• Development and Reproduction
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• v.27 no.1
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• pp.1-7
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• 2023

Small-cell lung cancer (SCLC) continues to be the deadliest of all lung cancer types. Its high mortality is largely attributed to the unchangeable development of resistance to standard chemo/radiotherapies, which have remained invariable for the past 30 years, underlining the need for new therapeutic approaches. Recent studies of SCLC genome revealed a large number of somatic alterations and identified remarkable heterogeneity of the frequent mutations except for the loss of both RB and P53 tumor suppressor genes (TSGs). Identifying the somatic alterations scattered throughout the SCLC genome will help to define the underlying mechanism of the disease and pave the way for the discovery of therapeutic vulnerabilities associated with genomic alterations. The new technique made it possible to determine the underlying mechanism for the discovery of therapeutic targets. To these ends, the techniques have been focused on understanding the molecular determinants of SCLC.

• Journal of Microbiology and Biotechnology
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• v.26 no.2
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• pp.213-225
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• 2016

To reduce attrition in drug development, it is crucial to consider the development and implementation of translational phenotypic assays as well as decipher diverse molecular mechanisms of action for new molecular entities. High-throughput fluorescence and confocal microscopes with advanced analysis software have simplified the simultaneous identification and quantification of various cellular processes through what is now referred to as high-content screening (HCS). HCS permits automated identification of modifiers of accessible and biologically relevant targets and can thus be used to detect gene interactions or identify toxic pathways of drug candidates to improve drug discovery and development processes. In this review, we summarize several HCS-compatible, biochemical, and molecular biology-driven assays, including immunohistochemistry, RNAi, reporter gene assay, CRISPR-Cas9 system, and protein-protein interactions to assess a variety of cellular processes, including proliferation, morphological changes, protein expression, localization, post-translational modifications, and protein-protein interactions. These cell-based assay methods can be applied to not only 2D cell culture but also 3D cell culture systems in a high-throughput manner.

• Korean Chemical Engineering Research
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• v.56 no.1
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• pp.79-84
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• 2018

In the present study, we performed electroporation to deliver protein into microalgae using previously developed digital electroporation system. Green fluorescence protein was successfully delivered into a live microalgae cell nucleus without cell wall removal. By investigating the effects of applied voltage on the protein delivery efficiency, optimal electroporation electric field condition was found (960 V/cm). We also investigated the delivery of Yo-Pro-1 into cell to examine the size effects of delivered materials and found that there is little size effects on the optimal condition. Finally, the implications of the present results and future work are discussed.

• Journal of Plant Biotechnology
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• v.42 no.3
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• pp.154-160
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• 2015

The plant breeding technology was developed with genetic engineering. Many researchers and breeders have turned from traditional breeding to molecular breeding. Genetically modified organisms (GMO) were developed via molecular breeding technology. Currently, molecular breeding technologies facilitate efficient plant breeding without introducing foreign genes, in virtue by of gene editing technology. Gene targeting (GT) via homologous recombination (HR) is one of the best gene editing methods available to modify specific DNA sequences in genomes. GT utilizes DNA repair pathways. Thus, DNA repair systems are controlled to enhance HR processing. Engineered sequence specific endonucleases were applied to improve GT efficiency. Engineered sequence specific endonucleases like the zinc finger nuclease (ZFN), TAL effector nuclease (TALEN), and CRISPR-Cas9 create DNA double-strand breaks (DSB) that can stimulate HR at a target site. RecQl4, Exo1 and Rad51 are effectors that enhance DSB repair via the HR pathway. This review focuses on recent developments in engineered sequence specific endonucleases and ways to improve the efficiency of GT via HR effectors in plants.

• Journal of Microbiology and Biotechnology
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• v.29 no.5
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• pp.667-686
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• 2019

Streptomyces are attractive microbial cell factories that have industrial capability to produce a wide array of bioactive secondary metabolites. However, the genetic potential of the Streptomyces species has not been fully utilized because most of their secondary metabolite biosynthetic gene clusters (SM-BGCs) are silent under laboratory culture conditions. In an effort to activate SM-BGCs encoded in Streptomyces genomes, synthetic biology has emerged as a robust strategy to understand, design, and engineer the biosynthetic capability of Streptomyces secondary metabolites. In this regard, diverse synthetic biology tools have been developed for Streptomyces species with technical advances in DNA synthesis, sequencing, and editing. Here, we review recent progress in the development of synthetic biology tools for the production of novel secondary metabolites in Streptomyces, including genomic elements and genome engineering tools for Streptomyces, the heterologous gene expression strategy of designed biosynthetic gene clusters in the Streptomyces chassis strain, and future directions to expand diversity of novel secondary metabolites.

• Journal of Marine Bioscience and Biotechnology
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• v.11 no.2
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• pp.52-61
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• 2019

Over the past few decades, microalgae-based biotechnology conjugated with innovative CRISPR/Cas9-mediated genetic engineering has been attracted much attention for the cost-effective and eco-friendly value-added compounds production. However, the discharge of reproducible living modified organism (LMO) into environmental condition potentially causes serious problem in aquatic environment, and thus it is essential to assess potential environmental risk for human health. Accordingly, in this study, we monitored discharged genetically modified microalgae (GMM) near the research complex which is located in Daejeon, South Korea. After testing samples obtained from 6 points of near streams, several green-colored microalgal colonies were detected under hygromicin-containing agar plate. By identification of selection marker genes, the GMM was not detected from all the samples. For the lab-scale environmental risk assessment of GMM, acute toxicity test using rotifer Brachionus calcyflorus was performed by feeding GMM. After feeding, there was no significant difference in mortality between WT and transformant Chlamydomonas reinhardtii. According to further analysis of horizontal transfer of green fluorescence protein (GFP)-coding gene after 24 h of incubation in synthetic freshwater, we concluded that the GFP-expressed gene not transferred into predator. However, further risk assessments and construction of standard methods including prolonged toxicity test are required for the accurate ecological risk assessment.

• Journal of Veterinary Science
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• v.21 no.2
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• pp.26.1-26.14
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• 2020

Pancreatic ductal adenocarcinoma is a lethal cancer type that is associated with multiple gene mutations in somatic cells. Genetically engineered mouse is hardly applicable for developing a pancreatic cancer model, and the xenograft model poses a limitation in the reflection of early stage pancreatic cancer. Thus, in vivo somatic cell gene engineering with clustered regularly interspaced short palindromic repeats is drawing increasing attention for generating an animal model of pancreatic cancer. In this study, we selected Kras, Trp53, Ink4a, Smad4, and Brca2 as target genes, and applied Campylobacter jejuni Cas9 (CjCas9) and Streptococcus pyogens Cas9 (SpCas9) for developing pancreatic cancer using adeno associated virus (AAV) transduction. After confirming multifocal and diffuse transduction of AAV2, we generated SpCas9 overexpression mice, which exhibited high double-strand DNA breakage (DSB) in target genes and pancreatic intraepithelial neoplasia (PanIN) lesions with two AAV transductions; however, wild-type (WT) mice with three AAV transductions did not develop PanIN. Furthermore, small-sized Cjcas9 was applied to WT mice with two AAV system, which, in addition, developed high extensive DSB and PanIN lesions. Histological changes and expression of cancer markers such as Ki67, cytokeratin, Mucin5a, alpha smooth muscle actin in duct and islet cells were observed. In addition, the study revealed several findings such as 1) multiple DSB potential of AAV-CjCas9, 2) peri-ductal lymphocyte infiltration, 3) multi-focal cancer marker expression, and 4) requirement of > 12 months for initiation of PanIN in AAV mediated targeting. In this study, we present a useful tool for in vivo cancer modeling that would be applicable for other disease models as well.

• Proceedings of the Korean Society of Crop Science Conference
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• 2022.10a
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• pp.207-207
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• 2022

Eating and cooking quality (ECQ) is one of the most complex quantitative traits in rice. The understanding of genetic regulation of transcript expression levels attributing to phenotypic variation in ECQ traits is limited. We integrated whole-genome resequencing, transcriptome, and phenotypic variation data from 84 Japonica accessions to build a transcriptome-wide association study (TWAS) based regulatory network. All ECQ traits showed a large phenotypic variation and significant phenotypic correlations among the traits. TWAS analysis identified a total of 285 transcripts significantly associated with six ECQ traits. Genome-wide mapping of ECQ-associated transcripts revealed 66,905 quantitative expression traits (eQTLs), including 21,747 local eQTLs, and 45,158 trans-eQTLs, regulating the expression of 43 genes. The starch synthesis-related genes (SSRGs), starch synthase IV-1 (SSIV-1), starch branching enzyme 1 (SBE1), granule-bound starch synthase 2 (GBSS2), and ADP-glucose pyrophosphorylase small subunit 2a (OsAGPS2a) were found to have eQTLs regulating the expression of ECQ associated transcripts. Further, in co-expression analysis, 130 genes produced at least one network with 22 master regulators. In addition, we developed CRISPR/Cas9-edited glbl mutant lines that confirmed the role of alpha-globulin (glbl) in starch synthesis to validate the co-expression analysis. This study provided novel insights into the genetic regulation of ECQ traits, and transcripts associated with these traits were discovered that could be used in further rice breeding.