팀 도일, 박사 및 bioluminescence 이미징과 지원 VIVO 이미징에 대한 스탠포드 센터에 감사드립니다. 또한 매트릭스 솔루션과 줄기 세포 공동 사출 그녀의 기술을 공유 Ngan 황, 박사 덕분에. 마지막으로, 스티브 덕분에 박사에게 펠트 동물 동물 보호와 지원.

<ol><li> 더블 퓨전 리포터 유전자의 건설 <ol><li> 인간 배아 줄기 세포의 bioluminescence 이미징을 수행하기 위해서는 먼저 안정 등 Ubiquitin이나 EF1a 같은 제정 발기인에 의한 반딧불 루시페라제로 루시페라제 리포터 유전자를 표현 세포를 얻기 위해 필요합니다. </li><li> 이 프로토콜의 초점은 기자의 유전자 응용 프로그램에, 자세한 절차를 여기에 제공되지 않습니다 때문에. 그러나, 우리 실험실의 일반적인 전략은 반딧불 루시페라제 (fluc)와 pCDNA 3.1 이내 스페이서 +로 구분하여 향상된 녹색 형광 단백질 (egfp)를 포함하는 이중 융합 구조 벡터를 사용하는 것입니다. </li><li> 간단히 우리 두 융합 유전자가 원래 pCDNA 3.1 사이토 메갈로 바이러스 프로 모터 (+)의 하류에 위치하고 있었기 때문에 3.3 kbp의 단편은 자체 운영을 중지시키지 (SIN의 여러 복제 사이트에 출혈도 잡았 NdeI와 노티 소화하고 무딘 엔드를 사용하여 excised되었습니다 Ubiquitin 모터의 제어하에) lentiviral 벡터. </li></ol></li><li> Lentiviral 형질 도입 <ol><li> hESCs 10의 감염의 다중성 (뫄) (106 전지 incubated 약 107의 바이러스 titer)의 통과 후 3~5일을 transduced 수 있습니다. </li><li> 해동 바이러스 주식은 신선한 hESC 미디어에 직접 추가할 수 있습니다. </li><li> 나중에 미디어 12 24 시간을 새로 고칩니다. </li><li> 48 H 후, 전달 효율에 의해 질적으로 형광 현미경을 사용하여 평가하실 수 있습니다. 그 후, 외과는 감염된 세포를 분리하는 데 사용할 수 있습니다. </li></ol></li><li> Culturing의 hESCs 및 설정 이미징 소개 <ol><li> 문화 귀하의 루시페라제 긍정적인 hESCs를 피더없는 조건입니다. 우리는 일반적으로 표준 WiCell 프로토콜에 따라 에어컨 미디어 또는 상업적 중 하나를 사용하여 Matrigel 감소 성장 인자에 코팅 6 자 접시, 피더없는 미디어를 사용합니다. </li><li> 루시페라제 긍정적인 세포를 감지하기 위해서는 기자 프로브 D - luciferin 이미 준비하셔야합니다. 45 MG / ML의 최종 농도에서 1-1.5 ML 준비에서, 나누어지는의 luciferin을 수행하려면. -20 ° C에서 aliquots을 유지하지 않을 때 종이 타월하지 않을 때 저장소에 함께 걸쳐 사용하고 조명에 노출을 피하 인치 </li><li> 루시페라제 긍정적인 세포를 시각화하기 위해, 우리는 IVIS 50 IVIS 200 이미징 시스템 (Xenogen 공사, 알라 메다, CA)을 사용합니다. 이 후자의 시스템은 작은 동물을 일시적으로 마취에 대한 통합 isoflourane 장치와 유도 챔버를 포함합니다. </li></ol></li><li> hESCs의 시험 관내 bioluminescence 이미징에 <ol><li> 시험 관내 bioluminescence 이미징의 경우, 그것은 멸균 상태를 유지하는 것이 중요합니다. 따라서, 영상 시스템은 선호 세포 배양 룸에서 멸균한다. </li><li> 이미징 전에 세포 미디어를 제거하고 세포를 충당하기 위해 충분 PBS를 추가합니다. 예를 들어, hESC 문화를 포함하는 6 자 판의 각 잘하는 1mL PBS를 추가합니다. </li><li> PBS로 D - Luciferin의 비율은 1:100해야하므로 6 잘 플레이트의 각 잘하는 해동 D - Luciferin 10 μL를 추가합니다. </li><li> 잠시만 기다려 다음 일초의 노출 시간을 사용하여 이미지를 가져가라. 신호가 약한 경우, 노출 간격을 증가하고 다시 시도하십시오. </li><li> 발광 생물 신호 휴대폰 번호를 반영, quantitation assays의 서로 다른 세포 숫자와 신호를 상관 관계 것을 수행할 수 있도록. </li></ol></li><li> 생체내 이미징에 대한 마우스 및 주입 세포의 준비 <ol><li> 리포터 유전자 영상은 일, 주, 심지어 몇 달 동안 이식 후 추적 세포에 대한 높은 처리량, 저렴하고 민감한 방법을 제공합니다. 이식 줄기 세포가 자주, 기형종 형성 숙주의 면역 체계에 의해 거부, 또는 단순히 죽고 때문에 이것은 특히 중요합니다. </li><li> 생체내 이미징에서 기형 종 형성을위한 가장 간단한 방법은 SCID 생쥐의 백업에 반딧불 루시페라제 리포터 유전자를 표현하고 IVIS 시스템 발광 생물 이미지를 획득 hESCs를 삽입하는 것입니다. </li><li> 당신이 세포를 이식하기 위해 준비가되면, 세포를 풀어 여러 번 씻어하고, 성장 인자 감소 - matrigel과 DMEM의 1:1 혼합물에서 그들을 정지 dispase 또는 collagenase를 사용합니다. 일반적으로 우리가 먼저 차게 DME​​M으로 세포를 섞어 다음 matrigel에 추가합니다. 각각의 피하 주입 사이트에, 우리는 matrigel / DMEM 혼합물의 50-100 UL 볼륨에서 정지 200,000 개 셀을 주입. 휴대폰 번호는 응용 프로그램에 따라 조정할 수 있습니다. 사전 주사로 얼음에있는 모든 유지되도록주의를 기울이십시오. </li><li> 세포가 준비가되면,이 켜져 isoflurane 흐름 유도 상자에 배치하여 마우스를 마취. 1-5분 후, 마우스 잠이이고 지속적인 isoflurane으로 수술대에 둘 수 있습니다. </li><li> 모피 자연스럽게 자동 fluoresces하고는 발광 생물 이미지를 모호한 수 있기 때문에, 우리는 마우스의 뒷면에서 머리를 제거해야합니다. 이 작업을 수행하는 가장 쉬운 방법은 전기 면도기를 사용하는 것입니다,하지만 당신은 머리 - 제거 젤을 사용할 수 있습니다. 소독에 알코올로 닦아나중에 피부 </li><li> 다음 주사기에 세포 현탁액을 그립니다. 그들은 세포와 방해할 않기 때문에 23-27 게이지 바늘은 최고의 작동합니다. 바늘이 (&lt;23 게이지) 너무 큰 경우 바늘의 넓이 세포가 다시 밖으로 누출 수있는을 통해 큰 구멍을 떠날 수 있습니다. </li><li> 마우스의 뒷면에 피부 아래에있는 세포를 주사. 피부가 매우 느슨한 때문에, 핀치에 엄지와 집게손가락을 사용하고 삽입하고자하는 영역을 스트레칭. 너무 깊이 주사로 치료하지 복용, 피부 아래에 주사를 막. 또한, 플런저를 우울 동안 사출 사이트의 밖으로 미끄러져에서 바늘을 유지하려고이 다시 주입하려고하면 세포가 누출 수있는을 통해 피부에 구멍을 만드는 것을 방지할 수 있습니다. </li><li> 세포 주입 후, 마우스 깨어있게하고 bioluminescence 이미징을위한 다시 anesthetizing 전에 주위를 실행하는 데 몇 시간을 기다립니다. 이렇게하면 isoflurane 독성을 방지합니다. </li></ol></li><li> 이식 세포의 생체내 bioluminescence 이미징에 <ol><li> 전체 동물 bioluminescence 이미징을 위해, 우리는 375 밀리그램 / kg 체중에서 D - luciferin의 intraperitoneal 주사를 않습니다. 사전 주사로 주사 량을 계산하기 위해 동물을 나가는거야. </li><li> 그냥 정중선에서, 복막에있는 D - luciferin을 주사. 너무 깊이 가지 않은 또는 내부 장기를 손상시킬 수주의하십시오. 마우스 깨어하기 시작하면, 녹아웃 상자에 다시 배치하고 몇 분만 기다립니다. 생쥐의 경우 최대 bioluminescence은 일반적으로 주사 후 15-40 분을 발생합니다. </li><li> hESCs가 배출되지 않은 빛을 흡수에 도움이되는 이미징 상자에 무광택 검은 종이를 삽입해야합니다. 검은 종이 위에, isoflurane과 이미징 챔버에서 마우스 (후면까지)을 놓으십시오. 10초의 노출 시간과 함께 시작합니다. 신호가 포화되면, 노출 시간을 감소 노력, 너무 약한 경우, 노출을 증가시킵니다. </li><li> 신호 노출 시간이 마우스에 최적화된되면, 신호가 최대에 도달할 때까지 이미지를 매 분 복용 시작합니다. 이것은 가장 사출 사이트를 커버 &quot;관심 영역 (ROI)&quot;을 선택 이미징 소프트웨어를 사용하여 이루어집니다. 각각의 투자 수익 (ROI)에서 신호 강도를 모니터링함으로써 쉽게 최대 신호 강도에 도달되었음을 나타내는 신호가 감소하기 시작하면 확인할 수 있습니다. 최대 신호는 최종 데이터로 사용해야합니다. </li><li> 당신이 이미지에 만족하는 경우, isoflurane에서 마우스를 제거하고는 새장에 깨어 수 있습니다. 보통 마우스는 15 분 이내에 깨어날 것입니다. </li><li> hESCs이 동물의 생존과 같은 Bioluminescence 기자의 유전자 이미징이있는 동안, 매일 주별 및 월별 반복 수 있습니다. hESCs가 세포 분열 따위에 의해 번식을 시작으로 발전 기형 종에서 신호는 일반적으로 주의 순서에, 시간이 지남에 따라 기하 급수적으로 증가합니다. </li><li> bioluminescence 이미지의 원하는 시간 코스 취득 후, 동물 희생 및 조직 섹션 조직학에 사용할 수 있습니다. </li></ol></li></ol>

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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+human+embryonic+stem+cells:+Small+and+macromolecule+loading+and+DNA+transfection.">Electroporation of human embryonic stem cells: Small and macromolecule loading and DNA transfection.</a> Biotechnol Prog. 22, 825-834 (2006).</li><li>Siemen, H., 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=Nucleofection+of+human+embryonic+stem+cells.">Nucleofection of human embryonic stem cells.</a> Stem Cells Dev. 14, 378-383 (2005).</li><li>Lakshmipathy, U., 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=Efficient+transfection+of+embryonic+and+adult+stem+cells.">Efficient transfection of embryonic and adult stem cells.</a> Stem Cells. 22, 531-543 (2004).</li><li>Ma, Y., Ramezani, A., Lewis, R., Hawley, R. G., Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Level+Sustained+Transgene+Expression+in+Human+Embryonic+Stem+Cells+Using+Lentiviral+Vectors.">High-Level Sustained Transgene Expression in Human Embryonic Stem Cells Using Lentiviral Vectors.</a> Stem Cells. 21, 111-117 (2003).</li><li>Menendez, P., Wang, L., Chadwick, K., Li, L., Bhatia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+transduction+of+hematopoietic+cells+differentiated+from+human+embryonic+stem+cell-derived+CD45(neg)PFV+hemogenic+precursors.">Retroviral transduction of hematopoietic cells differentiated from human embryonic stem cell-derived CD45(neg)PFV hemogenic precursors.</a> Molecular therapy. 10, 1109-1120 (2004).</li><li>Thyagarajan, B., 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=Creation+of+engineered+human+embryonic+stem+cell+lines+using+phiC31+integrase.">Creation of engineered human embryonic stem cell lines using phiC31 integrase.</a> Stem cells. 26, 119-126 (2008).</li><li>Liew, C. -. G., Draper, J. S., Walsh, J., Moore, H., Andrews, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Transient and Stable Transgene Expression in Human Embryonic Stem Cell. , .</li></ol>

이러한 PET와 MRI와 같은 다른 modalities 비교, bioluminescence 제한된 공간 해상도와 방출되는 광자의 상대적으로 약한 에너지 (2-3 EV)으로 인해 감소 조직 침투력을 가지고, 이러한 이유로 그것은 지금까지 큰 동물에서 적용되지 않았습니다. 그러나, bioluminescence 작은 동물의 생체내 추적의 줄기 세포에 대해 그것이 매우 바람직 만들기, 저렴한 비용, 높은 처리량, 그리고 비침습되는 장점이 있습니다. 이러한 PET 및 형광 ...

<table><tbody><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium (DMEM)</td><td></td><td>Hyclone</td><td></td><td></td></tr><tr><td>BD Matrigel&amp;#8482; Basement Membrane Matrix</td><td>Growth factor reduced (optional: phenol-red free)</td><td>BD Biosciences</td><td></td><td></td></tr><tr><td>mTeSR1 Maintenance Medium for Human Embryonic Stem Cells</td><td></td><td>Stem Cell Technologies</td><td></td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td></td><td></td><td></td><td></td></tr><tr><td>D-Luciferin Firefly, potassium salt</td><td></td><td>Biosynth International, Inc</td><td></td><td></td></tr><tr><td>Collagenase IV solution</td><td></td><td></td><td></td><td>Dissolve 30 mg Collagenase Type IV in 30 mL DMEM-F12 media. Sterile filter and store at 4 degrees (Celsius).</td></tr><tr><td>Baked Pasteur pipets</td><td></td><td></td><td></td><td></td></tr><tr><td>6-well tissue culture-treated plates</td><td></td><td>Techno Plastic Products</td><td>92006</td><td></td></tr></tbody></table>

in vitro and in vivo bioluminescence reporter gene imaging of human embryonic stem cells

인간 배아 줄기 세포 (hESCs)의 발견은 극적으로 재생 의료에 관심이 의료 과학자에 사용할 수있는 도구를 증가했습니다. 그러나, 살아있는 생물로, hESCs에서 차별 hESCs, 그리고 세포의 직접 분사는 지금까지 상당한 세포 사망, 기형 종의 형성, 및 호스트 면역 거부 반응에 의해 방해되었다. 이식 후 생체내 hESC 행동에을 이해하는 것은 새로운 이미징 기술은 길이 방향 hESC 현지화, 증식 및 생존을 모니터링해야합니다. 분자 영상은 조사에게 생체내 세포 일 동안 확산, 주, 심지어 개월 이내에 추적에 대한 높은 처리량, 저렴하고, 중요한 수단을 제공하고 있습니다. 이 발전은 크게 hESC의 engraftment, 확산, 생활 과목의 기형 종 - 형성 spatio - 시간적 동력학에 대한 이해를 증가하고 있습니다. 분자 이미징의 주요 사전은 생체내에서 다중 양상 이미징 플랫폼으로 분자 및 세포 생물학에서 비침 투 리포터 유전자의 assays의 확장되었습니다. 이 기자의 유전자는 숙주 세포의 transcriptional 기계를 활용 설계 발기인 및 강화의 통제하에, 벡터가 아닌 벡터 다양한 방법을 사용하여 세포에 도입하고 있습니다. 일단 세포에서 기자의 유전자를 사용 모터의 종류에 따라, 특정 생물 학적 또는 세포 조건 중 constitutively 또는 유일한 베꼈는데 수 있습니다. bioactive 단백질로 전사와 기자의 유전자의 번역은 다음 D - luciferin 같은 신호 생성 프로브를 사용하여 민감한, 비침 투 계측 (예 : CCD 카메라)와 검색입니다. 로 형광 이미징에 필요한 생체내에서 줄기 세포를 추적하는 흥분성의 조명의 필요성을 방지하기 위해, bioluminescence의 리포터 유전자 이미징 시스템은 발광을 유발에만 exogenously 관리 프로브가 필요합니다. 반딧불 Photinus pyralis에서 파생된 반딧불 루시페라제은, 광학 활성 대사, oxyluciferin에 D - luciferin을 catalyzes 효소를 인코딩합니다. 광학 활동 후 외부 CCD 카메라로 모니터링할 수 있습니다. 안정 기자 들고 세포들은 염색체 DNA 내에 구축 transduced 기자가 생체내에 hESC의 생존과 확산의 길이 모니터링있게 딸 세포에 DNA를 구성 전달합니다. 리포터 유전자 제품의 표현은 신호 생성을 위해 필요하기 때문에 또한, 오직 실용적 부모와 딸 세포는 bioluminescence 신호를 만들 것이다; apoptotic이나 죽은 세포가되지 않습니다. 이 동영상에서 특정 자료 및 bioluminescence 이미징과 함께 추적 줄기 세포 증식 및 기형 종의 형성에 필요한 방법은 설명한다.

Watch this Scientific Journal Video about In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells at JoVE.com

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

줄기 세포 요법에 대한 관심이 점점으로, 분자 이미징 기술은 이식 후 모니터링 줄기 세포 행동에 이상적입니다. 루시페라제 리포터 유전자는 세포 생존, 위치 및 생체내의 확산의 비침습, 반복적인 평가를 활성화합니다. 이 동영상은 살아있는 마우스의 hESC 확산을 추적하는 방법을 보여줍니다 것입니다.

체외에서 인간 배아 줄기 세포의 생체내의 Bioluminescence 리포터 진 영상에

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

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Research

JoVE Journal

Biology

21.7K 조회수.  Stanford University School of Medicine. 줄기 세포 요법에 대한 관심이 점점으로, 분자 이미징 기술은 이식 후 모니터링 줄기 세포 행동에 이상적입니다. 루시페라제 리포터 유전자는 세포 생존, 위치 및 생체내의 확산의 비침습, 반복적인 평가를 활성화합니다. 이 동영상은 살아있는 마우스의 hESC 확산을 추적하는 방법을 보여줍니다 것입니다.

비디오: In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

개발, 확대,<em&gt; 생체 내</em&gt; 인간 배아 줄기 세포 (hESCs에)에서 인간 NK 세포의 모니터링 및 및 유도 된 pluripotent 줄기 세포 (유도 만능)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

연구

생체공학

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

키메라 Zebrafish의 태아의 개별 망막 전구 세포의 형질 전환 유전자 발현의 생체 내 이미징에서 세포 비 자립적 영향을 연구하기 위해

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

발생학

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 ...

면역학 및 감염병학

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. The observation of Tribolium embryos with light sheet-based fluorescence microscopy has multiple advantages over conventional widefield and confocal fluorescence microscopy. Due to the unique properties of a light sheet-based microscope, three dimensional images of living specimens can be recorded with high signal-to-noise ratios and significantly reduced photo-bleaching as well as photo-toxicity along multiple directions over periods that last several days. With more than four years of methodological development and a continuous increase of data, the time seems appropriate to establish standard operating procedures for the usage of light sheet technology in the Tribolium community as well as in the insect community at large. This protocol describes three mounting techniques suitable for different purposes, presents two novel custom-made transgenic Tribolium lines appropriate for long-term live imaging, suggests five fluorescent dyes to label intracellular structures of fixed embryos and provides information on data post-processing for the timely evaluation of the recorded data. Representative results concentrate on long-term live imaging, optical sectioning and the observation of the same embryo along multiple directions. The respective datasets are provided as a downloadable resource. Finally, the protocol discusses quality controls for live imaging assays, current limitations and the applicability of the outlined procedures to other insect species.
This protocol is primarily intended for developmental biologists who seek imaging solutions that outperform standard laboratory equipment. It promotes the continuous attempt to close the gap between the technically orientated laboratories/communities, which develop and refine microscopy methodologically, and the life science laboratories/communities, which require 'plug-and-play' solutions to technical challenges. Furthermore, it supports an axiomatic approach that moves the biological questions into the center of attention.

Light Sheet-based Fluorescence Microscopy of Living or Fixed and Stained Tribolium castaneum Embryos

Imaging the morphogenesis of insect embryos with light sheet-based fluorescence microscopy has become state of the art. This protocol outlines and compares three mounting techniques appropriate for Tribolium castaneum embryos, introduces two novel custom-made transgenic lines well suited for live imaging, discusses essential quality controls and indicates current experimental limitations.

라이트 시트 기반의 형광 생활의 현미경 또는 고정 및 스테인드<em&gt; Tribolium castaneum</em&gt; 배아

The red flour beetle Tribolium castaneum has become an important insect model organism in developmental genetics and evolutionary developmental biology. ...

Multimodal Imaging of Stem Cell Implantation in the Central Nervous System of Mice

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, both in preclinical and clinical studies. However, to date results regarding functional outcome and/or tissue regeneration following stem cell transplantation are quite diverse. Generally, a clinical benefit is observed without profound understanding of the underlying mechanism(s)1. Therefore, multiple efforts have led to the development of different molecular imaging modalities to monitor stem cell grafting with the ultimate aim to accurately evaluate survival, fate and physiology of grafted stem cells and/or their micro-environment. Changes observed in one or more parameters determined by molecular imaging might be related to the observed clinical effect. In this context, our studies focus on the combined use of bioluminescence imaging (BLI), magnetic resonance imaging (MRI) and histological analysis to evaluate stem cell grafting.
BLI is commonly used to non-invasively perform cell tracking and monitor cell survival in time following transplantation2-7, based on a biochemical reaction where cells expressing the Luciferase-reporter gene are able to emit light following interaction with its substrate (e.g. D-luciferin)8, 9. MRI on the other hand is a non-invasive technique which is clinically applicable10 and can be used to precisely locate cellular grafts with very high resolution11-15, although its sensitivity highly depends on the contrast generated after cell labeling with an MRI contrast agent. Finally, post-mortem histological analysis is the method of choice to validate research results obtained with non-invasive techniques with highest resolution and sensitivity. Moreover end-point histological analysis allows us to perform detailed phenotypic analysis of grafted cells and/or the surrounding tissue, based on the use of fluorescent reporter proteins and/or direct cell labeling with specific antibodies.
In summary, we here visually demonstrate the complementarities of BLI, MRI and histology to unravel different stem cell- and/or environment-associated characteristics following stem cell grafting in the CNS of mice. As an example, bone marrow-derived stromal cells, genetically engineered to express the enhanced Green Fluorescent Protein (eGFP) and firefly Luciferase (fLuc), and labeled with blue fluorescent micron-sized iron oxide particles (MPIOs), will be grafted in the CNS of immune-competent mice and outcome will be monitored by BLI, MRI and histology (Figure 1).

This article describes an optimized sequence of events for multimodal imaging of cellular grafts in rodent brain using: (i) in vivo bioluminescence and magnetic resonance imaging, and (ii) post mortem histological analysis. Combining these imaging modalities on a single animal allows cellular graft evaluation with high resolution, sensitivity and specificity.

마우스의 중추 신경계에서 줄기 세포 주입의 Multimodal 이미징

During the past decade, stem cell transplantation has gained increasing interest as primary or secondary therapeutic modality for a variety of diseases, ...

신경과학

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate light-gated ion channels and pumps in neurons. While this bioluminescent optogenetics (BL-OG) approach confers a chemogenetic component to optogenetic tools, it is not limited to use in neuroscience. Rather, bioluminescence can be harnessed to activate any photosensory protein, thus enabling the manipulation of a multitude of light-mediated functions in cells. A variety of luciferase-luciferin pairs can be matched with photosensory proteins requiring different wavelengths of light and light intensities.
Depending on the specific application, efficient light delivery can be achieved by using luciferase-photoreceptor fusion proteins or by simple co-transfection. Photosensory proteins based on light-dependent dimerization or conformational changes can be driven by bioluminescence to effect cellular processes from protein localization, regulation of intracellular signaling pathways to transcription. The protocol below details the experimental execution of bioluminescence activation in cells and organisms and describes the results using bioluminescence-driven recombinases and transcription factors. The protocol provides investigators with the basic procedures for carrying out bioluminescent optogenetics in vitro and in vivo. The described approaches can be further extended and individualized to a multitude of different experimental paradigms.

Bioluminescent Optogenetics 2.0: Harnessing Bioluminescence to Activate Photosensory Proteins In Vitro and In Vivo

Bioluminescence-light emitted by a luciferase enzyme oxidizing a small-molecule substrate, a luciferin-can be harnessed to activate photosensory proteins, thereby adding another dimension to light stimulation and enabling the manipulation of a multitude of light-mediated functions in cells across temporal and spatial scales.

생체 발광 광유전학 2.0 : 생체 발광을 활용하여 체외 및 생체 내에서 광감각 단백질을 활성화 합니다.

Bioluminescence - light emitted by a luciferase enzyme oxidizing a small molecule substrate, a luciferin - has been used in vitro and in vivo to activate ...

생화학

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 ...

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

마우스 배아 줄기 세포 유지 보수 및 자체 갱신을 조절 유전자를 공부하는 Oct4GiP 리포터 분석

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

생물학

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 ...

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point measurements and can therefore only evaluate longitudinal changes of cell migration and survival to a limited extent. This paper provides a reliable, minimally invasive protocol to transplant and longitudinally track neural progenitor cells (NPCs) in the adult mouse brain. Before transplantation, cells are transduced with a lentiviral vector comprising a bioluminescent (firefly-luciferase) and fluorescent (green fluorescent protein [GFP]) reporter. The NPCs are transplanted into the right cortical hemisphere using stereotaxic injections in the sensorimotor cortex. Following transplantation, grafted cells were detected through the intact skull for up to five weeks (at days 0, 3, 14, 21, 35) with a resolution limit of 6,000 cells using in vivo bioluminescence imaging. Subsequently, the transplanted cells are identified in histological brain sections and further characterized with immunofluorescence. Thus, this protocol provides a valuable tool to transplant, track, quantify, and characterize cells in the mouse brain.

Intracerebral Transplantation and In Vivo Bioluminescence Tracking of Human Neural Progenitor Cells in the Mouse Brain

We describe the intraparenchymal transplantation of human neural progenitor cells transduced with a dual reporter vector expressing luciferase-green fluorescent protein (GFP) in the mouse brain. After transplantation, the luciferase signal is repeatedly measured using in vivo bioluminescence and GFP-expressing grafted cells identified in brain sections using fluorescence microscopy.

Intracerebral 이식 및 마우스 뇌에서 인간 신경 전구 세포의 생체 내 생체 발광 추적

Cell therapy has long been an emerging treatment paradigm in experimental neurobiology. However, cell transplantation studies often rely on end-point ...

체외에서 인간 배아 줄기 세포의 생체내의 Bioluminescence 리포터 진 영상에

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