• Plant Biotechnology Reports
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• 제5권1호
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• pp.9-17
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• 2011

With the development of next-generation sequencing technology, ever-expanding databases of genetic information from various organisms are available to researchers. However, our ability to study the biological meaning of genetic information and to apply our genetic knowledge to produce genetically modified crops and animals is limited, largely due to the lack of molecular tools to manipulate genomes. Recently, targeted cleavage of the genome using engineered DNA scissors called zinc finger nucleases (ZFNs) has successfully supported the precise manipulation of genetic information in various cells, animals, and plants. In this review, we will discuss the development and applications of ZFN technology for genome engineering and highlight recent reports on its use in plants.

• Asian-Australasian Journal of Animal Sciences
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• 제7권3호
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• pp.427-434
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• 1994

The primordial germ cells (PGCs) were transfected in vitro and expressed the exogenous RSVLTR/${\beta}G2$ plasmid, suggesting thaI PGC is a possible vector for direct gene transfer into the germ line. Transfection efficiency of cell suspensions containing PGCs was 1.5% by liposome mediated DNA transfection. By microinjection of the transfected PGCs into the host germinal crescent, PGCs migrated via blood vessel to the future gonad and these transfected PGCs resulted in the RSVLTR/${\beta}G2$ expression in the gonad. The results from the seeding of PGCs on the chorioallantoic membrane were insufficient to test the hypothesis that PGCs can penetrate or invade the chorioallantoic membrane for transport via the circulatory system.

• 한국식물학회:학술대회논문집
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• 한국식물학회 1987년도 식물생명공학 심포지움 논문집 Proceedings of Symposia on Plant Biotechnology
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• pp.283-307
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• 1987

Glycinin and $\beta$-conglycinin are the most abundant storage protein in soybean. These proteins are known to be synthesized predominantly during germination and cell expansion phase of seed development for short period, and synthesized not in other tissues. Genes encoding these storage proteins are useful system to study the mechanism of development stage and tissue specific gene expression in eukaryotes, especially plants, at the molecular level. The cDNA and genomic clones coding for glycinin have been isolated and regulation mechanism of the gene expression has been studied. Initially, development and tissue-specific expression of the glycinin gene is regulated at the level of transcription. Post-transcriptional processing is also responsible for delayed accumulation of the mRNA. Translational control of the storage protein gene has not been reported. Post-translational modification is another strategic point to regulate the expression of the gene. It is possible to identify positive and/or negative reguratory clements in vivo by producing transgenic plants agter gene manipulation. Elucidation of activation and repression mechanism of soybean storage protein genes will contribute to the understanding of the other plant and eukaryotic genes at molecular level.

• Journal of Microbiology and Biotechnology
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• 제19권10호
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• pp.1098-1102
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• 2009

Gamma-linolenic acid (GLA, C18:3 ${\Delta}^{6,9,12}$) is synthesized by a delta-6 fatty acid desaturase using linoleic acid (LA, C18:2 ${\Delta}^{9,12}$) as a substrate. To enable the production of GLA in the conventional yeast Pichia pastoris, we have isolated a cDNA encoding the delta-6 fatty acid desaturase from Cunninghamella echinulata MIAN6 and confirmed its function by heterogeneous expression in P. pastoris. Sequence analysis indicated that this cDNA sequence has an open reading frame of 1,404 bp, which encodes a 52 kDa peptide of 468 amino acids. This sequence has 64% identity to the previously reported delta-6 fatty acid desaturase from Rhizopus oryzae. The polypeptide has a cytochrome b5 domain at the N-terminus including the HPGG motif in the heme-binding region, as reported for other delta-6 fatty acid desaturases. In addition, this enzyme differs from other desaturases by the presence of three possible N-linked glycosylation sites. Analysis of the fatty acid composition demonstrated the accumulation of GLA to the level of 3.1% of the total fatty acids. Notably, the amounts of ginkgolic acid (C17:1) and palmitic acid (C16:0) were increased from 1.3% to 29.6% and from 15% to 33%, respectively. These results reveal that the modification of the fatty acid biosynthetic pathway by genetic manipulation in order to produce specific polyunsaturated fatty acids in P. pastoris is a promising technique.

• The Plant Pathology Journal
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• 제25권2호
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• pp.179-183
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• 2009

To facilitate the genetic manipulation of Cladosporium phlei, a causal agent of leaf spot disease in timothy (Phleum pretense), protoplast-mediated transformation of C. phlei has been developed and the resulting transformants were characterized in this study. Hygromycin B resistance was applied as a dominant selection marker due to the sensitivity of C. phlei to this antibiotic. The transformation efficiency ranged from approximately 20-100 transformants per experiment. Southern blot analysis of stable transformants revealed that transformation occurred by way of stable integration of the vector DNA into the fungal chromosome. PCR analysis and plasmid rescuing of randomly selected transformants suggested that integration of tandem repeat copies of vector DNA was common. In addition, multiple integrations of the transforming vector at different chromosomal sites were also observed. The establishment of a transformation method for C. phlei facilitates strain improvement of this fungus and can be applied as an initial step in the molecular analysis of pigment production in this fungus.

• 한국미생물·생명공학회지
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• 제19권3호
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• pp.215-221
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• 1991

YIp5를 사용하여 Cephalosporium acremonium ATCC 2033으로부터 Saccharomyces cerevisiae SHY3에서 발현되는 자율복제 기점 (ARS)를 분리하였다. 자율복제 기점을 갖는 40종의 vector 가운데 가장 안정성 (stability)이 높은 plasmid를 pCY-2라 명명하였으며 pCY-2는 C.acremonium에서 유래하는 3.7kb의 단편을 갖고 있었다. 또 Southern hybridization과 재형질전환을 통해 pCY-2가 효모내에서 자율적으로 복제되고 있다는 것을 확인하였다. 또한 pCY-2는 형질전환율과 안정성에 있어 효모의 ARS를 갖는 YRp7 vector보다 우수하였다.

• 미생물학회지
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• 제49권1호
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• pp.99-104
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• 2013

박테리오파지 P2 sir 변이체는 그것의 위성파지인 박테리오 파지 P4를 위해 비효율적인 도움파지로 알려졌다. 이러한 현상을 "P2 sir-관련 도움파지 비효율성"이라고 부르고 있으며, P2와 P4의 cos 지역에서의 DNA 염기배열 순서 차이가 이러한 현상을 나타나게 한다고 생각되었다. 이를 검증하기 위해, 여러 단계의 in vitro DNA 조작을 거쳐 P2의 cos 지역을 함유하는 유도체 P4인 P4 sid71 cosP2를 조성하였다. 일단계 생장 실험을 통해 그것의 후손 방출량을 결정하였다. 그 결과는, P4 sid71 cosP2에서 P4의 cos 지역을 P2의 cos 지역으로 대체한 것이 "P2 sir-관련 도움파지 비효율성"을 극복한 것으로 나타났다. P2 야생형과 P2 sir 돌연변이형을 도움파지로 삼아 준비한 P4 sid71 cosP2 파지 농축액들을 CsCl 부양 균등밀도 편차실험으로 분석하였다. 그 결과, 양 경우 모두 3개의 P4 유전체를 가진 파지 입자가 우세한 것으로 나타났다.

• 제어로봇시스템학회:학술대회논문집
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• 제어로봇시스템학회 2004년도 ICCAS
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• pp.196-201
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• 2004

In biometric and biomedical applications, a special transporting mechanism must be designed for the ${\mu}$TAS (micro total analysis system) to move samples and reagents through the microchannels that connect the unit procedure components in the system. An important issue for this miniaturization and integration is microfluid management technique, i.e., microfluid transportation, metering, and mixing. In view of this, this study presents an optimal fuzzy sliding-mode control (OFSMC) design based on the 8051 microprocessor and implementation of a complete microfluidic manipulated system implementation of biochip system with a pneumatic pumping actuator, a feedback-signal photodiodes and flowmeter. The new microfluid management technique successfully improved the efficiency of molecular biology reaction by increasing the velocity of the target nucleic acid molecules, which increases the effective collision into the probe molecules as the target molecules flow back and forth. Therefore, this hybridization chip was able to increase hybridization signal 6-fold and reduce non-specific target-probe binding and background noises within 30 minutes, as compared to conventional hybridization methods, which may take from 4 hours to overnight. In addition, the new technique was also used in DNA extraction. When serum existed in the fluid, the extraction efficiency of immobilized beads with solution flowing back and forth was 88-fold higher than that of free-beads.

• 한국수산과학회지
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• 제47권4호
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• pp.431-434
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• 2014

Cytogenetic analysis was conducted to obtain basic information for chromosome manipulation of starry flounder Platichthys stellatus. Nuclear surface area and volume of erythrocyte were $7.60{\pm}0.93{\mu}m^2$ and $12.80{\pm}1.75{\mu}m^3$, respectively. The haploid DNA content of the species was 0.66 pg/haploid cell which correspond to 93% of olive flounder Paralichthys olivaceus. A karyotype analysis was also carried out with the species using conventional staining and Ag-NOR banding techniques. It was consisted of 48 acrocentric chromosomes and inter-sex or intra-individual polymorphism was not detected in all specimens analyzed. The NOR regions, appearing a terminal position of the short arm of the smallest acrocentric pairs.

• The Plant Pathology Journal
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• 제32권3호
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• pp.173-181
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• 2016

Gene disruption by homologous recombination is widely used to investigate and analyze the function of genes in Fusarium fujikuroi, a fungus that causes bakanae disease and root rot symptoms in rice. To generate gene deletion constructs, the use of conventional cloning methods, which rely on restriction enzymes and ligases, has had limited success due to a lack of unique restriction enzyme sites. Although strategies that avoid the use of restriction enzymes have been employed to overcome this issue, these methods require complicated PCR steps or are frequently inefficient. Here, we introduce a cloning system that utilizes multi-fragment assembly by In-Fusion to generate a gene disruption construct. This method utilizes DNA fragment fusion and requires only one PCR step and one reaction for construction. Using this strategy, a gene disruption construct for Fusarium cyclin C1 (FCC1), which is associated with fumonisin B1 bio-synthesis, was successfully created and used for fungal transformation. In vivo and in vitro experiments using confirmed fcc1 mutants suggest that fumonisin production is closely related to disease symptoms exhibited by F. fujikuroi strain B14. Taken together, this multi-fragment assembly method represents a simpler and a more convenient process for targeted gene disruption in fungi.