This study was funded by Canadian Institutes for Health Research (CIHR) grant. STC holds a doctoral research award from the CIHR and the Michael Smith Foundation. EYS holds a CIHR scholarship. The CIHR Transplantation Training Program also provided graduate scholarships to STC, EYS and SS.

1. 플라스미드 DNA를 정제 <ol><li> 제조 업체의 프로토콜에 따라 maxiprep 키트를 사용하여 플라스미드 DNA를 추출합니다. TE 완충액 500 μL에 재현 탁 플라스미드 DNA. </li><li> 클로로포름 : 페놀 수행 아밀 알코올 추출 및 이소프로판올 침전 세균 잔류 오염물을 제거 하였다. LPS의 존재는 형질 9 방해한다. <ol><li> 플라스미드 DNA (1, pH가 8 25 : 24), 15 초 동안 격렬하게 흔들어 이소 아밀 알코올 : 클로로포름 : 페놀의 500 μl를 추가합니다. 페놀은 피부에 심한 화상을 일으키는 심각한 손상이 될 때까지 화상 항상 생각되지 않도록 마취입니다. 흄 후드에서 이소 아밀 추출 및주의하십시오 : 클로로포름 : 페놀을 수행합니다. </li></ol></li><li> RT에서 5 분 동안 혼합물을 인큐베이션. 실온에서 10 분 동안 13,000 XG에 원심 분리기. 새로운 1.5 ml의 수집 관에 위 수상의 350 μl를 전송합니다. </li><li> 하부 유기상을 포함하는 튜브에 TE 완충액 350 μl를 추가. 15 초 동안 격렬하게 흔들어. 실온에서 5 분 동안 13,000 XG에 원심 분리기. 같은 1.5 ml의 수집 관에 위 수상의 350 μl를 전송합니다. </li><li> 3 M 아세트산 나트륨 (PH 5.2)의 70 μl를 넣고 잘 섞는다. 100 % 이소프로판올 700 μl를 추가합니다. 잘 혼합하고 실온에서 10 분을 품어. 10 minat 4 ℃에서 13,000 XG에 원심 분리기. 상층 액을 제거합니다. </li><li> 펠렛에 75 % 에탄올 500 μl를 추가합니다. 소용돌이 샘플은 4 ℃에서 5 분 동안 5,000 XG에 혼합하고 원심 분리한다. 상층 액을 제거합니다. DNA는 펠렛입니다. </li><li> 튜브의 캡을 열고 공기가 5 실온에서 펠렛을 건조 - 10 분. 펠렛은 반투명하게한다. DNA 펠렛을 재현 탁하는 TE 버퍼의 500 μl를 추가합니다. 50 ℃에서 열 - 10 분 동안 60 °의 C는 DNA 펠렛을 용해. </li><li> 260 nm의 (A260) 및 분광계와 280 nm의 (A280)에서 흡광도를 측정한다. 50 NG / μL로 A260 값을 곱함으로써 DNA의 농도를 계산한다. calculatin 의한 DNA의 품질을 평가G A260 / A280 비율. 2.0 - 고품질의 DNA는 1.8 A260 / A280 비율을 제공해야합니다. </li></ol> 2. 세포 배양 및 시드 <ol><li> DMEM에서 배양 된 RAW264.7 세포는 37 ° C, 5 % CO 2 인큐베이터에서 9 % 태아 송아지 혈청 (DMEM / 9 % FCS)으로 보충. 각 통과하는 동안, 파스퇴르 피펫을 사용하여 연속 피펫으로 판에서 세포를 분리. 통로는 세포 2 일마다, 1.5 씨앗 - 주식으로 10cm 조직 배양 처리 접시에 2 백만 세포를. 6 주 - 셀마다 5의 새로운 주식을 해동. </li><li> 트랜 스펙 션 당일, DMEM / 9 % FCS 500 μL 부피의 24 웰 플레이트에 웰 당 200,000 세포를 시드. 37 ° C 배양기에서 4 시간 동안 세포를 품어. </li><li> 형질 세포 중 지질 계 시약 (단계 3.1) 또는 단백질 / 폴리아민 계 시약 (스텝 3.2)과. </li></ol> 3. 형질 <ol><li> 지질 기반 형질 : <ol><li> 형질 전환 reagen을 따뜻하게T는 어둠 속에서 실온까지. 무 혈청 배지와 DMEM / 9 % FCS에 37 ° C를 따뜻하게. </li><li> 1.5 ML 튜브에서 무 혈청 배지의 플라스미드 DNA 0.5 μg의 50 μl를 추가합니다. 액체의 표면 아래에 피펫 팁과 형질 전환 시약의 2 μl를 추가합니다. 6 초 동안 소용돌이. </li><li> 실온에서 30 분 동안 어두운 데에서 시약 / DNA 혼합물을 남기기. </li><li> 30 분 동안 배양, 우물에서 미디어를 제거하고 각 웰에 신선한 DMEM / 9 % FCS 250 μl를 추가합니다. </li><li> 30 분 후, 반응물 / DNA 혼합물에 DMEM / 9 % FCS 250 μl를 추가한다. 로 pipetting 아래로 잘 섞는다. </li><li> 각 웰에 희석 된 혼합물의 300 μl를 추가합니다. 37 ℃, 5 % CO 2 배양기에서 4 시간 - 2 품어. </li><li> 형질 전환 솔루션을 제거하고 DMEM / 9 % FCS 500 μl를 추가합니다. 세포 자극하기 전에 37 ℃, 5 % CO 2 배양기에서 48 시간 - 24 품어. </li></ol></li><li> 단백질 / 폴리아민 기반 형질 : <ol><li> 워밍업 무 혈청매체 및 37 ° C의 DMEM는 / 9 % FCS. </li><li> 무 혈청 배지의 18.75 μL에 형질 전환 시약의 0.75 μl를 추가합니다. 소용돌이는 간단히 혼합한다. 5 분 동안 실온에서 인큐베이션. 희석 된 형질 전환 시약에 1 μg의 / μL DNA의 0.5 μl를 추가합니다. 실온에서 10 분 동안 인큐베이션. </li><li> 10 분 배양 동안, 24- 웰 플레이트의 각 웰로부터 매체를 제거하고, 각 웰에 250 ㎕의 신선한 DMEM / 9 % FCS를 추가한다. </li><li> 10 분 후, 반응물 / DNA 혼합물에 DMEM / 9 % FCS 250 μl를 추가한다. </li><li> 우물에 희석 혼합물의 270 μl를 추가합니다. 4 시간 - 2, 37 ℃, 5 % CO 2 배양기에서 품어. </li><li> 형질 전환 솔루션을 제거하고 DMEM / 9 % FCS 500 μl를 추가합니다. 37 ℃, 5 % CO 2 배양기에서 48 시간 - 24 품어. </li></ol></li></ol> 4. 세포 자극과 루시 페라 제의 분석 <ol><li> 신선한 DMEM / 9 % FCS의 잘 250 μL 미디어를 교체합니다. </li><li> 50 LPS의 μL 또는 LPS + IL 추가우물에 원하는 농도 -10. 37 ℃, 5 % CO 2 배양기에 플레이트를 돌려줍니다. 6 시간 - 2 자극한다. </li><li> , 흡인에 의해 자극 솔루션을 제거 얼음 차가운 PBS로 세척하고, 1X 수동 용해 버퍼 200 μl를 추가합니다. 실온에서 30 분 동안 바위. 긁고 1.5 ml의 튜브에 해물을 전송하고 4 ℃에서 10 분 동안 20,000 XG에 원심 분리하여 세포 파편을 제거합니다. </li><li> 전송 제조 업체의 프로토콜에 따라 루시 페라 제 신호를 결정하기 위해 흰색, 분명 바닥 96 웰 플레이트에 분해물 (상층 액)의 40 μL. </li><li> 전체 가시 광선 스펙트럼에 걸쳐 루미와 루시 페라 제 신호를 읽어보십시오. </li></ol> 5. 데이터 분석 <ol><li> 각 우물에서 레 닐라 루시퍼의 값으로 반딧불 루시 페라 제의 개별 값을 나누어 레 닐라 비율 : 반딧불을 계산합니다. </li><li> 에 의해 잘 치료의 레 닐라 비율 : 반딧불을 나누어 배의 변화를 계산치료 잘하는. </li></ol>

<ol>
	<li>Maurisse, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+transfection+of+DNA+into+primary+and+transformed+mammalian+cells+from+different+lineages.">Comparative transfection of DNA into primary and transformed mammalian cells from different lineages.</a> BMC Biotechnol. 10 (1), 9-18 (2010).</li><li>Thompson, C. D., Frazier-Jessen, M. R., Rawat, R., Nordan, R. P., Brown, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+methods+for+transient+transfection+of+a+murine+macrophage+cell+line+RAW+264.7.">Evaluation of methods for transient transfection of a murine macrophage cell line RAW 264.7.</a> Biotechniques. 27 (4), 824-835 (1999).</li><li>Kim, T., Eberwine, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cell+transfection:+the+present+and+the+future.">Mammalian cell transfection: the present and the future.</a> Analytical and Bioanalytical Chemistry. 397 (8), 3173-3178 (2010).</li><li>Stacey, K. J., Sweet, M. J., Hume, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophages+ingest+and+are+activated+by+bacterial+DNA.">Macrophages ingest and are activated by bacterial DNA.</a> Journal of immunology. 157 (5), 2116-2122 (1996).</li><li>Jiang, W., Reich, I. C., Pisetsky, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+activation+of+the+RAW264.7+macrophage+cell+line+by+transfected+mammalian+DNA.">Mechanisms of activation of the RAW264.7 macrophage cell line by transfected mammalian DNA.</a> Cell Immunol. 229 (1), 31-42 (2004).</li><li>Jiang, W., Pisetsky, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+induction+of+HMGB1+release+from+RAW+264.7+cells+by+transfected+DNA.">The induction of HMGB1 release from RAW 264.7 cells by transfected DNA.</a> Mol Immunol. 45 (7), 2038-2046 (2008).</li><li>Hargreaves, D. C., Horng, T., Medzhitov, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+inducible+gene+expression+by+signal-dependent+transcriptional+elongation.">Control of inducible gene expression by signal-dependent transcriptional elongation.</a> Cell. 138 (1), 129-147 (2009).</li><li>Cheung, S. T., So, E. Y., Chang, D., Ming-Lum, A., Mui, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interleukin-10+inhibits+lipopolysaccharide+induced+miR-155+precursor+stability+and+maturation.">Interleukin-10 inhibits lipopolysaccharide induced miR-155 precursor stability and maturation.</a> PLoS One. 8 (8), e71336 (2013).</li><li>Weber, M., Moller, K., Welzeck, M., Schorr, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short+technical+reports.+Effects+of+lipopolysaccharide+on+transfection+efficiency+in+eukaryotic+cells.">Short technical reports. Effects of lipopolysaccharide on transfection efficiency in eukaryotic cells.</a> Biotechniques. 19 (6), 930-940 (1995).</li><li>Berghaus, L. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+responses+of+primary+murine+macrophage-lineage+cells+and+RAW+264.7+cells+to+ligands+of+Toll-like+receptors.">Innate immune responses of primary murine macrophage-lineage cells and RAW 264.7 cells to ligands of Toll-like receptors.</a> Comp Immunol Microbiol Infect Dis. 33 (5), 443-456 (2000).</li><li>Fiszer-Kierzkowska, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liposome-based+DNA+carriers+may+induce+cellular+stress+response+and+change+gene+expression+pattern+in+transfected+cells.">Liposome-based DNA carriers may induce cellular stress response and change gene expression pattern in transfected cells.</a> BMC Mol Biol. 12 (1), 27-36 (2011).</li><li>Jacobsen, L., Calvin, S., Lobenhofer, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+effects+of+transfection:+the+potential+for+misinterpretation+of+gene+expression+data+generated+from+transiently+transfected+cells.">Transcriptional effects of transfection: the potential for misinterpretation of gene expression data generated from transiently transfected cells.</a> Biotechniques. 47 (7), 617-626 (2009).</li></ol>

저자가 공개하는 게 없다.

프로토콜은 전적으로 형질 감염 효율에 집중하지 않고 여기에서 설명하지만, 세포의 생리 학적 상태의 효율과 보존 사이의 균형을 유지하는 것을 목표로하고있다. 구체적으로, 우리의 방법은 형질 전환 시약의 독성을 최소화하고 루시페라아제 신호를 최대화하는데 성공. 프로토콜의 하나의 중요한 단계는 셀의 상태이다. 자란 배양 그들의 생리 변화하고, 또한 셀 (10)의<...

세포의 핵산의 형질 과학 연구에서 다양한 응용 프로그램을 가지고있다. 예로는 (1) 리포터 유전자, 유전자 발현의 다른 유전자 요소의 역할을 연구하기 위해 (2) 단백 발현 플라스미드 관심 단백질을 과발현, 및 (3) 작은 RNA 간섭하는 유전자 발현을 하향 조절하기 위해 포함된다. 특정 유전자의 발현 량을 조작하는 등의 조작 차동 효과를 측정함으로써, 연구자들은 선택된 생물학적 시스템에서 유전자 기능을 추론 할 수있다. 모든 형질 전환 방법은 동일한 형질 전환 효율을 제공하고, 심지어 같은 형질 전환 방법은 균등 한 모든 유형의 세포를 형질 감염되지 않는다. 따라서, 다른 형질 전환 방법은 인산 칼슘 법, DEAE 덱스 트란, 양이온 성 지질 형질 감염, 비 양이온 성 지질 - 폴리머 형질 감염, 전기 천공, 및 2,3- nucleofection 등이 개발되었다. 대식 세포로 형질은 ESPE입니다cially 어려운 의한 대 식세포 (메틸화) DNA (4) 파생 된 박테리아를 포함하여 이물질에 매우 민감 전문 식세포가 있다는 사실에. 외래 DNA의 도입은 사이토 카인과 산화 질소 (5, 6)의 생산을 유도하는 수신자 같은 수용체 9 (TLR9) 경로를 활성화합니다. 이 활성화 된 대 식세포는 연구자가 조사하고자하는 치료에 덜 반응 할 수있다. 우리 연구소는 정기적으로 루시퍼 라제 리포터 유전자와 RAW264.7 대식 세포주를 transfects, 우리는 배경보다 훨씬 높은 루시 페라 제 신호가 충분히 강력 프로토콜을 개발하지만, 대 식세포가 휴식 상태로 유지하기에 충분한 또한 부드러운했다. 형질 감염된 세포의 행동은 IκBζ (pGL3- IκBζ)의 프로모터 영역을 은닉 반딧불이 루시퍼 라제 - 리포터 유전자에 의해 평가 하였다. IκBζ 발현 박테리아 세포벽 성분의 립에 의해 상향 조절되고opolyssacharide (LPS) 7,8, 및 항 염증성 사이토 카인에 의해 하향 조절, 인터루킨 10 (IL-10) 8. 웰 중에서 형질의 변화를 고려하기 위해 정규화 상업적 레 닐라 루시퍼 라제 유전자 (예 phRL-TK)를 포함하는 제어 플라스미드 - 형질 공동. 설명 프로토콜은 DNA 플라스미드의 다양한 형질 타이밍, 형질 전환 시약의 종류, 형질 전환 시약의 플라스미드 DNA의 양을 포함한 파라미터뿐만 아니라 형질 전환 시약의 비율 테스트 후 최적화된다. 이 프로토콜에 포함 된 두 개의 형질 전환 시약 (1) 지질 계 형질 전환 시약 및 (2) 단백질 / 폴리아민 계 형질 감염 시약이다.

<table><tbody><tr><td>PureLink HiPure Plasmid Maxiprep Kit</td><td>Life Technologies</td><td>K210007</td><td>Any maxiprep kit will work</td></tr><tr><td>Phenol:chloroform:isoamyl alcohol</td><td>Life Technologies</td><td>15593-049</td><td>Molecular Biology Grade. Phenol is toxic so work in the fume hood, if possible. Use the lower clear organic layer if two layers of liquid form in the container.</td></tr><tr><td>DMEM</td><td>Thermo Scientific</td><td>SH30243.01</td><td>Warm in 37 &amp;deg;C water bath before use.</td></tr><tr><td>Fetal bovine serum</td><td>Thermo Scientific</td><td>SH30396.03</td><td>Inactivated at 56 &amp;deg;C water bath for 45 min before use.</td></tr><tr><td>Opti-MEM</td><td>Life Technologies</td><td>31985-070</td><td>Warm to at least room temperature before use.</td></tr><tr><td>XtremeGene HP DBA transfection reagent</td><td>Roche</td><td>6366236001</td><td>Warm to room temperature before use.</td></tr><tr><td>GeneJuice</td><td>EMD Millipore</td><td>70967</td><td>Warm to room temperature before use.</td></tr><tr><td>5x Passive Lysis Buffer</td><td>Promega</td><td>E1941</td><td>30 ml is included in the Dual Luciferase Reporter Assay System</td></tr><tr><td>Dual Luciferase Reporter Assay System</td><td>Promega</td><td>E1910</td><td></td></tr></tbody></table>

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.

그림 1은 RAW264.7에있는 두 개의 형질 전환 시약의 형질 전환 효율을 비교합니다. 단백질 / 폴리아민 계 형질 감염은 약 5 %의 효율 (도 1a)의 결과 동안 지질 계 시약은 통상적으로 약 25 %의 형질 도입 율을 주었다. 형질 감염 효율에 차이는 pGL3-IκBζ 프로모터 리포터 (도 1b)로 형질 RAW264.7 세포에서 루시페라아제 신호가 관찰되었다. 이러한 형질 감염된 세포에 LPS?...

Watch this Scientific Journal Video about Transfecting RAW264.7 Cells with a Luciferase Reporter Gene at JoVE.com

Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

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 ...

transfecting-raw2647-cells-with-a-luciferase-reporter-gene

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JoVE Journal

Biology

20.6K 조회수.  Vancouver Costal Health Research Institute. Transfection into the macrophage cell line, RAW264.7, is difficult due to the cell’s natural response against foreign materials. We described here a gentle yet robust procedure for transfecting luciferase reporter genes into RAW264.7 cells. 

비디오: Transfecting RAW264.7 Cells with a Luciferase Reporter Gene

In vivo Dual Substrate Bioluminescent Imaging

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate since the advent of in vivo bioluminescent imaging technologies 1-3. Indeed, the ability to locate and quantify tumor growth longitudinally in a single cohort of animals to completion of the study as opposed to sacrificing individual groups of animals at specific assay times has revolutionized how researchers investigate breast cancer metastasis. Unfortunately, current methodologies preclude the real-time assessment of critical changes that transpire in cell signaling systems as breast cancer cells (i) evolve within primary tumors, (ii) disseminate throughout the body, and (iii) reinitiate proliferative programs at sites of a metastatic lesion. However, recent advancements in bioluminescent imaging now make it possible to simultaneously quantify specific spatiotemporal changes in gene expression as a function of tumor development and metastatic progression via the use of dual substrate luminescence reactions. To do so, researchers take advantage for two light-producing luciferase enzymes isolated from the firefly (Photinus pyralis) and sea pansy (Renilla reniformis), both of which react to mutually exclusive substrates that previously facilitated their wide-spread use in in vitro cell-based reporter gene assays 4. Here we demonstrate the in vivo utility of these two enzymes such that one luminescence reaction specifically marks the size and location of a developing tumor, while the second luminescent reaction serves as a means to visualize the activation status of specific signaling systems during distinct stages of tumor and metastasis development. Thus, the objectives of this study are two-fold. First, we will describe the steps necessary to construct dual bioluminescent reporter cell lines, as well as those needed to facilitate their use in visualizing the spatiotemporal regulation of gene expression during specific steps of the metastatic cascade. Using the 4T1 model of breast cancer metastasis, we show that the in vivo activity of a synthetic Smad Binding Element (SBE) promoter was decreased dramatically in pulmonary metastasis as compared to that measured in the primary tumor 4-6. Recently, breast cancer metastasis was shown to be regulated by changes within the primary tumor microenvironment and reactive stroma, including those occurring in fibroblasts and infiltrating immune cells 7-9. Thus, our second objective will be to demonstrate the utility of dual bioluminescent techniques in monitoring the growth and localization of two unique cell populations harbored within a single animal during breast cancer growth and metastasis.

In vivo Dual Substrate Bioluminescent Imaging

Herein we describe the methods to construct, visualize, and quantify the bioluminescent reactions of both firefly and renilla luciferase enzymes expressed in metastatic breast cancer cells during their growth and metastasis in vivo.

생체 듀얼 기판 발광 생물 이미징에

Our understanding of how and when breast cancer cells transit from established primary tumors to metastatic sites has increased at an exceptional rate ...

연구

의학

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor (LATS) Biosensor

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical challenges, it remains difficult to assess the activity of this signaling pathway and interpret it within a biological context. The existing literature on large tumor suppressor (LATS) relies on methods that are qualitative and cannot easily be scaled-up for screening. Recently, we have developed a bioluminescence-based biosensor to monitor the kinase activity of LATS-a core component of the Hippo kinase cascade. Here, we describe procedures for how this LATS biosensor (LATS-BS) can be used to characterize Hippo pathway regulators. First, we provide a detailed protocol for investigating the effect of an overexpressed protein candidate (e.g., VEGFR2) on LATS activity using the LATS-BS. Then, we show how the LATS-BS can be used for a small-scale kinase inhibitor screen. This protocol can feasibly be scaled-up to perform larger screens, which undoubtedly will identify novel regulators of the Hippo pathway.

Monitoring Hippo Signaling Pathway Activity Using a Luciferase-based Large Tumor Suppressor LATS Biosensor

Here we present a luciferase-based biosensor to quantify the kinase activity of large tumor suppressor (LATS)-a central kinase in the Hippo signaling pathway. This biosensor has diverse applications in basic and translational research aimed at investigating Hippo pathway regulators in vitro and in vivo.

Hippo 신호 Luciferase 기반 큰 종양 억제기 (라트) 바이오 센서를 사용 하 여 경로 활동 모니터링

The Hippo signaling pathway is a conserved regulator of organ size and has important roles in the development and cancer biology. Due to technical ...

암 연구학

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

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 ...

생물학

Identification of Transcription Factor Regulators using Medium-Throughput Screening of Arrayed Libraries and a Dual-Luciferase-Based Reporter

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for anti-cancer therapies. However, directly targeting transcription factors is often difficult and can cause adverse side effects if the transcription factor is necessary in one or more adult tissues. Identifying upstream regulators that aberrantly activate transcription factors in cancer cells offers a more feasible alternative, particularly if these proteins are easy to drug. Here, we describe a protocol that can be used to combine arrayed medium-scale lentiviral libraries and a dual-luciferase-based transcriptional reporter assay to identify novel regulators of transcription factors in cancer cells. Our approach offers a quick, easy, and inexpensive way to test hundreds of genes in a single experiment. To demonstrate the use of this approach, we performed a screen of an arrayed lentiviral RNAi library containing several regulators of Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ), two transcriptional co-activators that are the downstream effectors of the Hippo pathway. However, this approach could be modified to screen for regulators of virtually any transcription factor or co-factor and could also be used to screen CRISPR/CAS9, cDNA, or ORF libraries.

To identify novel regulators of transcription factors, we developed an approach to screen arrayed lentiviral or retroviral RNAi libraries using a dual-luciferase-based transcriptional reporter assay. This approach offers a quick and relatively inexpensive way to screen hundreds of candidates in a single experiment.

배열된 라이브러리의 중간 처리량 스크리닝및 이중 루시퍼라제 기반 리포터를 이용한 전사 계수 조절기 식별

Transcription factors can alter the expression of numerous target genes that influence a variety of downstream processes making them good targets for ...

Monitoring Breast Cancer Growth and Metastatic Colony Formation in Mice using Bioluminescence

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several animal models have been generated, including the widely used orthotopic mouse models, where cancer cells are injected into the mammary fat pad. However, these models cannot help monitor tumor growth kinetics and metastatic colonization. Cutting-edge tools to monitor cancer cells in real time in mice will significantly advance the understanding of tumor biology. 
Here, breast cancer cell lines stably expressing luciferase and green fluorescent protein (GFP) were established. Specifically, this technique contains two sequential steps initiated by measuring the luciferase activity in vitro and followed by the implantation of the cancer cells into mammary fat pads of nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice. After the injection, both the tumor growth and metastatic colonization are monitored in real time by the noninvasive bioluminescence imaging system. Then, the quantification of GFP-expressing metastases in the lungs will be examined by fluorescence microscopy to validate the observed bioluminescence results. This sophisticated system combining luciferase and fluorescence-based detection tools evaluates cancer metastasis in vivo, which has great potential for use in breast cancer therapeutics and disease management.

Here, we describe a noninvasive monitoring method involving luciferase and green fluorescent protein expression in various breast cancer cell lines. This protocol provides a technique to monitor tumor formation and metastatic colonization in real time in mice.

생체 발광을 사용하여 마우스의 유방암 성장 및 전이성 콜로니 형성 모니터링

Breast cancer is a frequent heterogeneous malignancy and the second leading cause of mortality in women, mainly due to distant organ metastasis. Several ...

Bioluminescence Imaging of an Immunocompetent Animal Model for Glioblastoma

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and histopathologic features of the human disease. When GL261 cells are implanted intracranially in syngeneic C57BL/6 mice, the model has the added advantage of maintaining an intact immune microenvironment. Stable expression of luciferase in GL261 cells allows non-invasive cost effective bioluminescence monitoring of intracranial tumor growth. We have recently demonstrated that luciferase expression in GL261 cells does not affect the tumor growth properties, tumor cell immunomodulatory cytokine expression, infiltration of immune cells into the tumor, or overall survival of animals bearing the intracranial tumor. Therefore, it appears that the GL261 luciferase glioma model can be useful in the study of novel chemotherapeutic and immunotherapeutic modalities. Here we report the technique for generating stable luciferase expression in GL261 cells and how to study the in vitro and in vivo growth of the tumor cells by bioluminescence imaging.

GL261 glioma cells provide a useful immunocompetent animal model of glioblastoma. The goals of this protocol are to demonstrate proper techniques for monitoring intracranial tumor growth using in vivo bioluminescence imaging, and to verify the utility of luciferase-modified GL261 cells for studying tumor immunology and immunotherapeutic approaches for treating glioblastoma.

교 모세포종에 대한 면역 적격 동물 모델의 생물 발광 영상

In contrast to commonly reported human glioma xenograft animal models, GL261 murine glioma xenografts recapitulate nearly all relevant clinical and ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

HELA 세포 Transfection 얹는 Gaussia의 루시페라제의 mRNA의 시험 관내 스크립트 작성 및 상한에

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Monitoring Cell-autonomous Circadian Clock Rhythms of Gene Expression Using Luciferase Bioluminescence Reporters

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere1,2. Individual cells are the functional units for generation and maintenance of circadian rhythms3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous5-7. Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects5,8. Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms5,8-13.
Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection13,16,17 or stable transduction5,10,18,19. Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells20. Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems.

Circadian clocks function within individual cells, i.e., they are cell-autonomous. Here, we describe methods for generating cell-autonomous clock models using non-invasive, luciferase-based real-time bioluminescence technology. Reporter cells provide tractable, functional model systems for studying circadian biology.

루시 페라 제 Bioluminescence 기자를 사용하여 유전자 발현의 셀 자치 Circadian 시계 리듬을 모니터링

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

집에서 만든 듀얼 글로우 루시 페라 제 분석 실험을 사용하여 높은 처리량 기능 검사

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder (ALLIGATOR)

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In circadian rhythms research, for example, clock gene fusions with firefly luciferase give rise to robust rhythms in cellular bioluminescence that persist over many days. Technical limitations associated with photomultiplier tubes (PMT) or conventional microscopy-based methods for bioluminescence quantification have typically demanded that cells and tissues be maintained under quite non-physiological conditions during recording, with a trade-off between sensitivity and throughput. Here, we report a refinement of prior methods that allows long-term bioluminescence imaging with high sensitivity and throughput which supports a broad range of culture conditions, including variable gas and humidity control, and that accepts many different tissue culture plates and dishes. This automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) also allows the observation of spatial variations in luciferase expression across a cell monolayer or tissue, which cannot readily be observed by traditional methods. We highlight how the ALLIGATOR provides vastly increased flexibility for the detection of luciferase activity when compared with existing methods.

Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR

Genetically encoded luciferase is a popular non-invasive reporter of gene expression. Use of an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (ALLIGATOR) enables longitudinal recording from bioluminescent cells under a wide range of conditions. Here we show how ALLIGATOR may be used in the context of circadian rhythms research.