduced into the bovine fibroblast by electroporation. The knock-in efficiencies of cEndo, tEndo I, and tEndo II vector were 4.6%, 2.2% and 4.8%, respectively. These results indicated that size of 3' arm in the knock-in vector is important for TALEN-mediated homologous recombination in the fibroblast. In conclusion, our knock-in system may help to create transgenic dairy cattle expressing human endostatin protein via the endogenous expression system of the bovine ${\beta}-casein$ gene in the mammary gland.

• 한국정보통신학회:학술대회논문집
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• 한국정보통신학회 2018년도 추계학술대회
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• pp.444-447
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• 2018

Baculovirus 발현 시스템은 박테리아 발현 시스템, 특히 복잡한 번역 후 변형을 필요로 하는 것과 비교하여 다량의 재조합 단백질을 생성하는 빠르고 비용 효율적인 방법을 포함하는 많은 알려진 장점을 갖는다. 특히 재조합 baculovirus는 광범위한 포유류 세포 유형에서 벡터를 전달하고 재조합 단백질을 발현 할 수 있다. 본 연구에서는 pcDNA3.1로부터 재구성된 baculovirus 벡터를 사용하였는데 이 벡터는 cytomegalovirus (CMV) 프로모터, uroplakin II promoter, polyhedron promoter, 수포 구내염 바이러스 G (VSVG), 녹색 형광 단백질 (EGFP), 단백질 전달 도메인 (PTD) 유전자 등 다양한 유전자들로 재조합 되어 개발되었다. 이러한 재구성 된 벡터를 다양한 세포 및 세포주에 감염시켰다. 이렇게 개발된 baculovirus 벡터는 재조합된 유전자들의 전이성 및 발현성을 기존의 일반적인 벡터와 비교하여 분석하였다. 본 연구결과로 이렇게 개발된 baculovirus 벡터는 기존의 대조군 벡터보다 전이성 및 발현성면에서 더 높은 효율을 갖는다는 것을 확인하였다.

• 한국음향학회지
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• 제36권5호
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• pp.338-344
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• 2017

본 논문에서는 머리전달함수 보간 알고리즘을 제안하며, 특히 작은 크기의 측정 데이터를 다루는 경우를 고려한다. 제안하는 알고리즘은 머리전달함수의 음향학적 모델링에 기초하며, 모델링 계수를 추정함으로써 머리전달함수를 보간한다. 이 때 측정 위치의 개수가 부족할 경우 모델링 계수를 추정하는 것은 매우 어려우며, 따라서 본 알고리즘은 벡터-기반 크기 패닝 기법을 이용하여 데이터를 확장함으로써 이러한 문제를 해결하려고 한다. 본 알고리즘은 벡터-기반 크기 패닝 기법 기반의 데이터 확장 단계와, 최소자승법 기반의 모델링 계수 추정 단계의 두 단계로 이루어져 있다. 제안하는 알고리즘의 성능을 확인하기 위하여 CIPIC(Center for Image Processing and Integrated Computing) 머리전달함수 데이터베이스의 측정 데이터 중 일부를 이용한 시뮬레이션을 진행하였으며, 시뮬레이션 결과 약 1.5 dB ~ 4 dB의 최소 자승 오차가 감소됨을 확인할 수 있었다.

• Reproductive and Developmental Biology
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• 제32권2호
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• pp.81-87
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• 2008

Production of transgenic animals for studying specific gene has been limited due to a low efficiency, lack of skilled researchers and the need for expensive equipment. Currently, the boar spermatozoa as a vector to deliver exogenous DNA into the oocyte were used to improve the efficiency of transfection rate. In this study, we revealed that the optimal conditions for DNA uptake in spermatozoa by liposome were to 90 min of incubation, $17^{\circ}C$, $10^5$ spermatozoa, 4 ng/ml of exogenous DNA and 0.5% (v/v) liposome, without damage to fertility. In addition, the developmental rate to the blastocyst stage of embryo in control group was significantly higher than those embryos with exogenous DNA and liposome, whereas there were no significant differences in embryo development between the liposome and type of DNA. The transfection rates of embryo using treated spermatozoa with both liposome and circular DNA were higher than those using linear DNA. These findings raise the possibility thattreated spermatozoa with liposome/DNA complexes could be used in in vitro fertilization, and the exogenous DNA transferred into the oocytes. Taken together, we demonstrated that liposome a vector for the uptake of exogenous DNA in boar spermatozoa could improve the efficiency of sperm-mediated gene transfer in creating transgenic pig and the other domestic transgenic animals.

• KIEAE Journal
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• 제16권1호
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• pp.89-94
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• 2016

This study is part of three-dimensional(3D) heat transfer analysis program developmental process. The program is being developed without it's own built in 3D-modeller. So 3D-model must be created from another 3D-modeller such as generic CAD programs and imported to the developed program. After that, according to the 3D-geometric data form imported model, 3D-mesh created for numerical calculation. But the 3D-model created from another 3D-modeller is likely to have errors in it's geometric data such as mismatch of position between vertexes or surfaces. these errors make it difficult to create 3D-mesh for calculation. These errors are must be detected and cured in the pre-process before creating 3D-mesh. So, in this study four kinds of filters and functions are developed and tested. Firstly, 'vertex error filter' is developed for detecting and curing for position data errors between vertexes. Secondly, 'normal vector error filter' is developed for errors of surface's normal vector in 3D-model. Thirdly, 'intersection filter' is developed for extracting and creating intersection surface between adjacent objects. fourthly, 'polygon-line filter' is developed for indicating outlines of object in 3D-model. the developed filters and functions were tested on several shapes of 3D-models. and confirmed applicability. these developed filters and functions will be applied to the developed program and tested and modified continuously for less errors and more accuracy.

• 한국수정란이식학회지
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• 제31권3호
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• pp.161-168
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• 2016

Nucleotide metabolism in endothelium is variable between different species. Recent studies demonstrated that this variability could contribute coagulation dysfunction, even though organs of the alpha 1,3-galactosyltransferase gene knockout pig were transplanted into the primate. CD73 (ecto-5'-nucelotidase) is an enzyme at cell surface catalyzing the hydrolysis of adenosine triphosphate to adenosine, which plays role on a substance for anti-inflammatory and anti-coagulant. Thus, overexpression of CD73 in endothelial cells of the pig is considered as an approach to reduce coagulopathy. In this study, we constructed a human CD73 expression vector under control of porcine Icam2 promoter (pIcam2-hCD73), which is expressed specifically at endothelial cells, and of CMV promoter as a control (CMV-CD73). First, we transfected the CMV-CD73 vector into HEK293 cells, and then confirmed CD73 expression at cell surface by flow cytometry analysis. Next, we transfected the pIcma2-CD73 and CMV-CD73 vectors into primary porcine fibroblasts and endothelial cells. Consequence was that the pIcma2-CD73 vector was expressed only at the porcine endothelial cells, meaning that the pIcam2 promoter lead to endothelial cell-specific expression of CD73 in vitro. Finally, we nucleofected the pIcam2-hCD73 vector into passage 3 fibroblasts, and enforced hygromycin selection of 400mg/ml. We were able to obtain forty three colonies harboring pIcam2-CD73 to provide donor cells for transgenic cloned porcine production.

• 한국수정란이식학회지
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• 제29권4호
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• pp.339-344
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• 2014

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein (Cas9) system can be applied to produce transgenic pigs. Therefore, we applied CRISPR/Cas9 system to generate FoxN1-targeted pig parthenogenetic embryos. Using single guided RNA targeted to pig FoxN1 genes was injected into cytoplasm of in vitro matured oocyte before electrical activation. In results, regardless of the concentrations of vector, the cleavage rate were significantly (p<0.05) decreased ($4ng/{\mu}l$, 51.24%; $8ng/{\mu}l$, 40.88%; and $16ng/{\mu}l$; 45.22%) compared to no injection group (70.44%). The blastocyst formation rates were also decreased in vector injected 3 groups ($4ng/{\mu}l$, 7.96%; $8ng/{\mu}l$, 6.4%; and $16ng/{\mu}l$; 9.04%) compared to no injection group (29.07%). In addition, the blastocyst formation rates between sham injected group (13.51%) and no injection group (29.07%) also showed significant difference (p<0.05). The mutation rates were comparable between groups ($4ng/{\mu}l$, 18.4%; $8ng/{\mu}l$, 12.5%; and $16ng/{\mu}l$; 20.0%). The sequencing analysis showed that blastocysts derived from each group were successfully mutated in FoxN1 loci regardless of the vector concentrations. However, the deletion patterns were higher than the patterns of point mutation and insertion regardless of the vector concentrations. In conclusion, we described that cytoplasmic microinjection of FoxN1-targeted CRISPR/Cas9 vector could efficiently generate transgenic pig parthenogenetic embryos in one-step.

• 인터넷정보학회논문지
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• 제20권4호
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• pp.91-102
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• 2019

자율주행 시스템에서, 카메라에 포착된 영상을 통하여 보행자를 분류하는 기능은 보행자 안전을 위하여 매우 중요하다. 기존에는 HOG(Histogram of Oriented Gradients)나 SIFT(Scale-Invariant Feature Transform) 등으로 보행자의 특징을 추출한 후 SVM(Support Vector Machine)으로 분류하는 기술을 사용했었으나, 보행자 특징을 위와 같이 수동(handcrafted)으로 추출하는 것은 많은 한계점을 가지고 있다. 따라서 본 논문에서는 CNN(Convolutional Neural Network)의 깊은 특징(deep features)과 전이학습(transfer learning)을 사용하여 보행자를 안정적이고 효과적으로 분류하는 방법을 제시한다. 본 논문은 2가지 대표적인 전이학습 기법인 고정특징추출(fixed feature extractor) 기법과 미세조정(fine-tuning) 기법을 모두 사용하여 실험하였고, 특히 미세조정 기법에서는 3가지 다른 크기로 레이어를 전이구간과 비전이구간으로 구분한 후, 비전이구간에 속한 레이어들에 대해서만 가중치를 조정하는 설정(M-Fine: Modified Fine-tuning)을 새롭게 추가하였다. 5가지 CNN모델(VGGNet, DenseNet, Inception V3, Xception, MobileNet)과 INRIA Person데이터 세트로 실험한 결과, HOG나 SIFT 같은 수동적인 특징보다 CNN의 깊은 특징이 더 좋은 성능을 보여주었고, Xception의 정확도(임계치 = 0.5)가 99.61%로 가장 높았다. Xception과 유사한 성능을 내면서도 80% 적은 파라메터를 학습한 MobileNet이 효율성 측면에서는 가장 뛰어났다. 그리고 3가지 전이학습 기법중 미세조정 기법의 성능이 가장 우수하였고, M-Fine 기법의 성능은 미세조정 기법과 대등하거나 조금 낮았지만 고정특징추출 기법보다는 높았다.