• Title/Summary/Keyword: genome engineering

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Optimization of a Multiplex DNA Amplification of Three Short Tandem Repeat Loci for Genetic Identification

  • Ryu, Jae-Song;Noh, Jae-Sang;Koo, Yoon-Mo;Lee, Choul-Gyun;So, Jae-Seong
    • Journal of Microbiology and Biotechnology
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    • v.10 no.6
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    • pp.873-876
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    • 2000
  • Short tendem repeat (STR) loci have been used in the field of forensic science. There are literally hundreds of STR systems which have been mapped throughout the human genome. These STR loci are found in almost every chromosome in the genome. They may be amplified using a variety of PCR primers. In this study, a DNA genotyping system based on the multiplex amplification of highly polymorphic STR loci was developed. Three STR loci with nonoverlapping allele size ranges have been utilized in the multiplex amplification including the Neurotensin receptor gene, D21S11, and Human tyrosine hydroxylase gene. The optimal condition for triplex PCr was obtained in a solution with a total volume of $25{\mu}l$ containing 2.0 U of Taq polymerase, 3 mM of $MgCl_2$, $300{\mu}M$ of dNTP, 10 pmole of each primer set, an annealing temperature of $62^{\circ}C$, and 35 cycles. The optimized condition was successfully employed in a family paternity test.

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Production of Mutated Porcine Embryos Using Zinc Finger Nucleases and a Reporter-based Cell Enrichment System

  • Koo, Ok Jae;Park, Sol Ji;Lee, Choongil;Kang, Jung Taek;Kim, Sujin;Moon, Joon Ho;Choi, Ji Yei;Kim, Hyojin;Jang, Goo;Kim, Jin-Soo;Kim, Seokjoong;Lee, Byeong-Chun
    • Asian-Australasian Journal of Animal Sciences
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    • v.27 no.3
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    • pp.324-329
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    • 2014
  • To facilitate the construction of genetically-modified pigs, we produced cloned embryos derived from porcine fibroblasts transfected with a pair of engineered zinc finger nuclease (ZFN) plasmids to create targeted mutations and enriched using a reporter plasmid system. The reporter expresses RFP and eGFP simultaneously when ZFN-mediated site-specific mutations occur. Thus, double positive cells ($RFP^+/eGFP^+$) were selected and used for somatic cell nuclear transfer. Two types of reporter based enrichment systems were used in this study; the cloned embryos derived from cells enriched using a magnetic sorting-based system showed better developmental competence than did those derived from cells enriched by flow cytometry. Mutated sequences, such as insertions, deletions, or substitutions, together with the wild-type sequence, were found in the cloned porcine blastocysts. Therefore, genetic mutations can be achieved in cloned porcine embryos reconstructed with ZFN-treated cells that were enriched by a reporter-based system.

Understanding Epistatic Interactions between Genes Targeted by Non-coding Regulatory Elements in Complex Diseases

  • Sung, Min Kyung;Bang, Hyoeun;Choi, Jung Kyoon
    • Genomics & Informatics
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    • v.12 no.4
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    • pp.181-186
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    • 2014
  • Genome-wide association studies have proven the highly polygenic architecture of complex diseases or traits; therefore, single-locus-based methods are usually unable to detect all involved loci, especially when individual loci exert small effects. Moreover, the majority of associated single-nucleotide polymorphisms resides in non-coding regions, making it difficult to understand their phenotypic contribution. In this work, we studied epistatic interactions associated with three common diseases using Korea Association Resource (KARE) data: type 2 diabetes mellitus (DM), hypertension (HT), and coronary artery disease (CAD). We showed that epistatic single-nucleotide polymorphisms (SNPs) were enriched in enhancers, as well as in DNase I footprints (the Encyclopedia of DNA Elements [ENCODE] Project Consortium 2012), which suggested that the disruption of the regulatory regions where transcription factors bind may be involved in the disease mechanism. Accordingly, to identify the genes affected by the SNPs, we employed whole-genome multiple-cell-type enhancer data which discovered using DNase I profiles and Cap Analysis Gene Expression (CAGE). Assigned genes were significantly enriched in known disease associated gene sets, which were explored based on the literature, suggesting that this approach is useful for detecting relevant affected genes. In our knowledge-based epistatic network, the three diseases share many associated genes and are also closely related with each other through many epistatic interactions. These findings elucidate the genetic basis of the close relationship between DM, HT, and CAD.

Development and Performance Evaluation of Parallel Sequence Analysis System on PC-Cluster (PC-Cluster 기반 병렬형 유전자 서열 검색 시스템의 개발 및 성능 평가)

  • Shin Yong-Won;Park Jeong-Seon
    • Journal of Biomedical Engineering Research
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    • v.25 no.6
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    • pp.617-621
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    • 2004
  • In recent, researchers in the field of Bioinformatics need to analyze thousands of genome sequences efficiently according to introduce of new analysis methods and technologies such as genome expression microchip. This rapid growth in the field of bio-engineering needs computing resources to analyze rapidly for genome sequences, but it does not introduce the computing resources due to an enormous investment expense. The core factor of this study is integrated environment based PC-Cluster system & high speed access rate up to 155Mbps, continuous collection system for bio-information at home and abroad. The results of the study are establishment & stabilization of information and communication infrastructure, establishment & stabilization of high performance computer network up to 155Mbps, development of PC-Cluster system with 32 nodes, a parallel BLAST on Cluster system, which can provides scalable speedup in terms of response time, and development of collection & search system for bio-information.

Draft Genome Analysis of Antimicrobial Streptomyces Isolated from Himalayan Lichen

  • Kim, Byeollee;Han, So-Ra;Lamichhane, Janardan;Park, Hyun;Oh, Tae-Jin
    • Journal of Microbiology and Biotechnology
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    • v.29 no.7
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    • pp.1144-1154
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    • 2019
  • There have been several studies regarding lichen-associated bacteria obtained from diverse environments. Our screening process identified 49 bacterial species in two lichens from the Himalayas: 17 species of Actinobacteria, 19 species of Firmicutes, and 13 species of Proteobacteria. We discovered five types of strong antimicrobial agent-producing bacteria. Although some strains exhibited weak antimicrobial activity, NP088, NP131, NP132, NP134, and NP160 exhibited strong antimicrobial activity against all multidrug-resistant strains. Polyketide synthase (PKS) fingerprinting revealed results for 69 of 148 strains; these had similar genes, such as fatty acid-related PKS, adenylation domain genes, PfaA, and PksD. Although the association between antimicrobial activity and the PKS fingerprinting results is poorly resolved, NP160 had six types of PKS fingerprinting genes, as well as strong antimicrobial activity. Therefore, we sequenced the draft genome of strain NP160, and predicted its secondary metabolism using antiSMASH version 4.2. NP160 had 46 clusters and was predicted to produce similar secondary metabolites with similarities of 5-100%. Although NP160 had 100% similarity with the alkylresorcinol biosynthetic gene cluster, our results showed low similarity with existing members of this biosynthetic gene cluster, and most have not yet been revealed. In conclusion, we expect that lichen-associated bacteria from the Himalayas can produce new secondary metabolites, and we found several secondary metabolite-related biosynthetic gene clusters to support this hypothesis.

Atorvastatin inhibits the proliferation of MKN45-derived gastric cancer stem cells in a mevalonate pathway-independent manner

  • Choi, Ye Seul;Cho, Hee Jeong;Jung, Hye Jin
    • The Korean Journal of Physiology and Pharmacology
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    • v.26 no.5
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    • pp.367-375
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    • 2022
  • Gastric cancer stem cells (GCSCs) are a major cause of radioresistance and chemoresistance in gastric cancer (GC). Therefore, targeting GCSCs is regarded as a powerful strategy for the effective treatment of GC. Atorvastatin is a widely prescribed cholesterol-lowering drug that inhibits 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a rate-limiting enzyme in the mevalonate pathway. The anticancer activity of atorvastatin, a repurposed drug, is being investigated; however, its therapeutic effect and molecular mechanism of action against GCSCs remain unknown. In this study, we evaluated the anticancer effects of atorvastatin on MKN45-derived GCSCs. Atorvastatin significantly inhibited the proliferative and tumorsphere-forming abilities of MKN45 GCSCs in a mevalonate pathway-independent manner. Atorvastatin induced cell cycle arrest at the G0/G1 phase and promoted apoptosis by activating the caspase cascade. Furthermore, atorvastatin exerted an antiproliferative effect against MKN45 GCSCs by inhibiting the expression of cancer stemness markers, such as CD133, CD44, integrin α6, aldehyde dehydrogenase 1A1, Oct4, Sox2, and Nanog, through the downregulation of β-catenin, signal transducer and activator of transcription 3, and protein kinase B activities. Additionally, the combined treatment of atorvastatin and sorafenib, a multi-kinase targeted anticancer drug, synergistically suppressed not only the proliferation and tumorsphere formation of MKN45 GCSCs but also the in vivo tumor growth in a chick chorioallantoic membrane model implanted with MKN45 GCSCs. These findings suggest that atorvastatin can therapeutically eliminate GCSCs.

Vitamin B6 Deficiency, Genome Instability and Cancer

  • Wu, Xia-Yu;Lu, Lin
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.11
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    • pp.5333-5338
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    • 2012
  • Vitamin B6 functions as a coenzyme in >140 enzymatic reactions involved in the metabolism of amino acids, carbohydrates, neurotransmitters, and lipids. It comprises a group of three related 3-hydroxy-2-methyl-pyrimidine derivatives: pyridoxine (PN), pyridoxal (PL), pyridoxamine (PM) and their phosphorylated derivatives [pyridoxal 5'-phosphate (PLP) and pyridoxamine 5'-phosphate (PMP)], In the folate metabolism pathway, PLP is a cofactor for the mitochondrial and cytoplasmic isozymes of serine hydroxymethyltransferase (SHMT2 and SHMT1), the P-protein of the glycine cleavage system, cystathionine ${\beta}$-synthase (CBS) and ${\gamma}$-cystathionase, and betaine hydroxymethyltransferase (BHMT), all of which contribute to homocysteine metabolism either through folate-mediated one-carbon metabolism or the transsulfuration pathway. Folate cofactors carry and chemically activate single carbons for the synthesis of purines, thymidylate and methionine. So the evidence indicates that vitamin B6 plays an important role in maintenance of the genome, epigenetic stability and homocysteine metabolism. This article focuses on studies of strand breaks, micronuclei, or chromosomal aberrations regarding protective effects of vitamin B6, and probes whether it is folate-mediated one-carbon metabolism or the transsulfuration pathway for vitamin B6 which plays critical roles in prevention of cancer and cardiovascular disease.

Mutational Data Loading Routines for Human Genome Databases: the BRCA1 Case

  • Van Der Kroon, Matthijs;Ramirez, Ignacio Lereu;Levin, Ana M.;Pastor, Oscar;Brinkkemper, Sjaak
    • Journal of Computing Science and Engineering
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    • v.4 no.4
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    • pp.291-312
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    • 2010
  • The last decades a large amount of research has been done in the genomics domain which has and is generating terabytes, if not exabytes, of information stored globally in a very fragmented way. Different databases use different ways of storing the same data, resulting in undesired redundancy and restrained information transfer. Adding to this, keeping the existing databases consistent and data integrity maintained is mainly left to human intervention which in turn is very costly, both in time and money as well as error prone. Identifying a fixed conceptual dictionary in the form of a conceptual model thus seems crucial. This paper presents an effort to integrate the mutational data from the established genomic data source HGMD into a conceptual model driven database HGDB, thereby providing useful lessons to improve the already existing conceptual model of the human genome.

Genomic Insights into Nematicidal Activity of a Bacterial Endophyte, Raoultella ornithinolytica MG against Pine Wilt Nematode

  • Shanmugam, Gnanendra;Dubey, Akanksha;Ponpandian, Lakshmi Narayanan;Rim, Soon Ok;Seo, Sang-Tae;Bae, Hanhong;Jeon, Junhyun
    • The Plant Pathology Journal
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    • v.34 no.3
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    • pp.250-255
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    • 2018
  • Pine wilt disease, caused by the nematode Bursaphelenchus xylophilus, is one of the most devastating conifer diseases decimating several species of pine trees on a global scale. Here, we report the draft genome of Raoultella ornithinolytica MG, which is isolated from mountain-cultivated ginseng plant as an bacterial endophyte and shows nematicidal activity against B. xylophilus. Our analysis of R. ornithinolytica MG genome showed that it possesses many genes encoding potential nematicidal factors in addition to some secondary metabolite biosynthetic gene clusters that may contribute to the observed nematicidal activity of the strain. Furthermore, the genome was lacking key components of avermectin gene cluster, suggesting that nematicidal activity of the bacterium is not likely due to the famous anthelmintic agent of wide-spread use, avermectin. This genomic information of R. ornithinolytica will provide basis for identification and engineering of genes and their products toward control of pine wilt disease.

Generation of mmp15b Zebrafish Mutant to Investigate Liver Diseases

  • Kim, Oc-Hee;An, Hye Suck;Choi, Tae-Young
    • Development and Reproduction
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    • v.23 no.4
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    • pp.385-390
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    • 2019
  • Upon gene inactivation in animal models, the zebrafish (Danio rerio) has become a useful model organism for many reasons, including the fact that it is amenable to various forms of genetic manipulation. Genome editing is a type of genetic engineering in which DNA is inserted, deleted, modified, or replaced in the genome of a living organism. Mainly, CRISPR (clustered regularly interspaced short palindromic repeats) Cas9 (CRISPR-associated protein 9) is a technology that enables geneticists to edit parts of the genome. In this study, we utilized this technology to generate an mmp15b mutant by using zebrafish as an animal model. MMP15 is the membrane-type MMP (MT-MMP) which is a recently identified matrix metalloproteinase (MMP) capable of degrading all kinds of extracellular matrix proteins as well as numerous bioactive molecules. Although the newly-established mmp15b zebrafish mutant didn't exhibit morphological phenotypes in the developing embryos, it might be further utilized to understand the role of MMP15 in liver-related diseases, such as liver fibrosis, and associated pathogeneses in humans.