이 작품은 NIH 감독의 새로운 혁신 상 프로그램 (ED)에서 부여에 의해 지원되었다. AA는 국립 암 연구소 (National Cancer Institute)와 국립 과학 재단 (National Science Foundation)에서 학부 상에 의해 부분적으로 지원되었다.

1. 바이러스 성 벡터를 만들기 목적 유전자뿐만 아니라, 양성 및 음성 대조군에 대한 코딩 서열은, 전통적인 클로닝 방법 (PCR 증폭, 제한 효소 및 라이 게이션)에 의하여 pBABEpuro 삽입된다. BamHI로, SnaBI로, EcoRI 및 SalI로 : DNA가 삽입 될 수있다 벡터 네 개의 제한 부위가있다. <ol><li> QIAGEN 플라스미드 미디 키트를 사용하여 유능한 세포에서 형질 급 플라스미드 DNA를 준비합니다. </li><li> NanoDrop2000 분광 광도계를 이용하여 DNA 농도를 측정한다. </li></ol> 2. 레트로 바이러스 생산 Plat의 E-패키징 세포주 NIH 3T3 세포가 관심 cDNA를 제공한다 ecotropic 레트로 바이러스를 생산하는 데 사용한다. <ol><li> 냉동 세포에서 Plat의-E 문화 구축 <ol><li> 빠르게 37 ° C의 물을 욕조에 Plat의-E 세포를 해동하고 15 ML 원뿔 튜브에 전송할 수 있습니다. 천천히 (뒤를 Plat의-E 매체의 9를 가하여lbecco 개변 이글 DMEM 배지 + 10 % 소 태아 혈청 (FBS) 및 스트렙토 마이신 +). </li><li> 180 X에서 튜브를 원심 분리기 g 5 분 상층 액을 버린다. </li><li> 10cm의 배양 접시에 작은지면-E 매체 및 전송 10 mL로 세포를 다시 일시 중지합니다. </li><li> 그들은 80 ~ 90 %의 합류 (약 2 일) 때까지 37 ° C, 5 % CO 2 배양기에서 세포를 품어. </li></ol></li><li> 셀 분할 <ol><li> 매체를 대기음 PBS로 한 번 세포를 씻으십시오. </li><li> 0.05 % 트립신 EDTA 2 ㎖를 추가하고 실온에서 1 분 동안 품어. </li><li> 도청 손가락으로 세포를 분리 Plat의-E 배지 10 mL를 넣고 15 ㎖의 튜브에 세포 현탁액을 전송. </li><li> 180 X에서 튜브를 원심 분리기 g 5 분 상층 액을 버린다. </li><li> 작은지면-E 매체의 10 ml의 세포를 재현 탁 1에서 새로운 요리를 시드 : 4-1 : 6 희석. </li></ol></li><li> 작은지면-E 시드 및 형질 <ol><li> 시드 2 × 10 (6)어떤 항생제없이 작은지면-E 매체를 이용하여 10cm 배양 접시 당 세포. </li><li> 37 ° C, 5 % CO 2 배양기에서 세포 O / N을 품어. </li><li> 다음 날, 1.5 ml의 미세 원심 분리 튜브에 옵티-MEM 300 μl를 전송합니다. </li><li> 옵티-MEM으로 준비 관, 폴리에틸렌 이민의 27 μL (PEI), 비용 효율적인 형질 전환 시약 (12)를 추가합니다. 도청 손가락으로 부드럽게 섞어 실온에서 5 분 동안 품어. </li><li> , 옵티-MEM / PEI 튜브에 형질 급 플라스미드 DNA의 9 μg의 추가 텍싱하여 부드럽게 혼합하고, 실온에서 15 분 동안 품어. </li><li> 추가 DNA / PEI Plat의-E 접시에, 37 ° C에서 O / N을 품어 복합 한 방울 씩, 5 % CO 2. </li><li> 다음 날, 형질 전환 시약을 포함하는 매체를 대기음 (항생제없이) 신선한 Plat의-E의 10 ml의 배지를 추가합니다. </li><li> 인큐베이터에 세포를 돌려줍니다. </li></ol></li></ol> 3. NIH 3T3 세포 및 감염 <ol><li> NIH 구축3T3 문화 및 시드 <ol><li> 빠르게 37 ° C의 물을 욕조에 NIH 3T3 세포를 해동하고 15 ML 튜브로 전송할 수 있습니다. 천천히 NIH 3T3 배지 (DMEM + 10 % FBS)의 구를 가하여. </li><li> 180 X에서 튜브를 원심 분리기 g 5 분 상층 액을 버린다. </li><li> 10cm 배양 접시 NIH 3T3 매체와 전사 10 mL로 세포를 재현 탁. </li><li> 37 ° C, 5 % CO 2 인큐베이터에서 세포를 배양한다. 그것은 자연 변환 (그리고 초점 형성 따라서 기초 수준)의 주파수로 문화 포화 상태에 도달하면 증가, NIH 3T3 세포는 항상 underconfluent (50~60%)을 유지하는 것이 매우 중요합니다. </li><li> 10cm 접시 당 종자 3 × 10 5 세포는 다음날 감염 O / N을 배양한다. </li></ol></li><li> 감염 <ol><li> 0.45 ㎛의 세공 크기를 나일론 멤브레인 필터로 여과 한, 10 ㎖의 일회용 주사기를 사용하고, 15 ㎖ 중에 전송해서 E-Plat의 접시에서 레트로 바이러스 상등액을 수확튜브. </li><li> (항생제없이) 세포에 신선한 Plat의-E 매체의 10 ML을 추가하고 인큐베이터에 반환. </li><li> 감염 될 수있는 NIH 3T3 세포 접시에서 매체를 대기음, 바이러스 함유 필터링 된 상층 액의 5 정기적 인 NIH 3T3 매체 ml의 5 mL를 추가합니다. </li><li> 6 ㎍ / ml의 농도로 폴리 브렌 접시에 추가. </li><li> 37 ° C, 5 % CO 2의 세포 O / N을 품어. </li><li> 감염의 두 번째 라운드에 대한 3.2.5 - 다음 날, 반복 3.2.1 단계를 반복합니다. </li><li> 일반 NIH 3T3 매체와 바이러스 함유 배지를 교체합니다. </li><li> 필요에 따라 매체를 교체, NIH 3T3 세포를 2 ~ 3 주 동안 성장하는 것을 허용합니다. 복제 판에서 전체 세포 용 해물에 기존의 단백질 전기 영동 및 면역에 의해 관심의 단백질의 발현을 확인합니다. </li></ol></li></ol> 4. 염색 및 정량화 <ol><li> 크리스탈 바이올렛 염색 <ol><li> NIH 3T3 매체를 대기음 요리를 배치얼음에. </li><li> 얼음처럼 차가운 PBS로 두 번 요리를 씻으십시오. </li><li> 10 분 동안 빙냉 메탄올로 세포를 고정. </li><li> 얼음에서 요리를 제거하고 메탄올을 대기음, 25 % 메탄올 (RT)에서 만든 0.5 % 크리스탈 바이올렛 용액 3 ㎖를 추가합니다. </li><li> RT에서 5 분 동안 요리를 품어. </li><li> 크리스탈 바이올렛 솔루션을 기음과 컬러가 린스 벗기하지 않을 때까지 조심스럽게 밀리-Q의 H 2 O와 접시를 씻어. </li><li> 요리는 벤치 탑에 O / N을 건조하도록 허용 (발견). </li></ol></li><li> 초점 정량화 <ol><li> 요리가 건조되면, 접시에 세포의 음울 색깔의 컬렉션을 측정하는 통치자를 사용합니다. 만 등의 직경보다 5mm 된 것을 계산 &quot;초점을.&quot;초점의 수를 기록하고 평균과 컨트롤에 비해 중요성을 계산합니다. </li></ol></li></ol>

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저자가 공개하는 게 없다.

FFA는 체외에서 악성 변화를 평가하기위한 빠르고 쉬운 방법을 제공합니다. 이는 후보 유전자의 상대적으로 큰 수의 검사 의무이며, 그 완만 한 기술적 요구는 비용 효율적인 만든다. 또한, 두 개 이상의 유전자를 공 발현 할 수있다 조합 발암 잠재력을 평가하는 (때때로 &quot;협동&quot;분석법이라 함). 이 분석의 장점은 간단 기술, 정량의 용이성과 상대적으로 짧은 턴어라운드 시간에 의존하...

종양 세포 유전자 발현의 패턴에서 증식 및 형태 학적 변화 후성 유전학에, 변경의 폭 넓은 정상 대조 구별 될 수있다. 후자 중에서, 궁극적으로 동물에 주입시 종양을 형성하는 능력은 악성화 (3)의 유용한 측정 지표 인 혈청에 의존하여, 접점 (밀도) 억제의 상실, 앵커리지 - 독립된 확산 인수를 감소. 생체 외 및 생체 내 분석 몇몇 셀룰러 변환을 위해 개발되었다. 시험 관내 분석법을 목표 (환원 혈청 성장)을 식별하고 배양 모폴로지 (초점 형성 어 세이), 문화 역학 (성장 속도, 포화 농도) 및 성장 인자의 변화가 측정 또는 요구 사항 앵커리지 (소프트 한천의 성장). 세포 유형의 악성 특성을 결정하기위한 금 표준 실험 동물에서 종양 형성 (이종) 남아있다. 그러나, t그는 비용 및 생체 내 연구의 길이는 항상 후보 종양 유전자의 첫 번째 검증 단계 또는 스크리닝 그들을 정당화하지 않습니다. 어떤 시험 관내 분석은 유전자의 발암 가능성의 명확한 평가를 제공하지 않지만, 그들은 생체 내 (in vivo) 연구가 미래를 좁힐 수 발암 가능성에 대한 통찰력을 제공 않습니다. 시험관 내에서 종양 발생 가능성을 평가하기위한 가장 널리 사용되는 시스템 중 하나는 초점 형성 분석법이있다. 이 접근법은 NIH 3T3 마우스 섬유 모세포, 강한 접촉 억제를 나타내고 형질 전환되지 않은 세포주의 사용에 의존한다. 밀도에 의존하는 성장의 손실 종양 유전자 결과의 과발현; 형질 전환 된 세포를 용이하게 형질 전환되지 않은 세포의 배경에 대해 단층 가시화 &quot;초점&quot;을 형성하는, 다층으로 성장할 수있다. 접점의 ON 인 손실에 의해 입증되는 바와 같이 포커스 정량법 형성 후, 악성 변환을 유도하는 후보의 종양 유전자 능력을 측정측정 표현형으로 ition. FFA (예를 들면, Src에 4 BRAF 5) 단백질 키나제의 과발현, 전사 인자 (예, N-myc의 6) (예 P2RY8 7), G는 단백질 - 결합 수용체와 GTP 아제 (예에 의해 변형을 평가하기 위해 사용되어왔다 , 라스 1), 다른 사람의 사이에서. 이 분석의 상대적 용이성은 유전자의 과발현은 체외에서 NIH 3T3 마우스 섬유 아세포를 변환 할만큼 충분한 지 여부에 대한 신속하고 시각적으로 명확한 답을 제공 할 것입니다 좋은 선택입니다. 이 프로토콜에서 설명 FFA 패키징 바이러스 단백질을 제공 Plat의 E-패키징 세포주 (8)를 사용하고, 레트로 바이러스 벡터 pBABEpuro 9 (Addgene 플라스미드 1764)는 레트로 바이러스를 생성한다. 관심의 유전자를 포함하는 구조체 pBABEpuro 형질 감염 후, E-Plat의 세포주는 NIH 3T3 세포를 감염시키기 위해 사용될 수 ecotropic 레트로 바이러스를 생성 할 것이다. 티유전자 전달에있어서의 그의 바이러스 전통적인 화학적 형질 전환 방법보다 더 효율적이며 지속적으로 10 유전자를 발현 할 수있는 방법을 제공한다. 일단 NIH 3T3 세포의 게놈 내로 혼입, 목적 유전자의 과발현은 바이러스 긴 말단 반복 (LTR) 프로모터 (11)에 의해 구동된다. 이 상수는 식 NIH 3T3 세포상의 초점의 형성에 의해 측정 된 관심 유전자, 종양 활성을 가지고 있는지 여부를 결정하는데 사용될 수있다.

<table><tbody><tr><td>pBABE-puro vector</td><td>Addgene</td><td>Plasmid 1764</td><td>cloning vector</td></tr><tr><td>Platinum-E Retroviral Packaging Cell Line, Ecotropic</td><td>Cell Biolabs, Inc.</td><td>RV-101</td><td>cell line for viral production</td></tr><tr><td>NIH 3T3 Cell Line murine</td><td>Sigma-Aldrich</td><td>93061524</td><td>cell line for focus formation assay</td></tr><tr><td>10 ml BD Luer-Lok tip syringe</td><td>BD Biosciences</td><td>309604</td><td>viral production reagent</td></tr><tr><td>0.45 &amp;mu;m Puradisc Syringe Filter</td><td>Whatman</td><td>6750-2504</td><td>viral production reagent</td></tr><tr><td>Polyethylenimine (PEI)</td><td>Polysciences, Inc.</td><td>23966-2</td><td>cell transfection reagent</td></tr><tr><td>Polybrene Infection / Transfection Reagent</td><td>EMD Millipore</td><td>TR-1003-G</td><td>cell transfection reagent</td></tr><tr><td>Crystal Violet</td><td>Fisher Scientific</td><td>C581-25</td><td>cell stain reagent</td></tr><tr><td>Plasmid Plus Midi Kit</td><td>QIAGEN</td><td>12945</td><td>plasmid purification</td></tr><tr><td>BD Falcon Tissue Culture Dishes</td><td>BD Biosciences</td><td>353003</td><td>cell culture supplies</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>11995-065</td><td>cell culture media</td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Gibco</td><td>25300-054</td><td>cell culture supplies</td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Gibco</td><td>31985-062</td><td>cell culture media</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>16000-044</td><td>cell culture media</td></tr></tbody></table>

focus formation: a cell-based assay to determine the oncogenic potential of a gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

MXD3는 MYC / MAX / MAD 네트워크의 구성원 인 염기성 헬릭스 - 루프 - 헬릭스 류신 지퍼 (bHLHZ) 전사 인자이다. 그것은 MAD 가족 13-15의 비정형 부재이며, 그것은 암화 (16, 17)에 관여하는 것으로보고되었다. pBABEpuro (대조군)과 MYC (양성 대조군)과 비교했을 때, MXD3가 과발현 된 NIH 3T3 요리는 훨씬 적은 초점 (그림 1A)를했다. 도 1b의 데이터는 중요성을 결정하기 위해 여러 실험으?...

Watch this Scientific Journal Video about Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene at JoVE.com

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

초점 형성의 분석은 후보 종양 유전자의 변화 가능성을 평가하기위한 간단한 방법을 제공한다.

형성 초점 : 세포 기반 분석은 유전자의 발암 성 가능성을 파악하기 위해

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

focus-formation-cell-based-assay-to-determine-oncogenic-potential

Research

JoVE Journal

Medicine

27.8K 조회수.  University of California, Davis.  초점 형성의 분석은 후보 종양 유전자의 변화 가능성을 평가하기위한 간단한 방법을 제공한다. 

비디오: Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery of the next generation of novel drug targets. It is becoming apparent that more complex models of cancer are required to fully appreciate the contributing factors that drive tumorigenesis in vivo and increase the efficacy of novel therapies that make the transition from pre-clinical models to clinical trials.
Here we present a methodology for generating uniform and reproducible tumor spheroids that can be subjected to siRNA functional screening. These spheroids display many characteristics that are found in solid tumors that are not present in traditional two-dimension culture. We show that several commonly used breast cancer cell lines are amenable to this protocol. Furthermore, we provide proof-of-principle data utilizing the breast cancer cell line BT474, confirming their dependency on amplification of the epidermal growth factor receptor HER2 and mutation of phosphatidylinositol-4,5-biphosphate 3-kinase (PIK3CA) when grown as tumor spheroids. Finally, we are able to further investigate and confirm the spatial impact of these dependencies using immunohistochemistry.

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

기능 유전체학에게 검사를 활용하여 암 세포 회전 타원체 문화에 잠재적으로 소설 약 표적을 식별하는

The identification of functional driver events in cancer is central to furthering our understanding of cancer biology and indispensable for the discovery ...

연구

암 연구학

Colony Forming Cell (CFC) Assay for Human Hematopoietic Cells

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and leukemogenesis. They have the capacity to differentiate into lymphoid and myeloid lineages. The colony forming cell (CFC) assay is used to study the proliferation and differentiation pattern of hematopoietic progenitors by their ability to form colonies in a semisolid medium. The number and the morphology of the colonies formed by a fixed number of input cells provide preliminary information about the ability of progenitors to differentiate and proliferate. Cells can be harvested from individual colonies or from the whole plate to further assess their numbers and differentiation states using flow cytometry and morphologic evaluation of Giemsa-stained slides. This assay is useful for assessing myeloid but not lymphoid differentiation. The term myeloid in this context is used in its wider sense to encompass granulocytic, monocytic, erythroid, and megakaryocytic lineages.

We have used this assay to assess the effects of oncogenes on the differentiation of primary human CD34+ cells derived from peripheral blood. For this purpose cells are transduced with either control retroviral construct or a construct expressing the oncogene of interest, in this case NUP98-HOXA9. We employ a commonly used retroviral vector, MSCV-IRES-GFP, that expresses a bicistronic mRNA that produces the gene of interest and a GFP marker. Cells are pre-activated by growing in the presence of cytokines for two days prior to retroviral transduction. After another two days, GFP+ cells are isolated by fluorescence-activated cell sorting (FACS) and mixed with a methylcellulose-containing semisolid medium supplemented with cytokines and incubated till colonies appear on the surface, typically 14 days. The number and morphology of the colonies are documented. Cells are then removed from the plates, washed, counted, and subjected to flow cytometry and morphologic examination. Flow cytometry with antibodies specific to the cell surface markers expressed during hematopoiesis provides information about lineage and maturation stage. Morphological studies of individual cells under a microscope after Wright- Giemsa staining provide further information with regard to lineage and maturation. Comparison of cells transduced with control empty vector to those transduced with an oncogene reveals the effects of the oncogene on hematopoietic differentiation.

Colony Forming Cell CFC Assay for Human Hematopoietic Cells

The colony forming cell (CFC) assay is an in vitro assay in which hematopoietic progenitors form colonies in a semi-solid medium. A combination of colony morphology, cell morphology, and flow cytometry are used to assess the ability of the progenitors to proliferate and differentiate along the different hematopoietic lineages.

인간 조혈 세포 콜로니 형성 세포 (CFC) 어세이

Human hematopoietic stem/progenitor cells are usually obtained from bone marrow, cord blood, or peripheral blood and are used to study hematopoiesis and ...

면역학 및 감염병학

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, a small population of cells does not respond to TKI treatment, resulting in minimal residual disease (MRD); even the most potent TKIs fail to eradicate these cells. These MRD cells serve as a reservoir to develop resistance to therapy. Why TKI treatment is ineffective against MRD cells is not known. Growth factor signaling is implicated in supporting the survival of MRD cells during TKI treatment, but a mechanistic understanding is lacking. Recent studies demonstrated that an elevated c-Fos and Dusp1 expression as a result of convergent oncogenic and growth factor signaling in MRD cells mediate TKI resistance. The genetic and chemical inhibition of c-Fos and Dusp1 renders CML exquisitely sensitive to TKIs and cures CML in both genetic and humanized mouse models. We identified these target genes using multiple microarrays from TKI-sensitive and -resistant cells. Here, we provide methods for target validation using in vitro and in vivo mouse models. These methods can easily be applied to any target for genetic validation and therapeutic development.

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

Oncogene 의존에 c Fos의 역할 및 Dusp1를 평가 하기 위한 방법

The demonstration of tyrosine kinase inhibitors (TKIs) in treating chronic myeloid leukemia (CML) has heralded a new era in cancer therapeutics. However, ...

Yeast As a Chassis for Developing Functional Assays to Study Human P53

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of different functional assays to study the impacts of 1) binding site [i.e., response element (RE)] sequence variants on P53 transactivation specificity or 2) TP53 mutations, co-expressed cofactors, or small molecules on P53 transactivation activity. Different basic and translational research applications have been developed. Experimentally, these approaches exploit two major advantages of the yeast model. On one hand, the ease of genome editing enables quick construction of qualitative or quantitative reporter systems by exploiting isogenic strains that differ only at the level of a specific P53-RE to investigate sequence-specificity of P53-dependent transactivation. On the other hand, the availability of regulated systems for ectopic P53 expression allows the evaluation of transactivation in a wide range of protein expression. Reviewed in this report are extensively used systems that are based on color reporter genes, luciferase, and the growth of yeast to illustrate their main methodological steps and to critically assess their predictive power. Moreover, the extreme versatility of these approaches can be easily exploited to study different TFs including P63 and P73, which are other members of TP53 gene family.

Presented here are four protocols to construct and exploit yeast Saccharomyces cerevisiae reporter strains to study human P53 transactivation potential, impacts of its various cancer-associated mutations, co-expressed interacting proteins, and the effects of specific small molecules.

인간 P53을 연구하기 위한 기능성 어세이 개발을 위한 섀시로 효모

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples from cancer patients often directly influences diagnosis, prognosis, and/or therapy selection. As a result, the accurate detection of gene fusions has become a critical component of clinical management for many disease types. Until recently, clinical gene fusion detection was predominantly accomplished through the use of single-gene assays. However, the ever-growing list of gene fusions with clinical significance has created a need for assessing fusion status of multiple genes simultaneously. Next generation sequencing (NGS)-based testing has met this demand through the ability to sequence nucleic acid in massively parallel fashion. Multiple NGS-based approaches that employ different strategies for gene target enrichment are now available for use in clinical molecular diagnostics, each with its own strengths and weaknesses. This article describes the use of anchored multiplex PCR (AMP)-based target enrichment and library preparation followed by NGS to assess for gene fusions in clinical solid tumor specimens. AMP is unique among amplicon-based enrichment approaches in that it identifies gene fusions regardless of the identity of the fusion partner. Detailed here are both the wet-bench and data analysis steps that ensure accurate gene fusion detection from clinical samples.

This article details the use of an anchored multiplex polymerase chain reaction-based library preparation kit followed by next-generation sequencing to assess for oncogenic gene fusions in clinical solid tumor samples. Both wet-bench and data analysis steps are described.

앵커드 멀티플렉스 폴리머라제 연쇄 반응을 이용한 종양발생 유전자 융합 검출 후 차세대 염기서열 분석

Gene fusions frequently contribute to the oncogenic phenotype of many different types of cancer. Additionally, the presence of certain fusions in samples ...

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these proteins contain an intrinsically disordered domain (IDD) fused with the DNA-binding domain (DBD) of another protein and orchestrate widespread transcriptional changes to promote malignancy. These fusions are often the sole recurring genomic aberration in the cancers they cause, making them attractive therapeutic targets. However, targeting oncogenic transcription factors requires a better understanding of the mechanistic role that low-complexity, IDDs play in their function. The N-terminal domain of EWSR1 is an IDD involved in a variety of oncogenic fusion transcription factors, including EWS/FLI, EWS/ATF, and EWS/WT1. Here, we use RNA-sequencing to investigate the structural features of the EWS domain important for transcriptional function of EWS/FLI in Ewing sarcoma. First shRNA-mediated depletion of the endogenous fusion from Ewing sarcoma cells paired with ectopic expression of a variety of EWS-mutant constructs is performed. Then RNA-sequencing is used to analyze the transcriptomes of cells expressing these constructs to characterize the functional deficits associated with mutations in the EWS domain. By integrating the transcriptomic analyses with previously published information about EWS/FLI DNA binding motifs, and genomic localization, as well as functional assays for transforming ability, we were able to identify structural features of EWS/FLI important for oncogenesis and define a novel set of EWS/FLI target genes critical for Ewing sarcoma. This paper demonstrates the use of RNA-sequencing as a method to map the structure-function relationship of the intrinsically disordered domain of oncogenic transcription factors.

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

전사 분석을 사용하여 무질서한 온코겐 전사 요인의 구조 기능 관계 매핑

Many cancers are characterized by chromosomal translocations which result in the expression of oncogenic fusion transcription factors. Typically, these ...

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

Genome-scale interrogation of gene function using RNA interference (RNAi) holds tremendous promise for the rapid identification of chemically tractable cancer cell vulnerabilities. Limiting the potential of this technology is the inability to rapidly delineate the mechanistic basis of phenotypic outcomes and thus inform the development of molecularly targeted therapeutic strategies. We outline here methods to deconstruct cellular phenotypes induced by RNAi-mediated gene targeting using multiplexed reporter systems that allow monitoring of key cancer cell-associated processes. This high-content screening methodology is versatile and can be readily adapted for the screening of other types of large molecular libraries.

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

배양 세포에서 암과 연관된 신호 전달을 심문위한 멀티 플렉스 루시 페라 기반 스크리닝 플랫폼

Genome-scale interrogation of gene function using RNA interference (RNAi)  holds tremendous promise for the rapid identification of chemically tractable  ...

의학

Proximity Ligation Assay to Study Oncogene-Derived Transcription-Replication Conflicts

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. Conflicts between the replication and transcription machinery, termed transcription-replication conflicts (TRCs), pose a considerable risk to genome stability, a critical factor in cancer development. While several factors regulating these collisions have been identified, pinpointing primary causes remains difficult due to limited tools for direct visualization and clear interpretation. In this study, we directly visualize TRCs using a proximity ligation assay (PLA), leveraging antibodies specific to PCNA and phosphorylated CTD of RNA polymerase II. This approach allows precise measurement of TRCs between replication and transcription processes mediated by RNA polymerase II. The method is further enhanced through DNA primers conjugated covalently to these antibodies, coupled with PCR amplification using fluorescent probes, providing a highly sensitive and specific means of detecting endogenous TRCs. Fluorescence microscopy enables the visualization of these conflicts, offering a powerful tool to study genome instability mechanisms associated with cancer. This technique addresses the gap in direct TRC visualization, allowing for a more comprehensive analysis and understanding of the underlying processes driving genome instability in cells.

Here, we present a sensitive and specific method to detect oncogene-induced transcription-replication conflicts (TRCs) using the proximity ligation assay (PLA). This approach leverages specific antibodies targeting PCNA and phospho-CTD RNAPII, enabling the assessment of TRC prevalence between RNA polymerase II transcription and DNA replication machinery.

종양유전자 유래 전사-복제 충돌을 연구하기 위한 근접 결찰 분석

Both DNA replication and RNA transcription utilize genomic DNA as their template, necessitating spatial and temporal separation of these processes. ...

Oncogene Expression Analysis with Alterations in pH in a Pancreatic Ductal Cell Line

The fourth leading cause of cancer-related death, pancreatic ductal adenocarcinoma (PDAC), has a 12% five-year survival rate. This disease has a poor prognosis and is characterized by a rigid stromal microenvironment, which represents a tangible challenge in its treatment. Chronic pancreatitis patients have a 10-fold greater risk of developing PDAC; in these patients, the ductal pH decreases from pH 8.0 to pH 6.0 due to bicarbonate insufficiency and the inflammatory milieu. Our goal was to understand the role of the acidic environment observed in chronic pancreatitis on oncogene expression in a pancreatic ductal cell line.
Therefore, 80% confluent human pancreatic ductal epithelial cells were incubated at pH 6.0 to pH 7.2 for 6 h. Total RNA from the cells was processed to enrich the total mRNA in the samples. Gene expression was evaluated via next-generation sequencing (NGS) of biological replicates. RNA-seq analysis was carried out with the aid of an online tool, and the differentially expressed genes (FCs &#60; &#177; 2.0) were identified; there were 90, 148, and 109 upregulated genes and 20, 14, and 23 downregulated genes at pH 6.0, 6.5, and 7.0, respectively. Four oncogenes were upregulated at pH 6.0, seven were upregulated at pH 6.5, and seven were upregulated at pH 7.0. The common genes that were upregulated at pH 6.0, pH 6.5, and pH 7.0 were lymphocyte cell-specific protein-tyrosine kinase (LCK) [pH 6.0, FC: 2.93; pH 6.5, FC: 2.93; pH 7.0, FC: 3.32], FGR proto-oncogene, Src family tyrosine kinase (FGR) [pH 6.0, FC: 4.17; pH 6.5, FC: 5.25; pH 7.0, FC: 5.09], and ArfGAP With SH3 domain, ankyrin repeat, and PH domain 3 (ASAP3) [pH 6.0, FC: 2.37; pH 6.5, FC: 3.84; pH 7.0, FC: 2.51]. The acidic environment triggers the activation of proto-oncogenes, which may trigger tumor initiation in chronic pancreatitis.

This study describes a protocol for exploring the effects of altered pH on oncogene expression via RNA-seq analysis of a pancreatic ductal cell line.

췌장 관 세포주에서 pH 변화를 이용한 종양유전자 발현 분석

The fourth leading cause of cancer-related death, pancreatic ductal adenocarcinoma (PDAC), has a 12% five-year survival rate. This disease has a poor ...

The Soft Agar Colony Formation Assay

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

소프트 한천 콜로니 형성 분석

Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft ...