22,667 research outputs found

    Capturing the β€˜ome’ : the expanding molecular toolbox for RNA and DNA library construction

    Get PDF
    All sequencing experiments and most functional genomics screens rely on the generation of libraries to comprehensively capture pools of targeted sequences. In the past decade especially, driven by the progress in the field of massively parallel sequencing, numerous studies have comprehensively assessed the impact of particular manipulations on library complexity and quality, and characterized the activities and specificities of several key enzymes used in library construction. Fortunately, careful protocol design and reagent choice can substantially mitigate many of these biases, and enable reliable representation of sequences in libraries. This review aims to guide the reader through the vast expanse of literature on the subject to promote informed library generation, independent of the application

    High-throughput, quantitative analyses of genetic interactions in E. coli.

    Get PDF
    Large-scale genetic interaction studies provide the basis for defining gene function and pathway architecture. Recent advances in the ability to generate double mutants en masse in Saccharomyces cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here we describe a method based on F factor-driven conjugation, which allows for high-throughput generation of double mutants in Escherichia coli. This method, termed genetic interaction analysis technology for E. coli (GIANT-coli), permits us to systematically generate and array double-mutant cells on solid media in high-density arrays. We show that colony size provides a robust and quantitative output of cellular fitness and that GIANT-coli can recapitulate known synthetic interactions and identify previously unidentified negative (synthetic sickness or lethality) and positive (suppressive or epistatic) relationships. Finally, we describe a complementary strategy for genome-wide suppressor-mutant identification. Together, these methods permit rapid, large-scale genetic interaction studies in E. coli

    ν™˜μžλ§žμΆ€ν˜• 치료λ₯Ό μœ„ν•œ 체외 ν•­μ•”μ œ μŠ€ν¬λ¦¬λ‹μš© λ°”μ΄μ˜€μΉ©

    Get PDF
    ν•™μœ„λ…Όλ¬Έ (박사) -- μ„œμšΈλŒ€ν•™κ΅ λŒ€ν•™μ› : κ³΅κ³ΌλŒ€ν•™ 전기·정보곡학뢀, 2020. 8. κΆŒμ„±ν›ˆ.μ •λ°€μ˜ν•™(Precision Medicine) ν˜Ήμ€ κ°œμΈλ§žμΆ€μ˜ν•™(Personalized Medicine)은 개개인의 μ΅œμ ν™”λœ μΉ˜λ£Œλ°©λ²•μ„ κ²°μ •ν•˜λŠ” 것을 λͺ©ν‘œλ‘œ ν•˜λŠ” μ˜ν•™μ˜ νŒ¨λŸ¬λ‹€μž„μ΄λ‹€. 특히, μž„μƒμ’…μ–‘ν•™μ—μ„œλŠ” μ°¨μ„ΈλŒ€μ—ΌκΈ°μ„œμ—΄λΆ„μ„(NGS), μ „μ‚¬μ²΄μ„œμ—΄λΆ„μ„, 그리고 μ§ˆλŸ‰λΆ„μ„λ²•λ“€μ„ ν†΅ν•œ ν™˜μžμ˜ λΆ„μž ν”„λ‘œνŒŒμΌ(molecular profile) 방법이 λ°œμ „ν•΄μ˜€κ³  있으며, 이λ₯Ό λ°”νƒ•μœΌλ‘œ ν™˜μžλ₯Ό μ„ΈλΆ„ν™”ν•˜μ—¬ λ§žμΆ€ν˜• 치료λ₯Ό κ΅¬ν˜„ν•˜λ €κ³  λ…Έλ ₯ν•΄μ˜€κ³  μžˆλ‹€. ν•˜μ§€λ§Œ, μ—¬μ „νžˆ ν˜„ μˆ˜μ€€μ—μ„œ μ΄ν•΄λ˜μ§€ λͺ»ν•˜λŠ” μˆ˜μ€€μ˜ μ’…μ–‘ μ΄μ§ˆμ„±(tumor heterogeneity)κ³Ό 였랜 μ²˜λ°©κΈ°λ‘μ„ 가진 ν™˜μžκ΅°λ“€μ˜ ν•­μ•”μ œ νšλ“λ‚΄μ„±(acquired resistance) λ“±μ˜ μ›μΈμœΌλ‘œ λ§žμΆ€ν˜• ν™˜μž μ²˜λ°©μ€ 쉽지 μ•Šμ€ κ²½μš°κ°€ λ§Žλ‹€. μ΄λŸ¬ν•œ 경우 ν™˜μžλ‘œλΆ€ν„° 얻어진 암세포, μ‘°μ§μœΌλ‘œλΆ€ν„° 얻어진 일차세포 ν˜Ήμ€ 체외 λ°°μ–‘λœ 세포, μŠ€νŽ˜λ‘œμ΄λ“œ, μž₯κΈ°μœ μ‚¬μ²΄ 등을 μ΄μš©ν•˜μ—¬ κ³ μ†λ‹€μ€‘μ•½λ¬ΌμŠ€ν¬λ¦¬λ‹κΈ°μˆ μ„ ν†΅ν•œ λ§žμΆ€ν˜• ν•­μ•”μ œλ₯Ό μ„ λ³„ν•΄λ‚΄λŠ” 체외 약물진단 κΈ°μˆ μ„ 생각해낼 수 μžˆλŠ”λ°, μ΄λŠ” 기쑴의 μœ μ „μ²΄ 기반의 μ‹œλ„μ™€ λ³‘ν–‰λ˜μ–΄ 개개의 ν™˜μžλ“€μ—κ²Œ λ”μš± μ ν•©ν•œ μΉ˜λ£Œλ°©λ²•μ„ μ°ΎλŠ” 것이 κ°€λŠ₯ν•˜κ²Œ ν•œλ‹€. ν•˜μ§€λ§Œ μ΄λŸ¬ν•œ λͺ©μ μ˜ κ³ μ†λ‹€μ€‘μ•½λ¬ΌμŠ€ν¬λ¦¬λ‹κΈ°μˆ μ€ 높은 ν™œμš©κ°€λŠ₯성에도 λΆˆκ΅¬ν•˜κ³ , κ΄‘λ²”μœ„ν•œ 보급과 ν™œμš©μ΄ λ˜κΈ°μ—λŠ” μ œμ•½μ μ΄ λ§Žμ•˜λ‹€. 기쑴의 κ³ μ†λ‹€μ€‘μ•½λ¬ΌμŠ€ν¬λ¦¬λ‹κΈ°μˆ μ€ λ§Žμ€ μ–‘μ˜ μƒ˜ν”Œμ΄ μ†Œλͺ¨λ˜κ³ , κ°’λΉ„μ‹Ό μ‹œμ•½μ˜ μ†Œλͺ¨λŸ‰λ„ 적지 μ•Šμ•˜λ‹€. κ²Œλ‹€κ°€, 수천 가지 μ΄μƒμ˜ μ„œλ‘œ λ‹€λ₯Έ λ¬Όμ§ˆλ“€μ„ νƒμƒ‰ν•˜κΈ° μœ„ν•΄ λ°˜λ“œμ‹œ ν•„μš”ν•œ κ³ κ°€μ˜ μžλ™ν™”λœ 앑체 운반기(liquid handler) 등이 ν•„μš”ν•˜μ˜€λŠ”λ°, μ΄λŸ¬ν•œ 문제둜 λŒ€ν˜• μ œμ•½μ‚¬, μ—°κ΅¬μ†Œ 등을 μ œμ™Έν•˜κ³ λŠ” λ„μž…μ΄ 쉽지가 μ•Šμ•„ κΈ°μˆ μ ‘κ·Όμ„±μ΄ μ œν•œλ˜μ–΄ μžˆμ—ˆλ‹€. λ³Έ μ—°κ΅¬μ—μ„œλŠ” λ°˜λ„μ²΄κ³΅μ •μ—μ„œμ˜ λ…Έκ΄‘κΈ°μˆ μ„ μ΄μš©ν•˜μ—¬ 개개의 식별할 수 μžˆλŠ” μ½”λ“œλ₯Ό 가지고 μžˆλŠ” μ½”λ“œν™”λœ ν•˜μ΄λ“œλ‘œμ € 기반의 광경화성폴리머 λ―Έμ„Έμž…μžλ₯Ό λ§Œλ“€μ–΄, 이λ₯Ό μ›ν•˜λŠ” 암세포에 μ•½λ¬Ό μŠ€ν¬λ¦¬λ‹μ„ ν•΄λ³΄κ³ μž ν•˜λŠ” λ‹€μ–‘ν•œ μ•½λ¬ΌλΌμ΄λΈŒλŸ¬λ¦¬λ₯Ό 이용 각각의 μ½”λ“œν™”λœ λ―Έμ„Έμž…μžμ— ν‘μˆ˜μ‹œμΌœ μ•½λ¬Ό-λ―Έμ„Έμž…μž 라이브러리λ₯Ό μ œμž‘ν•œλ‹€. κ·Έν›„, κ°’λΉ„μ‹Ό μ–΄λ ˆμ΄ μ œμž‘μš© μŠ€ν¬ν„° ν˜Ήμ€ λ””μŠ€νŽœμ„œ μž₯비없이 κ°„λ‹¨ν•œ μžκΈ°μ‘°λ¦½μ„ 톡해 λŒ€κ·œλͺ¨μ˜ λ‹€μ–‘ν•œ μ•½λ¬Ό-ν•˜μ΄λ“œλ‘œμ € μ–΄λ ˆμ΄λ₯Ό μ œμž‘ν•  수 μžˆλŠ” κΈ°μˆ μ„ κ°œλ°œν•˜μ˜€λ‹€. λ˜ν•œ, μ†ŒλŸ‰μ˜ 세포듀 λ§ŒμœΌλ‘œλ„ λ―Έμ„Έμš°λ¬Ό(microwell) 기반의 세포칩에 λ„ν¬ν•˜λŠ” 방식을 κ°œλ°œν•˜μ˜€μœΌλ©°, 이λ₯Όν†΅ν•΄ μ•½λ¬Ό-ν•˜μ΄λ“œλ‘œμ € μ–΄λ ˆμ΄μ™€ λ―Έμ„Έμš°λ¬ΌκΈ°λ°˜μ˜ μ„Έν¬μΉ©μ˜ κ²°ν•©μœΌλ‘œ 수백-수천의 λ‹€μ–‘ν•œ 어세이λ₯Ό 적은 수의 μƒ˜ν”Œλ§ŒμœΌλ‘œλ„ ν•œλ²ˆμ— μˆ˜ν–‰ν•  수 μžˆλŠ” κ³ μ†λ‹€μ€‘μ•½λ¬ΌμŠ€ν¬λ¦¬λ‹ κΈ°μˆ μ„ μˆ˜ν–‰ν•  수 있게 λ§Œλ“€μ—ˆλ‹€. λ³Έ μ—°κ΅¬μ—μ„œ μ œμ‹œν•œ μ†Œν˜•ν™”λœ 체외 ν•­μ•”μ œ μŠ€ν¬λ¦¬λ‹μš© μ•½λ¬Όν”Œλž«νΌμ€ λ‹€μŒκ³Ό 같은 의의λ₯Ό 가진닀. 적은 수의 ν™˜μžμ„Έν¬ ν˜Ήμ€ μƒ˜ν”Œμ˜ 양에 μ μš©ν•  수 μžˆλŠ”, μ‚¬μš©ν•˜κΈ° μ†μ‰¬μš΄ κΈ°μˆ λ‘œμ„œ, 기쑴의 κ°’λΉ„μ‹Ό μž₯λΉ„, μ‹œμ•½μ˜ μ‚¬μš©λŸ‰μ„ 획기적으둜 쀄일 수 μžˆλŠ” κΈ°μˆ μ΄λ‹€. λ³Έ μ—°κ΅¬μ—μ„œ μ œμ•ˆλœ κΈ°μˆ μ„ 톡해 기쑴의 μž₯λΉ„λ₯Ό μ‚¬μš©ν•  λ•Œ μ‹œμ•½μ˜ 값이 λΉ„μ‹Έκ±°λ‚˜, μž₯λΉ„μ˜ 가격이 λΉ„μ‹Έμ„œ, ν˜Ήμ€ λ‹€λ£¨κ³ μž ν•˜λŠ” μƒ˜ν”Œμ˜ 양이 μ œν•œμ μ΄μ–΄μ„œ 기쑴에 μ ‘κ·Όν•˜κΈ° νž˜λ“€μ—ˆλ˜ λ‹€μ–‘ν•œ ν•™μˆ μ—°κ΅¬μ— μ μš©ν•  수 있으며, λ³‘μ›μ—μ„œμ˜ μž„μƒμ—°κ΅¬ 및 μ‹€μ œ ν™˜μžλ§žμΆ€ν˜• μΉ˜λ£Œμ— μ‚¬μš© 될 수 μžˆλŠ” 접근성을 획기적으둜 높일 수 μžˆλ‹€. 특히, 비ꡐ적 쀑,μ†Œ 규λͺ¨μ˜ μ—°κ΅¬ν™˜κ²½μ—μ„œλ„ λ‹€μ–‘ν•œ ν¬κ·€ν•œ ν™˜μžμœ λž˜μ„Έν¬ ν˜Ήμ€ ν™˜μžμœ λž˜μ˜€κ°€λ…Έμ΄λ“œ λ“±κ³Ό μ ‘λͺ©ν•˜μ—¬ μ‚¬μš©λœλ‹€λ©΄ λ³Έ ν”Œλž«νΌμ˜ κ°€λŠ₯성을 λ”μš± κ·ΉλŒ€ν™” ν•  수 μžˆμ„ κ²ƒμœΌλ‘œ κΈ°λŒ€ν•œλ‹€.Precision or Personalized Medicine is a medical paradigm aimed to determine optimal therapy for individual patient. In particular, clinical oncology has been using methods of molecular profiling for each patient through next-generation sequencing (NGS), mRNA-sequencing, and mass spectrometry, and has been trying to implement personalized treatment. However, personalized treatment based on molecular profiling to each patient is not always possible due to the high level of heterogeneity of tumor that is still not fully understood at the current level and acquired resistance of anti-cancer drug due to cumulative targeted therapy. In such cases, in vitro drug testing platform using primary cells obtained from patients, or patient-derived cells, spheroids, and organoids can make it possible to find a more appropriate treatment for each individual patient. However, though high-throughput drug screening technology for this purpose is of the utmost importance in saving lives, there were many limitations to its wide use in many hospitals. The existing high-throughput drug combination screening technology consumes a large number of samples and consumes a considerable amount of expensive reagents. In addition, expensive automated liquid handlers, which were essential for exploring thousands of different pipetting, were not easy to introduce except for large-sized pharmaceutical companies and research institutes, which limited access to technology. In this study, I construct a heterogeneous drug-loaded microparticle library by fabricating encoded photocurable polymer particle that has individually identifiable codes to track loaded drug. and I load various drug molecules, which I want to test to target cells, into each coded microparticle. Then, I developed to produce heterogeneous drug-laden microparticle arrays through simple self-assembly without the need for a microarray spotter or dispensing machine for generating microarray. I also have developed cell seeding method of seeding small-volume samples into the microwell-based cell chip. By utilizing the drug-laden microparticle hydrogel array and microwell-based cell chip technology, hundreds to thousands of different assays can be done at once with just a small number of samples and low cost. Through the implemented platform, the anti-cancer drug sequential combination screening was conducted on the triple-negative breast cooler (TNBC) cells, which are generally known to be difficult to treat due to lack of known drug target, and the results of screening were analyzed by establishing a library of drugs in the EGFR inhibitory type and drugs in the genotoxin type. In addition, another study was conducted to find optimal drug combinations using patient-derived cells derived from tumors in patients with non-small cell lung cancer that have obtained acquired resistance. Finally, as the growing need for three-dimensional culture, such as spheroid and organoid for having a similar response to in vivo drug testing, it was also developed that microwell-based cell chip that is capable of 3D culture with low-cost and small-volume of cells. The miniaturized in vitro anticancer drug screening platform presented in this study has the following significance. An easy-to-use technique that can be applied to a small number of patient cells or samples, which can dramatically reduce the use of conventional expensive equipment, reagents. The proposed technology in this study can be applied to a variety of academic studies previously inaccessible to high-throughput screening due to the high cost of reagents, the high price of equipment, or the limited amount of samples in conventional drug screening. and this platform can also dramatically increase access to clinical research in hospitals for personalized treatments. In particular, it is expected that the possibility of this platform will be further maximized if it is used in a relatively small and medium-sized research environment by the combined use of various rare samples such as patient-derived cells or patient-derived organoids.Chapter 1 Introduction οΌ‘ 1.1 Motivation of this research οΌ’ 1.2 Competing technologies and Previous works 8 1.3 Main Concept: In vitro drug testing using miniaturized encoded drug-laden hydrogel array technology οΌ‘οΌ• Chapter 2 Platform Development of Drug Releasing Hydrogel Microarray 20 2.1 Encoded Drug-Laden Hydrogel & Library construction οΌ’οΌ‘ 2.2 Array generation of heterogenous drug-laden microparticles. οΌ“οΌ” 2.3 Cell Culturing on Cell Chip and bioassay οΌ“οΌ– Chapter 3 Sequential Drug Combination Screening Assy on TNBC 40 3.1 Background : Sequential Drug Combination as promising therapeutic option οΌ”οΌ‘ 3.2 Experimental design with sequential drug treatment assay οΌ”οΌ“ 3.3 Technical Issue & its engineering solution οΌ”οΌ” 3.4 Assay Result οΌ”οΌ™ Chapter 4 Drug Combination Assay on Patient-Derived Cells οΌ•οΌ˜ 4.1 Background : Simultaneous Combination Treatment using Patient-Derived Cells οΌ•οΌ™ 4.2 Improvement of Platform for facilitating translational study οΌ–οΌ’ 4.3 Study Design for small-volume drug combinatorial screening with NSCLC patient derived cell οΌ–οΌ• 4.4 Assay Result οΌ–οΌ™ Chapter 5 Development of platform for 3D culture model οΌ—οΌ’ 5.1 3D culturable platform οΌ—οΌ“ 5.2 Development of 3D culture platform based Matrigel scaffold. οΌ—οΌ˜ 5.3 Advantage over conventional 3D culture-based drug testing platform. οΌ˜οΌ• Chapter 6 Conclusion οΌ˜οΌ— Bibliography 90 Abstract in Korean οΌ™οΌ—Docto
    • …
    corecore