• 생명과학회지
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• 제34권8호
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• pp.567-575
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• 2024

New Breeding Technology (NBT)는 특정 DNA 서열을 인식하여 원하는 위치에서 DNA의 염기서열을 제거, 수정 또는 삽입할 수 있으며, 이는 인간의 유전병 치료나 가축 및 작물의 특성 향상에 사용될 수 있다. 본 연구에서는 모본인 벼(Oryza sativa L., cv Ilmi)와 CRISPR/Cas9의 SDN-1에 의해 T와 G염기가 들어간 3세대 유전자 교정 벼(OsCKq1-G₃)의 출수기, 간장, 수장, 수수, 수량, 유전자 전이 안전성, 발아율, 수발아율, 탈립성, 동토발아 활력, 독소 및 알레르겐 생성 여부를 분석했다. 목표형질인 출수기는 유전자 교정 벼가 5일 빨랐다. 간장, 수장, 수수, 수량은 Ilmi와 동일했다. 유전자 전이성을 확인하기 위한 T-DNA 밴드는 모두 확인되지 않았다. 3세대 유전자 교정 벼에서 자가 교배를 통한 세대가 지남에 따라 T-DNA가 사라진 것을 확인했다. 발아율, 수발아율, 탈립성과 동토발아 활력검증 분석을 통해 유전자 교정 벼의 잡초화 가능성은 없다는 것을 확인했다. 교정된 벼의 ORF 및 아미노산 서열에서 독소와 알레르겐은 발견되지 않았다. 본 실험을 통해 유전자 교정 벼의 안전성을 입증했고, 유전자 교정 벼의 환경 위해성 평가에 중요한 자료로 활용될 수 있을 것으로 사료된다.

• 한국동물생명공학회지
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• 제38권3호
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• pp.99-108
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• 2023

Background: Efficient gene editing technology is needed for successful knock-in. Homologous recombination (HR) is a major double-strand break repair pathway that can be utilized for accurately inserting foreign genes into the genome. HR occurs during the S/G2 phase, and the DNA mismatch repair (MMR) pathway is inextricably linked to HR to maintain HR fidelity. This study was conducted to investigate the effect of inhibiting MMR-related genes using CdCl₂, an MMR-related gene inhibitor, on HR efficiency in HC11 cells. Methods: The mRNA and protein expression levels of MMR-related genes (Msh2, Msh3, Msh6, Mlh1, Pms2), the HR-related gene Rad51, and the NHEJ-related gene DNA Ligase IV were assessed in HC11 cells treated with 10 μM of CdCl₂ for 48 hours. In addition, HC11 cells were transfected with a CRISPR/sgRNA expression vector and a knock-in vector targeting Exon3 of the mouse-beta casein locus, and treated with 10 μM cadmium for 48 hours. The knock-in efficiency was monitored through PCR. Results: The treatment of HC11 cells with a high-dose of CdCl₂ decreased the mRNA expression of the HR-related gene Rad51 in HC11 cells. In addition, the inhibition of MMR-related genes through CdCl₂ treatment did not lead to an increase in knock-in efficiency. Conclusions: The inhibition of MMR-related gene expression through high-dose CdCl₂ treatment reduces the expression of the HR-related gene Rad51, which is active during recombination. Therefore, it was determined that CdCl₂ is an inappropriate compound for improving HR efficiency.

• Asian-Australasian Journal of Animal Sciences
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• 제29권10호
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• pp.1500-1507
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• 2016

Myostatin (MSTN) can negatively regulate the growth and development of skeletal muscle, and natural mutations can cause "double-muscling" trait in animals. In order to block the inhibiting effect of MSTN on muscle growth, we transferred zinc-finger nucleases (ZFN) which targeted sheep MSTN gene into cultured fibroblasts. Gene targeted colonies were isolated from transfected fibroblasts by serial dilution culture and screened by sequencing. Two colonies were identified with mono-allele mutation and one colony with bi-allelic deletion. Further, we introduced the MSTN-ZFN mRNA into sheep embryos by microinjection. Thirteen of thirty-seven parthenogenetic embryos were targeted by ZFN, with the efficiency of 35%. Our work established the technical foundation for generation of MSTN gene editing sheep by somatic cloning and microinjection ZFN into embryos.

• 식물병연구
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• 제25권2호
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• pp.49-61
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• 2019

식물 바이러스는 작물 생산량 손실을 일으키는 주요 병원체 중 하나로, 돌연변이 발생이 빈번하고 치료 약제가 개발되어 있지 않아 방제가 매우 어렵다. 이러한 바이러스병을 방제하기 위한 가장 효과적인 방법은 저항성 품종을 재배하는 것이며, 바이러스 저항성 품종을 개발하기 위해서는 바이러스와 기주 식물 간의 다양한 유전자적 상호작용에 대한 정확한 이해가 필요하다. 열성 저항성은 병원체가 살아가는데 필요한 식물 유전자가 결핍되었을 때 획득되는데, 저항성 유전자(R gene)에 의해 유도되는 우성 저항성에 비해 넓은 범위의 저항성을 발현하고 돌연변이 출현에 쉽게 저항성이 깨지지 않는 특성을 보인다. 현재까지 알려진 바이러스병에 대한 열성 저항성 유전자는 대부분 순행유전학(forward genetics)를 통해 밝혀졌으나, 최근 CRISPR/Cas9 등을 이용한 유전자 교정 기술의 급속한 발전에 힘입어 역유전학(reverse genetics)을 통한 열성 저항성 작물개발의 가능성이 열리고 있다. 이러한 역유전학적 접근을 통한 열성 저항성 작물 개발은 먼저 바이러스 단백질과 상호작용하는 기주 인자를 밝히고 이들간의 상호작용을 억제하도록 하는 기주 인자에 대한 유전자 교정을 통해 이루어 질 수 있다. 본 논문에서는 열성 저항성에 대한 소개와 새로운 열성 저항성 후보 유전 소재 발굴을 위한 기주 인자 연구의 중요성 및 방법을 소개하고, 열성 저항성 작물 개발에 적용할 수 있는 유전자 교정기술의 최신 동향에 관해 정리하였다.

• Journal of Plant Biotechnology
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• 제48권1호
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• pp.34-43
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• 2021

CRISPR/Cas9에 의한 유전자 교정 기술은 유용 형질을 갖는 작물 및 임목의 육성에 있어 널리 사용되고 있는 핵심 기술이다. 유전자 교정 임목 육성에는 아그로박테리움에 의한 형질전환 방법이 높은 효율로 시행된 연구가 많았고 따라서 형질전환에 사용된 플라스미드 서열이 식물 유전체 안에 존재한다는 문제가 남아 있었다. 본 연구에서는 CRISPR/Cas9을 사용하여 유전자 교정 임목을 육성하는 데 기존에 알려진 벡터 도입 기술이 아닌, 단일 가닥 가이드 RNA (sgRNA)와 Cas9 단백질을 혼합하여 만든 리보핵산단백질을 현사시나무 원형질체에 도입하는 방법을 기술하였다. 염 스트레스 내성 관련 인자 PagSAP1 유전자를 표적으로 하는 3종류의 sgRNA를 디자인하고, 각 sgRNA와 Cas9 단백질을 혼합하여 만든 리보핵산단백질을 원형질체에 도입하였다. 표적화 딥시퀀싱을 통해 리보핵산단백질 형성 시 sgRNA와 Cas9 단백질을 혼합하고 일정 시간 배양하여 안정화되는 시간이 필요한 것을 확인하였다. 또한 sgRNA3의 리보핵산단백질이 sgRNA1, sgRNA2의 리보핵산단백질보다 높은 교정 효율을 보이는 것을 확인하였다. 본 실험을 통해 리보핵산단백질을 이용한 유전자 교정 기술이 임목에도 적용될 수 있음이 확인되었고, 이는 외래 유전자 없이 유전자 교정 임목을 육성하는 데 활용할 수 있을 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제33권6호
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• pp.1023-1033
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• 2020

Objective: The efficiency of the knock-in process is very important to successful gene editing in domestic animals. Recently, it was reported that transient loosening of the nucleosomal folding of transcriptionally inactive chromatin might have the potential to enhance homologous recombination efficiency. The objective of this study was to determine whether histone deacetylases (HDAC) inhibitor and RAD51 recombinase (RAD51) expression were associated with increased knock-in efficiency on the β-casein (bCSN2) gene locus in mammary alveolar-large T antigen (MAC-T) cells using the transcription activator-like effector nucleases (TALEN) system. Methods: MAC-T cells were treated with HDAC inhibitors, valproic acid, trichostatin A, or sodium butyrate for 24 h, then transfected with a knock-in vector, RAD51 expression vector and TALEN to target the bCSN2 gene. After 3 days of transfection, the knock-in efficiency was confirmed by polymerase chain reaction and DNA sequencing of the target site. Results: The level of HDAC 2 protein in MAC-T cells was decreased by treatment with HDAC inhibitors. The knock-in efficiency in MAC-T cells treated with HDAC inhibitors was higher than in cells not treated with inhibitors. However, the length of the homologous arm of the knock-in vector made no difference in the knock-in efficiency. Furthermore, DNA sequencing confirmed that the precision of the knock-in was more efficient in MAC-T cells treated with sodium butyrate. Conclusion: These results indicate that chromatin modification by HDAC inhibition and RAD51 expression enhanced the homologous recombination efficiency on the bCSN2 gene locus in MAC-T cells.

• 대한예방한의학회지
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• 제14권2호
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• pp.1-12
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• 2010

The individual differences in disease development and susceptibility have been researched primarily on the subject of genes, environment or the interaction between genes and the environment respectively. However, there have been limitations in explaining complex diseases, and the differences in health and diseases in monozygotic and dizygotic twins. Fortunately, thanks to active research on the relationship between genes and the environment, and epigenetics, there has been much progress in the understanding of body's reactions and changes. Epigenetics is referred to as a study of gene expression through the interactions of DNA methylation, chromatin's histone and the change of structure in tail, RNA editing without any change in DNA sequence. In this paper, we introduce the basic concepts and mechanisms of epigenetics. The result of the epigenetics is heritable ; can regulate gene expressions ; is reversible ; and has many variable forms depending on cell types. The influences of epigenetics occur throughout life, but it is mainly determined in utero during early pregnancies. Diseases occur or the risk rises if these influences continue after birth until adult life when problems occur in excess/lack of nutrition, environmental plasticity, or already inputted data. Therefore, there is a need for change and innovation, especially in interest and investment in health education for young women near pregnancies and correct treatment of epigenetic-related diseases.

• Journal of Genetic Medicine
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• 제20권1호
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• pp.6-14
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• 2023

Fabry disease (FD), a rare X-linked lysosomal storage disorder, is caused by mutations in the α-galactosidase A gene gene encoding α-galactosidase A (α-Gal A). The functional deficiency of α-Gal A results in progressive accumulation of neutral glycosphingolipids, causing multi-organ damages including cardiac, renal, cerebrovascular systems. The current treatment is comprised of enzyme replacement therapy (ERT), oral pharmacological chaperone therapy and adjunctive supportive therapy. ERT has been introduced 20 years ago, changing the outcome of FD patients with proven effectiveness. However, FD patients have many unmet needs. ERT needs a life-long intravenous therapy, inefficient bio-distribution, and generation of anti-drug antibodies. Migalastat, a pharmacological chaperone, augmenting α-Gal A enzyme activity only in patients with mutations amenable to the therapy, is now available for clinical practice. Furthermore, these therapies should be initiated before the organ damage becomes irreversible. Development of novel drugs aim at improving the clinical effectiveness and convenience of therapy. Clinical trial of next generation ERT is underway. Polyethylene glycolylated enzyme has a longer half-life and potentially reduced antigenicity, compared with standard preparations with longer dosing interval. Moss-derived enzyme has a higher affinity for mannose receptors, and seems to have more efficient access to podocytes of kidney which is relatively resistant to reach by conventional ERT. Substrate reduction therapy is currently under clinical trial. Gene therapy has now been started in several clinical trials using in vivo and ex vivo technologies. Early results are emerging. Other strategic approaches at preclinical research level are stem cell-based therapy with genome editing and systemic mRNA therapy.

• 한국초지조사료학회지
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• 제43권3호
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• pp.168-176
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• 2023

Silicon (Si) has the potential to improve plant growth and stress tolerance. The study aimed to explore Si-involving plant responses and molecular characterization of different Si-responsive genes in alfalfa. In this study, the exogenous supplementation of Si enhanced plant growth, and biomass yield. Si-acquisition in alfalfa root and shoot was higher in Si-supplemented compared to silicon deficient (-Si) plants, implying Si-acquisition has beneficial on alfalfa plants. As a consequence, the quantum efficiency of photosystem II (Fv/Fm) was significantly increased in silicon-sufficient (+Si) plants. The quantitative gene expression analysis exhibited a significant upregulation of the Lsi1, Lsi2, Lsi3, NIP5;1, and NIP6;1 genes in alfalfa roots, while BOR1, BOR4, NIP2, and NIP3 showed no significant variation in their expression. The MEME results further noticed the association of four motifs related to the major intrinsic protein (MIP). The interaction analysis revealed that NIP5;1 and Lsi1 showed a shared gene network with NIP2, BOR1, and BOR4, and Lsi2, Lsi3 and NIP3-1, respectively. These results suggest that members of the major intrinsic proteins (MIPs) family especially Lsi1, Lsi2, Lsi3, NIP5;1, and NIP6;1 genes helped to pass water and other neutral solutes through the cell membrane and those played significant roles in Si uptake and transport in plants. Together, these insights might be useful for alfalfa breeding and genome editing approaches for alfalfa improvement.

• 한국동물생명공학회지
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• 제34권2호
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• pp.65-69
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• 2019

Since the development of the first genetically-modified mouse, transgenic animals have been utilized for a wide range of industrial applications as well as basic research. To date, these transgenic animals have been used in functional genomics studies, disease models, and therapeutic protein production. Recent advances in genome modification techniques such zinc finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRIPSR)-Cas9, have led to rapid advancement in the generation of genome-tailored livestock, as well as experimental animals; however, the development of genome-edited poultry has shown considerably slower progress compared to that seen in mammals. Here, we will focus primarily on the technical strategies for production of transgenic and gene-edited chickens, and their potential for future applications.