저자는 기본 Rac1 flox / flox 마우스 배아 섬유아 세포의 자신의 선물 토론토, 온타리오 마운트시나이 병원에있는 닥터 Rizaldy 스콧 감사하고 싶습니다. 이 작품은 건강 연구 (CIHR, 청소 2009-93526)와 자연 과학 및 캐나다 공학 연구위원회 (NSERC, RG 327372-06)의 캐나다 연구소에서 뉴저지에 보조금을 운영하여 지원했다. SH와 MW는 CGSMA 각각 CIHR과 NSERC에서 CGS - M 보너스에 의해 지원됩니다. 스티브 홀리와 멜라니 윌스 마찬가지로이 문서에 기여.

해동 세포 <ol><li> 액체 질소에서 냉동 배아 섬유아 세포 마우스 (MEFs)을 (개인 배아 차 문화에서 이전에 생성한) 구합니다. </li><li> 37 병을 immersing로 녹여 세포 컨텐츠가 완전히 해동되기 전까지는 소용돌이와 함께 ° C 물. </li><li> 10cm의 세포 배양 접시 10 % 태아 소 혈청, 1 % 페니실린 - 스트렙토 마이신과 보완되었습니다 DMEM의 9mL를 추가합니다. </li><li> 판 드롭 현명한 해동 세포 현탁액을 추가, 37 ° C, 5 % CO 2 배양기에서 하룻밤 세포 및 장소 플레이트를 해산하기 위해 부드럽게 접시 바위. 세포 DMSO (냉동 과정에서)으로 구분하는 경우 세포 접시 (약 4 시간 게시물 도금)을 준수 후, 초기 미디어 신선한 매체로 대체될 수 있습니다. 또한 해동 후 세포의 합류를 참고하면 동결 세포의 숫자에 따라, 동결 후 세포의 회복 속도, 그리고 세포의 성장 속도. </li></ol> Passaging 세포 <ol start="5"><li> 세포 confluency 본질적으로 100 % (예 : 다음날)로 성장하면, 미디어를 기음과 멸균 PBS의 5mL와 세포를 씻으십시오. </li><li> PBS를 제거하고 세포가 접시에서 분리 수 있도록 약 5 분 동안 인큐베이터에서 10mM EDTA (에틸렌 다이아 민 테트라 초산)과 장소 플레이트를 포함하는 트립신의 1mL를 추가합니다. </li><li> 세포가 분리되면, 대단히 짧은 시간을 해산 미디어 9mL를 포함하는 새 접시에 드롭 현명이 정지 1mL을 추가하고 37 ° C 배양기 하룻밤에이 곳으로 trypsinized 세포 미디어 9mL를 추가, 피펫 2-3 회 . 미디어의 FBS의 존재 또는 트립신을 inactivate되며, 세포는 트립신을 희석 먼저 씻어 수 있습니다. </li></ol> 커버 전표에 세포를 계산하고 도금 <ol start="8"><li> 인큐베이터에서 세포를 제거, 이번에는 대신 9mL의 trypsinized 세포에 다시 5 6mL 미디어를 추가할 제외 씻어 및 이전과 trypsinize. </li><li> 피펫 세포는 철저하게 하나의 세포 현탁액에 완전한 분리를 보장하고, 별도의 microfuge 튜브에 trypan 블루 15μL와 결합 15μL 제거합니다. </li><li> 몇 시간을 아래와 pipetting하여 세포 / trypan 파랑 정지를 혼용하고 hemocytometer에 15μL 추가할 수 있습니다. </li><li> 현미경, 죽은 세포 파란색 나타납니다. hemocytometer에서 4X4 격자 중 하나에 (생활) 무색 / 취소 세포의 수를 계산합니다. 다른 세 4X4의 격자 (표시된 A, B, C 및 D)에서이 계산을 반복하고 정지 μL 당 세포의 수를 계산하려면 다음 공식을 사용 <img src="/files/ftp_upload/2160/2160eq0.jpg" alt="방정식 1" /></li><li> 프로토콜 최적화를 통해, 우리는 실험의 결론에서 시각화에 적합한 세포 밀도 30,000 전지 결과를 도금 것으로 나타났습니다. </li><li> 30000 세포에 필요한 세포 현탁액의 볼륨을 계산하려면 다음 공식을 사용 <img src="/files/ftp_upload/2160/2160eq1.jpg" alt="방정식 2" /></li><li> 지금 여섯 잘 요리의 각 우물에 멸균 유리 커버 슬립을 배치하고 각 물론 미디어의 2mL를 추가하고, 다음 세포 현탁액 30000 세포에 필요한 볼륨에서 각 우물에 드롭 현명 추가할 수 있습니다. 우리가 나중에 지점에 형광 현미경을 사용하여 세포를 시각화되므로 유리 커버 풀렸는데이 실험에 사용되는 것에주의하라; 모든 애플 리케이션은 반드시 현미경을 필요로하고, 따라서 전표가 필요하지 않을 수 있습니다 다룹니다. </li></ol> 바이러스 전달 <ol start="15"><li> 몇 시간 또는 하루 (세포 커버 풀렸는데을 준수하는지 확인하는 데 충분한 시간) 후, 미디어를 제거하고 혈청없는 DMEM의 1mL와 세포를 씻으십시오. </li><li> 바이러스의뿐만 아니라 각 잘하는 혈청이없는 미디어를 1mL를 추가합니다. 본 실험에서는, adeno - Cre 호텔 바이러스 벡터 Biolabs에서 상업적으로 얻은 것입니다. </li><li> 이전 프로토콜 최적화 500 감염의 다중성이 (뫄) 세포 생존 능력에 거의 혹은 전혀 영향과 기본 MEFs으로 고효율 유전자 전달을 제공하는 것으로 나타났습니다. 뫄은 다음과 같이 계산 수 있습니다 <img src="/files/ftp_upload/2160/2160eq2.jpg" alt="방정식 3" /> <img src="/files/ftp_upload/2160/2160eq3.jpg" alt="방정식 4" /></li><li> 감염된 것으로 각 물론 직접 바이러스의 계산된 볼륨을 추가 부드럽게 하루 37 ° C 배양기에 바이러스 및 장소 세포를 해산하기 위해 접시 바위. </li><li> 다음날 아침, 제거 바이러스가 들어있는 세포에서 미디어과 1mL 멸균 PBS로 세포를 세척하고 다음 각 잘하기 위해 정기적으로 미디어의 2mL 추가할 수 있습니다. </li><li> 이 실험에서 exemplified Rac1 세포의 경우, 형태학의 변화는 약 72시간 게시물 전달의 세포에서 발생했습니다. </li></ol> 대표 결과 세포는 시각화했다D 굴지 염료 phalloidin (이미징 목적만을 위해)와 얼룩과 겔프 대학에서 고급 분석 센터에서 Leica TCS SP5 - multiphoton 공촛점 레이저 스캐닝 현미경을 사용하여 몇 군데. 그림 1은 바이러스에 노출되지 동일한 세포에 비해 adeno - Cre 호텔 바이러스에 노출되었던 Rac1 flox / flox 세포의 계약과 길쭉한 형태를 보여줍니다. <img src="/files/ftp_upload/2160/2160fig1.jpg" alt="그림 1" /> 그림 1. 같은 소스 (오른쪽)에서 감염되지 않은 세포에 비해 adeno - Cre 호텔 바이러스 (왼쪽)에 감염된 Rac1 flox / flox 세포 사이의 비교.

<ol>
	<li>Guo, F., Debidda, M., Yang, L., Williams, D. A., Zheng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+Deletion+of+Rac1+GTPase+Reveals+Its+Critical+Role+in+Actin+Stress+Fiber+Formation+and+Focal+Adhesion+Complex+Assembly.">Genetic Deletion of Rac1 GTPase Reveals Its Critical Role in Actin Stress Fiber Formation and Focal Adhesion Complex Assembly.</a> J. Biol. Chem. 281, 18652-18659 (2006).</li><li>Glogauer, M., Marchal, C. C., Zhu, F., Worku, A., Clausen, B. E., Foerster, I., Marks, P., GP, D. o. w. n. e. y., Dinauer, M., Kwiatkowski, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac1+deletion+in+mouse+neutrophils+has+selective+effects+on+neutrophil+functions.">Rac1 deletion in mouse neutrophils has selective effects on neutrophil functions.</a> J. Immunol. 170, 5652-5657 (2003).</li><li>Prost, S., Sheahan, S., Rannie, D., Harrison, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-mediated+Cre+deletion+of+floxed+sequences+in+primary+mouse+cells+is+an+efficient+alternative+for+studies+of+gene+deletion.">Adenovirus-mediated Cre deletion of floxed sequences in primary mouse cells is an efficient alternative for studies of gene deletion.</a> Nucleic Acids Res. 29, E80-E80 (2001).</li><li>Rijnkels, M., Rosen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus-Cre-mediated+recombination+in+mammary+epithelial+early+progenitor+cells.">Adenovirus-Cre-mediated recombination in mammary epithelial early progenitor cells.</a> J. Cell. Sci. 114, 3147-3153 (2001).</li><li>Vidali, L., Chen, F., Cicchetti, G., Ohta, Y., Kwiatkowski, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rac1-null+mouse+embryonic+fibroblasts+are+motile+and+respond+to+platelet-derived+growth+factor.">Rac1-null mouse embryonic fibroblasts are motile and respond to platelet-derived growth factor.</a> Mol. Biol. Cell. 17, 2377-2390 (2006).</li><li>Wang, H., Xie, H., Zhang, H., Das, S. K., Dey, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+gene+recombination+by+adenovirus-driven+Cre+in+the+mouse+uterus.">Conditional gene recombination by adenovirus-driven Cre in the mouse uterus.</a> Genesis. 44, 51-56 (2006).</li></ol>

동물 실험 동물 관리에있는 캐나다 협의회가 정한 지침과 규정에 따라 수행되었다.

함께 비디오는 재조합 바이러스가 Cre 호텔 recombinase를 사용하여 체외에서 소비세 특정 유전자하는 데 사용할 수있는 방법을 exemplifies. 이 프로토콜의 개발은 그러나 번지 가치가 어떤 특정 지점을 밝히지 않았다. <ul><li> adenoviruses 작업을 할 때 적절한 안전 절차를 항상 고용해야합니다. 바이러스와 함께 작업하면서 실험 복과 장갑은 항상 착용해야합니다. 미디어 플라스틱 도자기는 적어도 30 분 동안 10 % 표백제와 함께 치료해야하며, 플라스틱 도자기는 그 이후 autoclaved해야합니다. </li><li> 피하은 반복 동결 - 해동이 바이러스 titer 영향을 따라서 전달의 효율성을 줄일 수로 바이러스 주식. 바이러스 주식의 작은 볼륨이 처음 별도의 튜브로 aliquoted해야합니다. </li><li> 50-500까지 MOIs는 방법을 개발하는 동안 테스트되었습니다 효율적인 전달 속도는 세포 생존 능력에 부정적인 영향없이, 대부분의 경우에서 관찰되었다. </li><li> Adeno - GFP는 신속하게 전달 효율성을 화면으로 방법을 개발하는 동안 사용되었다. 이 컨트롤 (또는 빈 벡터)는 세포 변화 발생하지 않습니다 혼자 바이러스 감염을 보장하기 위해 실험 기간 동안 실시한다. 마찬가지로, 당신의 세포에 대한 관심의 floxed 유전자를 절개하는 모든 세포 변화 (형태 또는 생존에 즉 변화)에 따라서하지 않는 경우, 그때 adeno - GFP와 동일한 셀 유형을 감염하면 해당 바이러스 전달이 입증하는 데 사용할 수있는 중요한 컨트롤입니다 발생. </li></ul> 문화 캐스케이즈 신호의 심사 넘어, adeno - Cre 호텔 접근법은 또한 조건 실험 동물 3,4에서 유전자를 제거하고, 유기체 6 안에서 세포의 특정 인구 라벨 생체내에 채용되었습니다. 아데노 바이러스 시스템은 또한 호스트 세포에 Cre 호텔 recombinase 이외의 다른 유전자를 도입하기 위해 적용할 수 있습니다. 이 배달 벡터를 사용하는 두 가지 주요 장점은 높은 전달 및 유전자 전달 효율, 및 호스트 DNA와 통합의 부족입니다. 그러나, 소설 재조합 adenoviruses의 세대 집중적인 노동 수 있습니다. 프로토콜 여기서 설명하는 것은 이전에 개발 adeno - Cre 호텔 바이러스를 이용하여 adenoviral 시스템의 전원을 장비하고, 커플링 관심, Rac1 우리의 floxed allele과 함께. 실험 아웃이 조건부 노크를 통해, 우리는 MEFs가 floxed Rac1의 allele를 들고의 adeno - Cre 호텔 바이러스를 사용하여 전달 실제로 Rac1 - 결함 세포 2,5의 세포 형태 특성의 변화로 이어지는 Rac1의 절단을 일으킬 않는 것으로 확인되었습니다.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>Ad-CMV-Cre virus</td>
<td>&nbsp;</td>
<td>Vector Biolabs</td>
<td>1045</td>
<td>&nbsp;</td>
<tr>
<td>DME High glucose media 500mL</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SH3002201</td>
<td>&nbsp;</td>
<tr>
<td>FBS Canadian origin 500mL</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>CAA15-701</td>
<td>&nbsp;</td>
<tr>
<td>PEN-STREP 1X solution 100mL</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SV30010</td>
<td>&nbsp;</td>
<tr>
<td>Texas Red-X phalloidin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>T7471</td>
<td>&nbsp;</td>
<tr>
<td>Trypsin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>T4549</td>
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adenovirus-mediated genetic removal of signaling molecules in cultured primary mouse embryonic fibroblasts

유전자 다양한 세포 신호 폭포의 특정 구성 요소를 제거하는 능력은 현대적인 신호 전달 분석에 필수적인 도구가되었습니다. 이 조건 삭제를 달성하기 위해 하나의 특정 방법은 Cre 호텔 - loxP 시스템의 사용을 통해이다. 이 방법은 Cre 호텔의 recombinase 단백질에 대한 구체적인 인식 시퀀스 아르 loxP 사이트와 관심의 유전자를 측면이 포함됩니다. Cre 호텔 - 중재 재조합 loxP 사이트에서 이벤트 및 개입 유전자 3 후속 절단이 효소 결과 소위 floxed (loxP 사이트 둘러싸인) DNA의 노출. 여러 가지 방법은 관심의 사이트에 Cre 호텔 recombinase를 관리할 수 존재합니다. 이 비디오에서는, 우리는 floxed Rac1 allele 1 포함 배아에서 얻은 기본 마우스 배아 섬유아 세포 (MEFs)를 감염하는 Cre 호텔 recombinase 유전자를 포함하는 아데노 바이러스의 사용을 보여줍니다. 우리의 실험 Rac1 MEFs 선택을위한 우리의 근거는 명확 형태학의 변경 사항이 굴지 cytoskeleton 2,5의 변경으로 인해 Rac1의 삭제,시 볼 수 있습니다. 바이러스 전달 및 Cre 호텔 식을 다음과 72시간은 세포가 굴지 염료 phalloidin를 사용하여 스테인드 및 공촛점 레이저 스캐닝 현미경을 사용하여 몇 군데 있었다. 이것은 adeno - Cre 호텔 바이러스에 노출되었던 MEFs가 감염되지 않은 세포에 비해 이전 보고서 2,5와 일치하는 계약을 체결하고 형태의 길쭉한 나타난 것을 관찰했다. Cre 호텔 recombinase 전달의 아데노 바이러스 방법은 adeno - Cre 호텔 바이러스가 쉽게 사용할 수로 유익하고, 세포의 거의 100 %에 Cre 호텔을 통해 유전자 삭제는 최적화된 adenoviral 감염 얻을 수 있습니다.

Watch this Scientific Journal Video about Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts at JoVE.com

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

이 비디오에서 우리는 floxed Rac1 allele를 갖고 기본 마우스 배아 섬유아 세포를 감염시킬 Cre 호텔 recombinase 유전자를 갖고 아데노 바이러스를 사용합니다.

교양 차 마우스 배아 섬유아 세포의 신호 분자의 아데노 바이러스 - 매개 유전자 제거

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

adenovirus-mediated-genetic-removal-signaling-molecules-cultured

Research

JoVE Journal

Biology

24.8K 조회수.  University of Guelph. 이 비디오에서 우리는 floxed Rac1 allele를 갖고 기본 마우스 배아 섬유아 세포를 감염시킬 Cre 호텔 recombinase 유전자를 갖고 아데노 바이러스를 사용합니다.

비디오: Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic 
analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3].  Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons.  One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, 
and maintaining general cell health.  Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons.  A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde.  Our approach to imaging 
GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently 
labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events.  The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.

Live cell imaging is of particular utility when studying the dynamics of organelle trafficking. Here we describe a protocol for live imaging of dense-core vesicles in cultured neurons using wide-field fluorescence microscopy. This protocol is flexible and can be adapted to image other organelles such as mitochondria, endosomes, and peroxisomes.

기본 교양 Hippocampal 신경에 덴스 코어 Vesicles의 라이브 영상

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images.  ...

연구

생물학

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

4 전사 인자와 마우스 배아 섬유아 세포를 프로그래밍하여 유도 Pluripotent 줄기 세포의 생성, Doxycycline는 Lentiviral 전달 시스템 Inducible

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals. Here, we describe an in vitro assay, "the reorientation assay", that has been used extensively to study the effect of extrinsic and intrinsic signals on the orientation of commissural axons2. This assay was developed by numerous people in the laboratories of Jane Dodd, Thomas Jessell and Andrew Lumsden (see acknowledgements for more details) and versions of this assay were used to demonstrate the reorientation activities of key axon guidance molecules, including the BMP chemorepellent in the roof plate3,4 and the chemoattractive activities of Netrin15 and Sonic Hedgehog (Shh)6 in the floor plate in the spinal cord.
Explants comprising 2-3 segments of the dorsal two-thirds of spinal cord are dissected from embryonic day (E) 11 rats and cultured in three dimensional collagen gels7. E11 dorsal spinal explants contain newly born commissural neurons, which can be identified by their axonal expression of the glycoprotein, Tag18. Over the course of 30-40 hours in culture, the commissural axon trajectory is recapitulated in these dorsal explants with a time course similar to that seen in vivo. This axonal trajectory can be challenged by placing either test tissues or a COS cell aggregate expressing a candidate signaling molecule in contact with one of the lateral edges of the dorsal explant. Commissural axons extending in the vicinity of the appended tissue will grow under the influence of both the endogenous roof plate and signals from the ectopic lateral tissue. The degree to which commissural axons are reoriented under these circumstances can be quantified. Using this assay, it is possible both to examine the sufficiency of a particular signal to reorient commissural axons3,4 as well the necessity for this signal to direct the commissural trajectory9.

This assay assesses the ability of a signaling molecule, here Bone Morphogenetic Protein 7 (BMP7), to reorient commissural axons. An explant of embryonic dorsal spinal cord is cultured adjacent to an aggregate of COS cells secreting the candidate growth factors. Reoriented commissural axons growing within the explant are visualized by immunohistochemistry.

배아 척수 Commissural Axons을 방향을 Diffusible 신호 분자의 능력을 시금

Dorsal commissural axons in the vertebrate spinal cord1 have been an invaluable model system in which to identify axon guidance signals.  Here, we ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

생체내 이미징 및 난소암의 Orthotopic 마우스 모델에서 치료 트리 트먼트

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

Rapid Fibroblast Removal from High Density Human Embryonic Stem Cell Cultures

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system maintains hESCs from undergoing spontaneous differentiation during cell expansion. However, this co-culture method is labor intensive, requires highly trained personnel, and yields low hESC purity4. Many laboratories have attempted to minimize the number of feeder cells in hESC cultures (i.e. incorporating matrix-coated dishes or other feeder cell types5-8). These modified culture systems have shown some promise, but have not supplanted the standard method for culturing hESCs with mitomycin C-treated mouse embyronic fibroblasts in order to retard unwanted spontaneous differentiation of the hESC cultures. Therefore, the feeder cells used in hESC expansion should be removed during differentiation experiments. Although several techniques are available for purifying the hESC colonies (FACS, MACS, or use of drug resistant vectors) from feeders, these techniques are labor intensive, costly and/or destructive to the hESC. The aim of this project was to invent a method of purification that enables the harvesting of a purer population of hESCs. We have observed that in a confluent hESC culture, the MEF population can be removed using a simple and rapid aspiration of the MEF sheet. This removal is dependent on several factors, including lateral cell-to-cell binding of MEFs that have a lower binding affinity to the styrene culture dish, and the ability of the stem cell colonies to push the fibroblasts outward during the generation of their own "niche". The hESC were then examined for SSEA-4, Oct3/4 and Tra 1-81 expression up to 10 days after MEF removal to ensure maintenance of pluripotency. Moreover, hESC colonies were able to continue growing from into larger formations after MEF removal, providing an additional level of hESC expansion.

Despite ongoing efforts to transition cultures to feeder-free conditions, the derivation and culture of human embryonic stem cells (hESC) remain largely dependent on co-cultures with mouse embryonic feeders (MEFs). Here, we show a novel methodology for rapidly removing feeders from hESC cultures prior to experimentation.

고밀도의 인간 배아 줄기 세포 문화의 급속한 섬유 아세포 제거

Mouse embryonic fibroblasts (MEFs) were used to establish human embryonic stem cells (hESCs) cultures after blastocyst isolation1. This feeder system ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

위한 시스템<em&gt; 예 생체</em배아 췌장의&gt; 배양

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

헤어 재생시 상피 - 중간 엽 시그널링의 빠른 유전 분석

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized microtubules. Organelles move along these processes by microtubule motors. Easy maintenance, high sensitivity to RNAi-mediated protein knock-down and efficient procedure for creating stable cell lines make Drosophila S2 cells an ideal model system to study cargo transport by live imaging. The results obtained with S2 cells can be further applied to a more physiologically relevant system: axonal transport in primary neurons cultured from dissociated Drosophila embryos. Cultured neurons grow long neurites filled with bundled microtubules, very similar to S2 processes. Like in S2 cells, organelles in cultured neurons can be visualized by either organelle-specific fluorescent dyes or by using fluorescent organelle markers encoded by DNA injected into early embryos or expressed in transgenic flies. Therefore, organelle transport can be easily recorded in neurons cultured on glass coverslips using living imaging. Here we describe procedures for culturing and visualizing cargo transport in Drosophila S2 cells and primary neurons. We believe that these protocols make both systems accessible for labs studying cargo transport.

Organelle Transport in Cultured Drosophila Cells: S2 Cell Line and Primary Neurons.

Drosophila S2 cells and cultured neurons are great systems for imaging of motor-driven organelle transport in vivo. Here we describe detailed protocols for culturing both cell types, their imaging and analysis of transport.

교양있는 세포 기관 전송<em&gt; 초파리</em&gt; 셀 : S2 세포주 및 기본 뉴런.

Drosophila S2 cells plated on a coverslip in the presence of any actin-depolymerizing drug form long unbranched processes filled with uniformly polarized ...

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle cells is of major relevance for all studies dedicated to exploring muscle glucose metabolism and characterizing metabolic alterations. In muscle cells, glucose transporter type 4 (GLUT4) proteins translocate to the plasma membrane in response to insulin, thus allowing massive entry of glucose into the cell. The ability of muscle cells to respond to insulin by increasing the rate of glucose uptake is one of the standard readouts to quantify muscle cell sensitivity to insulin. Human primary myotubes are a suitable in vitro model, as the cells maintain many features of the donor phenotype, including insulin sensitivity. This in vitro model is also suitable for the test of any compounds that could impact insulin responsiveness. Measurements of the glucose uptake rate in differentiated myotubes reflect insulin sensitivity.
In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes, and glucose uptake rates with and without insulin stimulation are measured. We provide a detailed protocol to quantify passive and active glucose transport rates using radiolabeled [3H] 2-deoxy-D-Glucose ([3H]2dG). Calculation methods are provided to quantify active basal and insulin-stimulated rates, as well as stimulation fold.

Glucose Uptake Measurement and Response to Insulin Stimulation in In Vitro Cultured Human Primary Myotubes

In this method, human primary muscle cells are cultured in vitro to obtain differentiated myotubes and glucose uptake rates are measured. We provide a detailed protocol to quantify rates in basal and insulin-stimulated states using radiolabeled [3H] 2-deoxy-D-Glucose.

포도당 섭취량 측정 및 인슐린 자극에 대한 반응<em&gt; In Vitro</em&gt; 배양 된 사람의 주요 근육 세포

Skeletal muscle is the largest glucose deposit in mammals and largely contributes to glucose homeostasis. Assessment of insulin sensitivity of muscle ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

트랜스-내부 세포 배아 줄기 세포의 대량 주입 리드 더 높은 Chimerism 요금

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...