Name: flexible measurement of bioluminescent reporters using an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (alligator)
Uploaded: 2017-12-13T14:00:00.000+00:00
Duration: 10 min 33 s
Description: Watch this Scientific Journal Video about Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR at JoVE.

우리는 케 른 연구 특히 마크 헨 슨, 제레미 그레이엄과 호 코 레이 아에서에서이 시스템을 개발 하기 위해 우리와 함께 작업 하는 것을 감사 하 고 싶습니다. 우리 또한 감사 데이비드 웨일스어 및 Akhilesh 레디 (하 마)의 이전 피터 Laskey 뿐 아니라 설계 단계 동안 귀중 한 토론에 대 한 원고를 그의 중요 한 입력에 대 한 데모 카메라와 데이비드 웡의 대출을 정리.

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아니 이해 충돌 존재 한다.

여기에 설명 된 프로토콜 끼얹는다 고 정적 조건 모두 포유류 세포 배양입니다. 그러나, 악어 쉽게 다른 모델 시스템에 적용할 수 있습니다. 실제로, 그것은 이미 표시 되었습니다 동시 운동, 수 면, 그리고 주변 유전자 초파리 melanogaster 상수 어둠15. 유지에 식 리듬의 모니터링을 위한 뛰어난 플랫폼을 제공 하 그것은 또한 응용 프로그램에 따라 여기에 언급 된 그 외 카메라 유형을 적절 한 있을 수 있습니다, 지적 했다. 우리는 적절 한 필터와 함께 현재 설치의 수정된 된 버전 보안 주체에 사용 될 수 형광 정량화 정시.
악어 적절 한 않을 수 있습니다 유일한 응용 프로그램은 특히 높은 공간 해상도 필요할는 포유류의 organotypic 조각에 PER2::LUC 식의 spatiotemporal 조직의 이미징 suprachiasmatic 핵, 또는 다른 작은 조직 조각입니다.
악어는 지금 까지는 되지 않은 기존의 레코딩 기술에 의해 쉽게 달성 많은 실험을을 수행할 수 있습니다. 생물 발광의 측정을 위한 현재 방법과 비교해, 악어 셀 문화 접시 또는 사용할 수 있는 슬라이드, 외부 미디어 조건, 감도, 및 processivity 두 유형의 증가 유연성을 제공 합니다.
이것은 특히 적합 한 번에 3D organoid 문화 시스템이 향해 문화 모델은 표준 2D 셀에서 이동이 있을 때. 이와 같이, 악어는 생물 발광은 많은 일 및 광범위 한 조건 아래 주에 측정 될 수 있다 적응 방법을 제공할 것입니다 예상 된다.

유전자 발현 및 단백질 활동의 기자로 luciferases 사용 하 여 분자 및 세포 생물학 연구에서 인기 있는 기술 되고있다. 이것은 사실이 circadian 필드, 반딧불 luciferase 합성 및 촉매 비활성화의 속도 약 24 h circadian 주기 동안 발생 하는 유전자 발현의 경도 변화를 보고 하는 데 특히 적합. 이와 같이, luciferase 생물, 균 류, 식물, 파리, 그리고 포유류1,2,,34를 포함 하 여의 넓은 범위에 걸쳐 circadian 기자로 채택 된다.
Circadian 유전자 식에 체 외에서측정 될 때 광 전 증폭 관 관 (PMT) 발광 신호를 기록 하는 데 주로 사용 됩니다. 그러나 PMT 기반 측정 제한 유연성, 일반적으로 미리 정해진된 접시 또는 접시 크기에 제한 되 고. 그것은 불가능도 luciferase 식에서 공간 변화를 보여 주는 샘플을 이미징 하는 경우 정보의 손실에 지도할 수 있는 PMT를 사용 하 여 모니터링 샘플에서 공간 정보를 수집 합니다. 또한, PMT와 관련된 전자는 표준 셀 문화 인큐베이터의 습도 환경에 노출 되 면 오작동 하는 경향이, Pmt를 사용 하 여 경도 luciferase 녹음은 항상 비 습도 인큐베이터에서 수행 됩니다. 결과, 셀 문화 요리 공기 증발을 통해 수 분 손실을 방지 하기 위해 꽉 밀봉 해야 합니다 및 3-따라서 문화 미디어를 버퍼링 해야 합니다 (N-morpholino) propanesulfonic 산 (MOPS) 또는 4-(2-hydroxyethyl)-1- piperazineethanesulfonic 산 (HEPES), vivo 기능 그리고 포유류 조직 문화에서 일상적으로 사용 하는 시스템을 버퍼링 CO2/bicarbonate 보다는.
이러한 제한의 결과로 Pmt에 의해 생물 발광의 측정은 일반적으로 세포 실험 동안 유지 되는 조건에 꽉 제한을 장소. 이러한 문제를 극복 하 고 또한 가능한 실험 조건의 범위를 증가, 우리는 표준 공동2/N2 170 L 조직 문화 인큐베이터는 물 냉장 전자-멀티의 추가 의해 적응 되었습니다 사용 하 여 전 하 결합 소자 (EMCCD) 카메라 anti-mist 광학 및 디지털 제어 온도 가스 레벨의. 이 가스 이미징 Luciferase 자동 경도 및 Temperature-Optimized 레코더, 또는 악어 별명은. 악어 발광 이미징, 모두 표준 조직 배양 플레이트의 높은 처리량 이미징의 유연성을 크게 증가 수 있습니다 (동시에 최대 6 x 96-또는 384-잘 접시)와 또한 비표준 조직 문화 시스템에 대 한 이러한 끼얹는다 셀으로 미세 장치에서 성장. 이 악기는 또한 가변 제어 모두 CO2 와 O2 부분 압력으로 온도 습도 조건 하에서 발생 하는 화상 진 찰에 대 한 수 있습니다.
프로토콜 아래 포유류 세포와 악어 (이제부터 '생물 발광 인큐베이터' 라고도 함)를 사용 하 여 조직 문화 시스템의 발광 녹음 하는 방법을 설명 합니다. 그러나 그것은,, 시스템 발광 영상 하 고 또한, 일부 수정, 형광 이미징, 다른 생물 학적 시스템 및 컨텍스트 수에 적합 될 것 이라고 주목 한다.

<table><tbody><tr><td>DMEM (1x) + GlutaMAX</td><td>Gibco</td><td>31966-021</td><td></td></tr><tr><td>Hyclone FetalClone III Serum</td><td>GE Healthcare</td><td>SH30109.03</td><td></td></tr><tr><td>Neurobasal medium</td><td>Thermofisher</td><td>21103049</td><td>basal medium</td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma</td><td>A4919</td><td></td></tr><tr><td>Catalase</td><td>Sigma</td><td>C40</td><td></td></tr><tr><td>Glutathione</td><td>Sigma</td><td>G6013</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I1882</td><td></td></tr><tr><td>Superoxide Dismutase</td><td>Sigma</td><td>S5395</td><td></td></tr><tr><td>Holo-transferrin</td><td>Calbiochem</td><td>616424</td><td></td></tr><tr><td>T3 (triiodo-L-thyronine</td><td>Sigma</td><td>T6397</td><td></td></tr><tr><td>L-Carnitine</td><td>Sigma</td><td>C7518</td><td></td></tr><tr><td>Ethanolamine</td><td>Sigma</td><td>E9508</td><td></td></tr><tr><td>D (+)-Galactose</td><td>Sigma</td><td>G0625</td><td></td></tr><tr><td>Putrescine</td><td>Sigma</td><td>P5780</td><td></td></tr><tr><td>Sodium Selenite</td><td>Sigma</td><td>S9133</td><td></td></tr><tr><td>Corticosterone</td><td>Sigma</td><td>C2505</td><td></td></tr><tr><td>Linoleic Acid</td><td>Sigma</td><td>L1012</td><td></td></tr><tr><td>Linolenic Acid</td><td>Sigma</td><td>L2376</td><td></td></tr><tr><td>Lipoic Acid</td><td>Sigma</td><td>T1395</td><td></td></tr><tr><td>Progesterone</td><td>Sigma</td><td>P8783</td><td></td></tr><tr><td>Retinol Acetate</td><td>Sigma</td><td>R7882</td><td></td></tr><tr><td>Retinol, all trans</td><td>Sigma</td><td>95144</td><td></td></tr><tr><td>D,L-alpha-Tocopherol</td><td>Sigma</td><td>95240</td><td></td></tr><tr><td>D,L-alpha-Tocopherol acetate</td><td>Sigma</td><td>T3001</td><td></td></tr><tr><td>Sodium Bicarbonate Solution</td><td>Sigma</td><td>S8761-100ML</td><td></td></tr><tr><td>GlutaMAX (100x)</td><td>Gibco</td><td>35050-038</td><td></td></tr><tr><td>Penicillin-Streptomycin</td><td>Sigma</td><td>P4333</td><td></td></tr><tr><td>Galaxy 170R incubator&amp;nbsp;</td><td>Eppendorf</td><td>CO17301001</td><td></td></tr><tr><td>Luciferin</td><td>Biosynth</td><td>L-8220</td><td></td></tr><tr><td>D -(+)-Glucose solution</td><td>Sigma</td><td>G8644-100ML</td><td></td></tr><tr><td>DMEM powder</td><td>Sigma</td><td>D5030</td><td></td></tr><tr><td>MOPS</td><td>Sigma</td><td>PHG0007</td><td></td></tr><tr><td>1 mm I.D. silicone tubing&amp;nbsp;</td><td>GE Healthcare</td><td>19-4692-01</td><td></td></tr><tr><td>Elbow luer connector</td><td>Ibidi</td><td>10802</td><td></td></tr><tr><td>Male luer fittings</td><td>Ibidi</td><td>10826</td><td></td></tr><tr><td>Female luer fittings</td><td>Ibidi</td><td>10825</td><td></td></tr><tr><td>&amp;micro;-slide luer I 0.6</td><td>Ibidi</td><td>80196</td><td></td></tr><tr><td>BD plastipak 20ml syringe</td><td>Becton Dickinson</td><td>300613</td><td></td></tr><tr><td>1mm I.D. ETFE tubing</td><td>GE Healthcare</td><td>18-1142-38</td><td></td></tr><tr><td>PF670462</td><td>Sigma</td><td>SML0795</td><td></td></tr><tr><td>B27 Supplement (50x)</td><td>ThermoFisher</td><td>17504044</td><td></td></tr><tr><td>iXon Ultra EMCCD camera</td><td>Andor</td><td>iXon 888</td><td></td></tr><tr><td>Fiji</td><td>ImageJ</td><td>N/A</td><td></td></tr><tr><td>Prism 7.0</td><td>Graphpad Software</td><td>N/A</td><td></td></tr><tr><td>Trypan blue</td><td>Sigma</td><td>T8154</td><td></td></tr><tr><td>Deltaphase Isothermal Pad</td><td>Braintree Scientific</td><td>39DP</td><td></td></tr><tr><td>Heated neutral density filter&amp;nbsp;</td><td>Cairn Research</td><td>Custom item</td><td></td></tr><tr><td>Osmomat 030</td><td>Gonotech</td><td>Discontinued</td><td></td></tr><tr><td>300 mOsmol/kg calibration standard</td><td>Gonotech</td><td>30.9.0020</td><td></td></tr><tr><td>Measuring vessel</td><td>Gonotech</td><td>30.9.0010</td><td></td></tr><tr><td>Focusing cylinder</td><td>Cairn Research</td><td>Custom item</td><td></td></tr><tr><td>NE-1600 programmable syringe pump</td><td>Pump Systems inc.</td><td>NE-1600</td><td></td></tr><tr><td>Andor Solis Software</td><td>Andor</td><td>N/A</td><td></td></tr></tbody></table>

flexible measurement of bioluminescent reporters using an automated longitudinal luciferase imaging gas- and temperature-optimized recorder (alligator)

세포 유전자 발현의 기자 luciferase 기반 둘 다 경도 대 한 광범위 한 사용에 있고 끝점 생물 학적 활동의 분석 실험. Circadian 리듬 연구, 예를 들어 반딧불 luciferase와 시계 유전자 융해 일으키 강력한 리듬에 많은 일 동안 유지 세포 생물 발광. 광 전 증폭 관 관 (PMT)와 관련 된 기술 제한 또는 생물 발광 정량화에 대 한 기존의 현미경-기반 방법 일반적으로 세포와 조직 중 아주 비 생리 적 조건 하에서 유지 될 요구 했다 녹음, 감도 처리량 사이 교환. 여기, 우리 수 있도록 장기적인 생물 발광 영상 높은 감도와 처리량을 문화 조건, 습도 제어, 가변 가스 등의 광범위 한 범위를 지원 하 고 많은 종류를 원하는 다른 이전 방법의 상세 보고 조직 문화 접시 그리고 요리입니다. 또한 가스 온도 최적화 레코더 (악어) 이미징이 자동화 경도 luciferase 셀 단층 또는 전통에 의해 쉽게 관찰 될 수 없습니다. 있는 조직, luciferase 식 공간 변이의 관측 수 방법입니다. 우리는 악어 기존의 방법에 비해 luciferase 활동의 탐지에 대 한 대폭 증가 유연성을 제공 하는 방법을 강조 표시 합니다.

이 문서는 악어 (생물 발광 인큐베이터)를 사용 하 여 포유류 세포의 발광 영상에 대 한 프로토콜을 설명 합니다. 이 기술은 발광 시스템 이미징 extracellular 조건과 실제 설치의 유연성에 대 한 수 있습니다. 간단한 정적 조직 문화 (그림 1, 보조 비디오 1) 및 (그림 2, 보조 비디오 2) 끼얹는다 세포 배양 방법을 설명 하지만 다른 많은 설정을이 시스템을 사용 하 여 이미지 수 있습니다. 모든 데이터 섹션 7에 설명 된 방법을 사용 하 여 정량 했다.
그림 1A PER2::LUC fibroblasts4를 포함 하는 6 x 96 잘 접시에서 녹음 하는 생물 발광의 비디오는 예를 보여 줍니다. 이들은이 실험에 필요한으로 바깥쪽 웰 스 셀을 포함 하지 않습니다. 녹음에 대 한 지속적인 37 ° C에서 개최 되 고 전에 셀 차동 온도 유입, 그것에 의하여 그들은 32 ° C 12 h 72 h 또는 대화 (12 h, 12 h 72 h에 32 ° C에 뒤 37 ° C에서), 37 ° C에 의해 다음에 12 h의 어느 온도 사이클을 받게 받은 합니다. 60 분 노출 매 시간을 촬영 했다. 이러한 조건 중 두 개는 그림 1B에서 정량.
그림 2A 끼얹는다 조직 문화에 대 한 시스템의 설치의 회로도 보여준다. 배관으로 연결 하는 두 개의 채널 슬라이드 이것에 의하여 이루어져 있다. 미디어 셀에 걸쳐 주사기 펌프에 의해 구동 됩니다. 이 슬라이드의 첫 번째 가스 투과성 거품 트랩 (버퍼 슬라이드) 역할을 하며, 두 번째 셀 포함 생물 발광은 기록 하는 셀을 포함 하. 이 시스템에서 비디오를 녹화 하는 대표는 그림 2B에 표시 됩니다. 15 분 노출 매 15 분을 촬영 했다. 표준 관류 조건 또는 카 세 인 키 니 아 제 억제 물 PF670462, 이전 circadian 기간을 길게 하 고 경작에 시계 유전자 식 리듬의 진폭을 감소를 보여왔다 존재 셀의 유지 관리 하는 자, 포유류 세포14. PER2::LUC 식에 미치는 영향의 정량화와 그림 2C (하단 패널)에 표시 된 표준 정적 셀 문화 조건 하에서 약물의 동일한 농도로 처리 하는 셀에 대 한 그림 2C (상단 패널)에 표시 됩니다. 그림 2D에 나와 있습니다. 그것은 이것에서 PF670462 치료 조건의 두 세트에서 PER2::LUC 식 영향입니다. 그러나, 동안 셀 끼얹는다 조건 표시 기간 길어 약 3 h (3 ±0.9 h)에서 PF670462로 치료, 약물으로 치료 하는 정적 조건에서 셀 표시의 기간에 실질적으로 더 큰 기간 길이 &#62; 48 h. 이 섹션 7에 설명 된 대로 감쇠 코사인으로 들어갈 수 있는 (사각형의 여분 합계 F 대 직선 p 테스트 &#60; 0.0001). 흥미롭게도, 관류에서 기간 연장의 진도 훨씬 vivo에서14관찰에 가까운 이다.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56623/56623fig1v2.jpg"> 
그림 1: 데이터 예제. (A) 예를 들어 6 x 96에서 불멸 하 게 PER2::LUC에서 생물 발광의 비디오 스냅샷 잘 생물 발광 인큐베이터에 접시. 웰 스를 측정할 수 있다 보조 비디오 1에서 노란색으로 강조 되었다. A.에서 생물 발광의 부 량 (B) 두 셀의 3 판 서로 반대 PER2 단백질 식의 단계 생산을 반대로 phasic 했다 온도 사이클 (32 ° c h 12; 12 h, 37 ° C에서)를 사용 하 여 개입 했다 (n = 6, ± SEM을 의미). <a href="//ecsource.jove.com/files/ftp_upload/56623/56623fig1v2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56623/56623fig2v2.jpg"> 
그림 2: CK1δ 억제제와 관류. 관류 시스템의 (A) 회로도 (B) 예를 들어 PER2::LUC에서 기록 하는 생물 발광의 비디오 스냅샷 관류에서 세포. 정량화의 샘플 영역 노란색으로 강조 표시 됩니다. PER2::LUC 섬유 아 세포 관류에서 고 3 µ M CK1 억제제 PF670462 없이 정적 조건에서의 생물 발광의 (C) 정량화 (n = 3, ± SEM을 의미). 생물 발광은 최소 및 최대 값을 정규화 되었습니다. (D) 분석 기간 (양방향 ANOVA, Holm Sidak 여러 비교 테스트).<a href="//ecsource.jove.com/files/ftp_upload/56623/56623fig2v2large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>구성 요소</td>
			<td>최종 중간 농도 (μ M)</td>
			<td>주식 (mg/mL)</td>
			<td>400 mL NS21에 대 한</td>
		</tr>
		<tr>
			<td>소 민</td>
			<td>37</td>
			<td>-</td>
			<td>50 g</td>
		</tr>
		<tr>
			<td>카 탈 라 제</td>
			<td>0.01</td>
			<td>-</td>
			<td>50 mg</td>
		</tr>
		<tr>
			<td>티</td>
			<td>3.2</td>
			<td>-</td>
			<td>20 mg</td>
		</tr>
		<tr>
			<td>인슐린</td>
			<td>0.6</td>
			<td>10</td>
			<td>8 mL</td>
		</tr>
		<tr>
			<td>Superoxide dismutase</td>
			<td>0.077</td>
			<td>-</td>
			<td>50 mg</td>
		</tr>
		<tr>
			<td>홀로 처리가</td>
			<td>0.062</td>
			<td>-</td>
			<td>100 mg</td>
		</tr>
		<tr>
			<td>T3 (triiodo-L-thyronine)</td>
			<td>0.0026</td>
			<td>2</td>
			<td>20 Μ L</td>
		</tr>
		<tr>
			<td>L-카 르 니 틴</td>
			<td>12</td>
			<td>-</td>
			<td>40 mg</td>
		</tr>
		<tr>
			<td>방법</td>
			<td>16</td>
			<td>액체 (1 g/ml)</td>
			<td>20 Μ L</td>
		</tr>
		<tr>
			<td>D (+)-갈 락 토스</td>
			<td>83</td>
			<td>-</td>
			<td>300 밀리 그램</td>
		</tr>
		<tr>
			<td>Putrescine</td>
			<td>183</td>
			<td>-</td>
			<td>322 밀리 그램</td>
		</tr>
		<tr>
			<td>나트륨 Selenite</td>
			<td>0.083</td>
			<td>1</td>
			<td>280 Μ L</td>
		</tr>
		<tr>
			<td>Corticosterone</td>
			<td>0.058</td>
			<td>2</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>리 놀 레 산</td>
			<td>3.5</td>
			<td>100</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>리 놀 렌 산</td>
			<td>3.5</td>
			<td>100</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>리 포 산</td>
			<td>0.2</td>
			<td>4.7</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>황 체 호르몬</td>
			<td>0.02</td>
			<td>3.2</td>
			<td>0.04 mL</td>
		</tr>
		<tr>
			<td>레 티 놀 아세테이트</td>
			<td>0.2</td>
			<td>20</td>
			<td>0.1 mL</td>
		</tr>
		<tr>
			<td>Retinol, 모든 트랜스</td>
			<td>0.3</td>
			<td>10</td>
			<td>0.2 mL</td>
		</tr>
		<tr>
			<td>D, L-알파-토 코 페 롤</td>
			<td>2.3</td>
			<td>100</td>
			<td>0. 2 mL</td>
		</tr>
		<tr>
			<td>D, L-알파-토 코 페 롤 아세테이트</td>
			<td>2.1</td>
			<td>100</td>
			<td>0.2 mL</td>
		</tr>
	</tbody>
</table>
표 1: NS21 준비입니다.
보충 비디오 1: 예제 비디오 잘 플레이트에서 생물 발광의. 예를 들어 생물 발광 인큐베이터에서 6 x 96 잘 접시에서 불멸 하 게 PER2::LUC에서 생물 발광의 비디오 스냅샷. 웰 스 측정할 수를 노란색으로 강조 표시 됩니다. <a href="http://ecsource.jove.com/files/ftp_upload/56623/Video_1A_Revision-1.avi">이 파일을 다운로드 하려면 여기를 클릭 하십시오.</a>
보충 비디오 2: PER2::LUC에서 기록 하는 생물 발광의 예제 비디오 스냅샷 세포 관류에서. 정량화의 샘플 영역 노란색으로 강조 표시 됩니다. <a href="http://ecsource.jove.com/files/ftp_upload/56623/Video_2B_Revision-1.avi">이 파일을 다운로드 하려면 여기를 클릭 하십시오.</a>

Watch this Scientific Journal Video about Flexible Measurement of Bioluminescent Reporters Using an Automated Longitudinal Luciferase Imaging Gas- and Temperature-optimized Recorder ALLIGATOR at JoVE.

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

유전자 인코딩된 luciferase 유전자 발현의 인기 있는 비-침략 적 기자입니다. 자동된 경도 luciferase 가스 온도 최적화 레코더 (악어) 이미징의 사용에는 다양 한 조건에서 발광 세포에서 경도 녹음 수 있습니다. 여기 우리가 어떻게 악어 circadian 리듬 연구의 맥락에서 사용할 수 있습니다 보여줍니다.

자동된 경도 Luciferase 가스 온도 최적화 레코더 (악어) 이미지를 사용 하 여 발광 기자 들의 유연한 측정

Luciferase-based reporters of cellular gene expression are in widespread use for both longitudinal and end-point assays of biological activity. In ...

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Biology

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

7.8K 조회수.  MRC Laboratory of Molecular Biology. 유전자 인코딩된 luciferase 유전자 발현의 인기 있는 비-침략 적 기자입니다. 자동된 경도 luciferase 가스 온도 최적화 레코더 (악어) 이미징의 사용에는 다양 한 조건에서 발광 세포에서 경도 녹음 수 있습니다. 여기 우리가 어떻게 악어 circadian 리듬 연구의 맥락에서 사용할 수 있습니다 보여줍니다.

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

Establishing Intracranial Brain Tumor Xenografts With Subsequent Analysis of Tumor Growth and Response to Therapy using Bioluminescence Imaging

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most commonly used procedure for human tumor xenograft establishment consisted of the collection of cells from culture flasks, followed by the subcutaneous injection of the collected cells in immunocompromised mice. Whereas this approach still sees frequent use in many laboratories, there has been a significant shift in emphasis over the past decade towards orthotopic xenograft establishment, which, in the instance of brain tumors, requires tumor cell injection into appropriate neuroanatomical structures. Because intracranial xenograft establishment eliminates the ability to monitor tumor growth through direct measurement, such as by use of calipers, the shift in emphasis towards orthotopic brain tumor xenograft models has necessitated the utilization of non-invasive imaging for assessing tumor burden in host animals. Of the currently available imaging methods, bioluminescence monitoring is generally considered to offer the best combination of sensitivity, expediency, and cost. Here, we will demonstrate procedures for orthotopic brain tumor establishment, and for monitoring tumor growth and response to treatment when testing experimental therapies.

Luciferase-modified human brain tumor xenografts can be established intracranially in athymic mice, with subsequent monitoring of tumor growth and response to therapy using bioluminescence imaging. In combination with survival analysis, bioluminescence monitoring is an essential research tool for pre-clinical testing of therapies being considered for treating brain tumors.

Bioluminescence 이미징을 사용하여 종양 성장과 치료에 반응 이후 분석 Intracranial 뇌종양의 Xenografts 구축

Transplantation models using human brain tumor cells have served an essential function in neuro-oncology research for many years. In the past, the most ...

연구

신경과학

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

마우스의 이미지 세균 감염에 루시페라제를 사용하여

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

면역학 및 감염병학

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 ...

의학

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

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

발광 생물 세균 이미징<em&gt; 생체 내</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

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  ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

뮤린 신경 발생 시 노치 신호 역학의 실시간 생물 발광 이미징

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

발생학

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 ...

Autonomously Bioluminescent Mammalian Cells for Continuous and Real-time Monitoring of Cytotoxicity

Mammalian cell-based in vitro assays have been widely employed as alternatives to animal testing for toxicological studies but have been limited due to the high monetary and time costs of parallel sample preparation that are necessitated due to the destructive nature of firefly luciferase-based screening methods. This video describes the utilization of autonomously bioluminescent mammalian cells, which do not require the destructive addition of a luciferin substrate, as an inexpensive and facile method for monitoring the cytotoxic effects of a compound of interest. Mammalian cells stably expressing the full bacterial bioluminescence (luxCDABEfrp) gene cassette autonomously produce an optical signal that peaks at 490 nm without the addition of an expensive and possibly interfering luciferin substrate, excitation by an external energy source, or destruction of the sample that is traditionally performed during optical imaging procedures. This independence from external stimulation places the burden for maintaining the bioluminescent reaction solely on the cell, meaning that the resultant signal is only detected during active metabolism. This characteristic makes the lux-expressing cell line an excellent candidate for use as a biosentinel against cytotoxic effects because changes in bioluminescent production are indicative of adverse effects on cellular growth and metabolism. Similarly, the autonomous nature and lack of required sample destruction permits repeated imaging of the same sample in real-time throughout the period of toxicant exposure and can be performed across multiple samples using existing imaging equipment in an automated fashion.

Mammalian cells expressing the bacterial bioluminescence gene cassette (lux) produce light autonomously. The resulting bioluminescent dynamics upon chemical exposure have been demonstrated to reflect the treatment effects on cellular growth and metabolism, making these cells an inexpensive, continuous, real-time toxicity screening tool that can easily be adapted for high-throughput automation.

세포 독성의 지속적인 실시간 모니터링을위한 자율적으로 바이오 루미 네 센트 포유 동물 세포

Mammalian  cell-based in vitro assays have been  widely employed as alternatives to animal testing for toxicological studies but  have been limited due to ...

일주기 리듬 연구를 위한 인간 장 장내 생물 최적화

Monitoring Circadian Oscillations with a Bioluminescence Reporter in Organoids

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse repertoire of cellular and physiological processes ranging from sleep-wake cycles to metabolism. This clock mechanism entrains the organism based on environmental changes and coordinates the temporal regulation of molecular and physiological events. Previously, it was demonstrated that autonomous circadian rhythms are maintained even at the single-cell level using cell lines such as NIH3T3 fibroblasts, which were instrumental in uncovering the mechanisms of circadian rhythms. However, these cell lines are homogeneous cultures lacking multicellularity and robust intercellular communications. In the past decade, extensive work has been performed on the development, characterization, and application of 3D organoids, which are in vitro multicellular systems that resemble in vivo morphological structures and functions. This paper describes a protocol for detecting circadian rhythms using a bioluminescent reporter in human intestinal enteroids, which enables the investigation of circadian rhythms in multicellular systems in vitro.

저자 스포트라이트: In vitro Investigations of Circadian Rhythms in Multicellular Systems(다세포 시스템의 일주기 리듬에 대한 시험관 내 조사)

Circadian rhythms, which exist in most organisms, regulate the temporal organization of biological processes. 3D organoids have recently emerged as a physiologically relevant in vitro model. This protocol describes the use of bioluminescent reporters to observe circadian rhythms in organoids, enabling in vitro investigations of circadian rhythms in multicellular systems.

Organoids에서 Bioluminescence Reporter를 사용한 Circadian Oscillations 모니터링

Most living organisms possess circadian rhythms, which are biological processes that occur within a period of approximately 24 h and regulate a diverse ...