세포 배양 <ol><li> 인간의 배아 신장 293 (HEK293) 세포의 재고 문화가 항생제의 칵테일 (페니실린의 ML 당 100 단위 및 100 μg와 보충 Dulbecco의 수정된 이글의 중간 (DMEM)으로 구성된 &#39;매체&#39;에 10 cm 문화 요리에 재배 당 스트렙토 마이신 황산의 ML)와 10 % 태아 소 혈청 (FBS). </li><li> 실험 세포를 설정하려면, 매체가 해제 빨려이며, 세포는 인산 5 ML로 두 번 씻어하는 것은 생리 (PBS) 솔루션을 버퍼. </li><li> PBS 그런 다음 제거하고 0.25 %의 트립신을 포함하는 솔루션 0.6 ML로 대체됩니다. </li><li> 세포 분리하기 시작했다 때까지 요리는 37 ˚ C 약 2 분 incubated입니다. </li><li> 매체의 5.5 ML은이 트립신을 중화에 추가되고 세포는 hemocytometer로 계산됩니다. </li><li> 전지는 ML 당 320,000 세포에 희석하고 polylysine - 코팅 96 - 웰 플레이트 문화에 잘 당 0.1 ML에 적절하고 있습니다. </li><li> 37 조직 문화의 인큐베이터로 접시를 놓고 ˚ C 8.8 %의 CO2를 포함하는 분위기와 24 시간을위한 성장. </li></ol> Transfections <ol><li> transfections 준비에서, 그것은 실험 조건에 따라 수행과 플라스미드의 양의 어떤 transfected 될 수 얼마나 많은 복제로 간주되어야합니다. 우리는 일상적으로 세중의 모든 조건을 수행하고 잘 따라 플라스미드 DNA의 50-100 NG 총 transfect. 플라스미드 믹스 적어도 기자 반딧불 루시페라제 및 제정 모터의 제어 플라스미드 표현 Renilla의 루시페라제 표현을 구성 포함해야합니다. </li><li> 플라스미드 솔루션은 1.5 ML microcentrifuge 튜브에 pipetted 및 혈청 - 무료 항생제없는 DMEM의 샘플 당 10 μl로 보충하고 있습니다. 20 우물 각 50 μg의 DNA와 transfected 수있다면 예를 들어, 1 μg의 DNA 200 μl DMEM를 추가합니다. </li><li> DMEM / DNA 솔루션 μg의 DNA 당 3 μl &#39;트랜짓 293 시약&#39;(Mirus)을 추가할 수 있습니다. &amp;을 아래로 pipetting으로 혼합하고 최소 10 분 상온에서 샘플 서 보자. </li><li> 이 기간 동안, 96 - 웰 문화 판에서 미디어를 제거하고 신선한 중간 A. 90 μl로 교체 </li><li> 각 잘 DMEM / DNA / 운송 293 혼합 10 μl를 추가합니다. </li><li> 37 조직 문화의 인큐베이터로 접시를 놓고 ˚ C 8.8 %의 CO2를 포함하는 분위기와 24 시간을위한 성장. </li></ol> Phagocytosis <ol><li> 미디어 B를 플러스 (DMEM과 F12 햄의 중간 플러스 항생제와 10 % FBS의 1:1 혼합물) ML 당 제로 1 MG에서 0.75 - μm의 라텍스 구슬이 들어 suspensions를 준비합니다. </li><li> 96 - 웰 문화 판에서 미디어를 제거하고 중간 B.의 비드 suspensions의 0.1 ML로 교체 </li><li> 1000 X G. 2 분 플레이트 스핀 </li><li> 세포는 다음 37 ˚ C 6-16 시간에 대한 양식입니다. 조건 및 루시페라제 증가 리포터 유전자 발현이 1로 3 시간 후에 감지 수 있습니다 운전하는 데 사용되는 모터에 따라 다릅니다. </li><li> 같은 경우에는 그것이 30-60 분 구슬로 세포를 부화 필요할 수 있습니다 다음, 비법인 구슬을 제거하고, 시간의 추가 기간을 위해 세포를 잠복기 전에 새로운 미디어를 추가하는 PBS 2 세척 할. </li></ol> 듀얼 GloTM 루시페라제 어세이 <ol><li> 참고 :이 키트를 사용을위한 프로토콜은 제조 업체에서 권장하는 것과 몇 가지 측면에서 다릅니다. 이것은 샘플 당 필요한 시약의 양을 줄이기 위해 수정되었습니다. </li><li> -20 ˚ C. 여러 작은 aliquots 및 저장에 1 M DTT, 분할의 솔루션을 준비 </li><li> 20 MM 트리스 - HCL (산도 7.8), 10 % (V / V) 글리세롤, 그리고 0.5 % (V / V) 트리톤 X - 100을 포함하는 용해 버퍼를 준비합니다. 사용하기 전에, 나누어지는 실험에 필요한 금액과 1X로 0.5의 최종 농도 1 M DTT 플러스 테아제 억제제 칵테일의 ML 당 1 μl를 추가합니다. 남은 DTT 솔루션을 재사용하지 마십시오. </li><li> 처음으로 듀얼 GloTM 키트를 사용할 경우, 듀얼 GloTM 루시페라제 기판 한 병 (제공하는 듀얼 GloTM 루시페라제 버퍼 한 병 (키트와 함께 제공)의 전체 내용을 전송하여 (반딧불) 루시페라제 시약 준비 키트와 함께). -20 ˚ C. 10 - ML aliquots 및 저장에 사용하지 않는 루시페라제 시약 디비드 </li><li> 조직 문화의 인큐베이터에서 96 - 웰 플레이트를 제거하고 얼음에 놓으십시오. 흡인하여 미디어를 제거, 40 μl 당 우물의 용해 버퍼를 추가한 다음 30 분 얼음 접시를 두십시오. </li><li> 잘 루시페라제 시약의 다음 당 흰색, 불투명한에서 96 - 웰 플레이트 (OptiplateTM - 96, 퍼어킨 엘머 카탈로그 번호 6005290) 나누어지는 15 μl은 세포 lysate 15 μl를 추가합니다. </li><li> 10 분 실온에서 접시를 부화 후 microplate 리더의 발광을 읽어보십시오. </li><li> 즉시 사용하기 전에, 1 일부 듀얼 GloTM 스톱 &amp; 글로을 추가하여 Renilla 루시페라제 기판 솔루션의 적절한 금액을 준비 ®; 시약 (키트와 함께 제공) 100 부품 듀얼 GloTM 스톱 &amp; 글로 ® 버퍼 (키트와 함께 제공)합니다. </li><li> 각 물론 10 분에 대해 실온에서 품어 Renilla 루시페라제 기판 솔루션을 15 μl 당 우물을 추가 후 다시 발광을 측정합니다. </li></ol>

The authors have nothing to disclose.

studying membrane biogenesis with a luciferase-based reporter gene assay

멤브레인 biogenesis 동안 다른 지질 합성 경로의 조정을 연구하기 위해 그것은 멤브레인 biogenesis 빠르게하고 inducible 방식에서 발생하는 실험 시스템을 작동하는 데 유용합니다. 우리는 세포가 빠르게 입자 engulfment 동안 자료를 배치로 손실 막 풀을 보충 막 lipids를 종합으로 라텍스 구슬의 phagocytosis가 이러한 목적을 위해 실용적인 것으로 나타났습니다. 여기, 우리는 Promega에서 듀얼 글로 루시페라제 분석 시스템을 사용 루시페라제 기반 리포터 유전자 접근 방식과 phagocytosis 유발 유전자 발현의 변화를 공부하고 절차를 설명합니다.

Watch this Scientific Journal Video about Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay at JoVE.com

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

여기, 우리는 Promega에서 듀얼 GloTM 루시페라제 분석 시스템을 사용 루시페라제 기반 리포터 유전자 접근 방식과 phagocytosis 유발 유전자 발현의 변화를 공부하고 절차를 설명합니다.

루시페라제 기반 리포터 유전자 분석과 함께 막 Biogenesis 유학

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

studying-membrane-biogenesis-with-luciferase-based-reporter-gene

Research

JoVE Journal

Biology

12.8K 조회수.  Harvard. 여기, 우리는 Promega에서 듀얼 GloTM 루시페라제 분석 시스템을 사용 루시페라제 기반 리포터 유전자 접근 방식과 phagocytosis 유발 유전자 발현의 변화를 공부하고 절차를 설명합니다.

비디오: Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

A Microfluidic Device with Groove Patterns for Studying Cellular Behavior

We describe a microfluidic device with microgrooved patterns for studying cellular behavior. This microfluidic platform consists of a top fluidic channel and a bottom microgrooved substrate. To fabricate the microgrooved channels, a top poly(dimethylsiloxane) (PDMS) mold containing the impression of the microfluidic channels was aligned and bonded to a microgrooved substrate. Using this device, mouse fibroblast cells were immobilized and patterned within microgrooved substrates (25, 50, 75, and 100 &mu;m wide). To study apoptosis in a microfluidic device, media containing hydrogen peroxide, Annexin V, and propidium iodide was perfused into the fluidic channel for 2 hours. We found that cells exposed to the oxidative stress became apoptotic. These apoptotic cells were confirmed by Annexin V that bound to phosphatidylserine at the outer leaflet of the plasma membrane during the apoptosis process. Using this microfluidic device with microgrooved patterns, the apoptosis process was observed in real-time and analyzed by using an inverted microscope containing an incubation chamber (37&deg;C, 5% CO2). Therefore, this microfluidic device incorporated with microgrooved substrates could be useful for studying the cellular behavior and performing high-throughput drug screening.

We describe a protocol for the fabrication of microfluidic devices that can enable cell capture and culture. In this approach patterned microstructures such as grooves within microfluidic channels are used to create low shear stress regions within which cell can dock.

세포 행동 유학을위한 그루브 패턴 Microfluidic 장치

We describe a microfluidic device with microgrooved patterns for studying cellular behavior.  This microfluidic platform consists of a top fluidic channel ...

연구

생물학

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

고효율 기본 셀을 Transfect하는 진 Pulser Electroporation MXcell의 시스템을 사용하여

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

의 꽃 - 물놀이 변환 Arabidopsis thaliana 검사 pTSO2 : β - glucuronidase 리포터 유전자 발현

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Agrobacterium-Mediated Virus-Induced Gene Silencing Assay In Cotton

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties. The large-scale gene functional analysis in cotton has been lagged behind most of the modern plant species, likely due to its large size of genome, gene duplication and polyploidy, long growth cycle and recalcitrance to genetic transformation1. To facilitate high throughput functional genetic/genomic study in cotton, we attempt to develop rapid and efficient transient assays to assess cotton gene functions.
Virus-Induced Gene Silencing (VIGS) is a powerful technique that was developed based on the host Post-Transcriptional Gene Silencing (PTGS) to repress viral proliferation2,3. Agrobacterium-mediated VIGS has been successfully applied in a wide range of dicots species such as Solanaceae, Arabidopsis and legume species, and monocots species including barley, wheat and maize, for various functional genomic studies3,4. As this rapid and efficient approach avoids plant transformation and overcomes functional redundancy, it is particularly attractive and suitable for functional genomic study in crop species like cotton not amenable for transformation.
In this study, we report the detailed protocol of Agrobacterium-mediated VIGS system in cotton. Among the several viral VIGS vectors, the tobacco rattle virus (TRV) invades a wide range of hosts and is able to spread vigorously throughout the entire plant yet produce mild symptoms on the hosts5. To monitor the silencing efficiency, GrCLA1, a homolog gene of Arabidopsis Cloroplastos alterados 1 gene (AtCLA1) in cotton, has been cloned and inserted into the VIGS binary vector pYL156. CLA1 gene is involved in chloroplast development6, and previous studies have shown that loss-of-function of AtCLA1 resulted in an albino phenotype on true leaves7, providing an excellent visual marker for silencing efficiency. At approximately two weeks post Agrobacterium infiltration, the albino phenotype started to appear on the true leaves, with 100% silencing efficiency in all replicated experiments. The silencing of endogenous gene expression was also confirmed by RT-PCR analysis. Significantly, silencing could potently occur in all the cultivars we tested, including various commercially grown varieties in Texas. This rapid and efficient Agrobacterium-mediated VIGS assay provides a very powerful tool for rapid large-scale analysis of gene functions at genome-wide level in cotton.

We present the detailed protocol for Agrobacterium-mediated virus-induced gene silencing (VIGS) assay in cotton. The tobacco rattle virus (TRV)-derived VIGS vectors were deployed to induce RNA silencing of cotton GrCLA1, Cloroplastos alterados 1 gene. The albino phenotype caused by silencing GrCLA1 was observed at the seedling stage within 2 weeks after inoculation.

커튼에서 Agrobacterium - 중재 바이러스 유발 유전자 입을 분석

Cotton (Gossypium hirsutum) is one of the most important crops worldwide. Considerable efforts have been made on molecular breeding of new varieties.  The ...

A Chemical Screening Procedure for Glucocorticoid Signaling with a Zebrafish Larva Luciferase Reporter System

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids can lead to severe side effects. Test systems are needed to search for novel compounds influencing glucocorticoid signaling in vivo or to determine unwanted effects of compounds on the glucocorticoid signaling pathway. We have established a transgenic zebrafish assay which allows the measurement of glucocorticoid signaling activity in vivo and in real-time, the GRIZLY assay (Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY). The luciferase-based assay detects effects on glucocorticoid signaling with high sensitivity and specificity, including effects by compounds that require metabolization or affect endogenous glucocorticoid production. We present here a detailed protocol for conducting chemical screens with this assay. We describe data acquisition, normalization, and analysis, placing a focus on quality control and data visualization. The assay provides a simple, time-resolved, and quantitative readout. It can be operated as a stand-alone platform, but is also easily integrated into high-throughput screening workflows. It furthermore allows for many applications beyond chemical screening, such as environmental monitoring of endocrine disruptors or stress research.

We describe the procedure and data analysis of a chemical screening system for glucocorticoid stress hormone signaling using zebrafish larvae: the Glucocorticoid Responsive In vivo Zebrafish Luciferase activitY (GRIZLY) assay. The assay sensitively and specifically detects effects on glucocorticoid signaling by compounds that require metabolization or affect endogenous glucocorticoid production.

Zebrafish의 유충 루시 페라 제 리포터 시스템과 글루코 코르티코이드 신호에 대한 화학적 스크리닝 절차

Glucocorticoid stress hormones and their artificial derivatives are widely used drugs to treat inflammation, but long-term treatment with glucocorticoids ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

pTIRFM와 이미징 플라즈마 막 변형

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

단백질 분해의 체계적인 분석을위한 기자 기반의 성장 분석

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been described, certain types of cells are resistant to many of these methods and yield low transfection efficiency1, potentially hindering research in those cell types. In this protocol, we present an optimized transfection procedure to introduce luciferase reporter genes as a plasmid DNA into the RAW264.7 macrophage cell line. Two different types of transfection reagents (lipid-based and polyamine-based) are described, and important notes are given throughout the protocol to ensure that the RAW264.7 cells are minimally altered by the transfection procedure and any experimental data obtained are the direct results of the experimental treatment. While transfection efficiency may not be higher compared to other transfection methods, the described procedure is robust enough for detecting luciferase signal in RAW264.7 without changing the physiological response of the cells to stimuli.

Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell&#8217;s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells.

루시퍼 라제 리포터 유전자 RAW264.7 세포를 형질

Transfection of desired genetic materials into cells is an inevitable procedure in biomedical research studies. While numerous methods have been ...

Visualizing Stromule Frequency with Fluorescence Microscopy

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but whose functions remain mysterious. Alongside growing attention on the role of chloroplasts in coordinating plant responses to stress, interest in stromules and their relationship to chloroplast signaling dynamics has increased in recent years, aided by advances in fluorescence microscopy and protein fluorophores that allow for rapid, accurate visualization of stromule dynamics. Here, we provide detailed protocols to assay stromule frequency in the epidermal chloroplasts of Nicotiana benthamiana, an excellent model system for investigating chloroplast stromule biology. We also provide methods for visualizing chloroplast stromules in vitro by extracting chloroplasts from leaves. Finally, we outline sampling strategies and statistical approaches to analyze differences in stromule frequencies in response to stimuli, such as environmental stress, chemical treatments, or gene silencing. Researchers can use these protocols as a starting point to develop new methods for innovative experiments to explore how and why chloroplasts make stromules.

Protocols to investigate the dynamics of chloroplast stromules, the stroma-filled tubules that extend from the surface of chloroplasts, are described.

형광 현미경으로 Stromule 주파수를 시각화

Stromules, or "stroma-filled tubules", are narrow, tubular extensions from the surface of the chloroplast that are universally observed in plant cells but ...

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and develops most often in the setting of immunosuppression. Despite decades of research, optimal treatment of KS remains poorly defined and clinical outcomes are especially unfavorable in resource-limited settings. KS lesions are driven by pathological angiogenesis, chronic inflammation, and oncogenesis, and various in vitro cell culture models have been developed to study these processes. KS arises from KSHV-infected cells of endothelial origin, so EC-lineage cells provide the most appropriate in vitro surrogates of the spindle cell precursor. However, because EC have a limited in vitro lifespan, and as the oncogenic mechanisms employed by KSHV are less efficient than those of other tumorigenic viruses, it has been difficult to assess the processes of transformation in primary or telomerase-immortalized EC. Therefore, a novel EC-based culture model was developed that readily supports transformation following infection with KSHV. Ectopic expression of the E6 and E7 genes of human papillomavirus type 16 allows for extended culture of age- and passage-matched mock- and KSHV-infected EC and supports the development of a truly transformed (i.e., tumorigenic) phenotype in infected cell cultures. This tractable and highly reproducible model of KS has facilitated the discovery of several essential signaling pathways with high potential for translation into clinical settings.

An In Vitro Model for Studying Cellular Transformation by Kaposi Sarcoma Herpesvirus

Kaposi sarcoma (KS) is a tumor induced by infection with the oncogenic virus human herpesvirus-8/KS herpesvirus (HHV-8/KSHV). The endothelial cell culture model described here is uniquely suited for studying the mechanisms by which KSHV transforms host cells.

공부 하는 Kaposi 육 종 Herpesvirus에 의해 세포 변환에 대 한 생체 외에서 모델

Kaposi sarcoma (KS) is an unusual tumor composed of proliferating spindle cells that is initiated by infection of endothelial cells (EC) with KSHV, and ...