연구는 NIH에 의해 투자 1R15GM094770했다.

1. 콜로니 분리 및 고정 <ol><li> 표시된 시간을 한천 배지에 300 식민지 (격리 식민지가 직경 1~2mm한다) 알을 품다. </li><li> 콜로니 (얼굴까지)와 좁은 주걱을 사용하여 기본 매체를 제거합니다. </li><li> 2 % 한천의 여러 방울을 플레이스 42 ° C가 굳은 한천 전에 얼굴 1 ML pipetman 팁 즉시 장소 식민지를 사용하여 현미경 슬라이드에. </li><li> 42 ° C 식민지에서 2 % 한천의 여러 방울을 넣고 응고 수 있습니다. </li><li> 이 프로토콜의 나머지 부분을 통해 모든 단계에 대한 장갑을 착용 </li><li> 트림 면도날로 블록과 4에서 7 일 동안 2 % paraformaldehyde / 2 % 글루 타 알데히드 정착액을 포함하는 3.5 ML borosilicate 나사 캡 병에 넣어 ° C. </li></ol> 2. 세척 및 오스뮴 트리 트먼트 <ol><li> 약 1 주 후, 완전히 동일한 볼륨 5 분 후 식민지 (약 1.5 ML) 두 번 이상을 감추기에 충분 할만큼 0.15M 나트륨 cacodylate (산도 7.2)로 15 분 동안 두 번 잠복기에 의해 화학 퓸 후드 아래에 얼음 한천 블록을 씻어 1X OS 버퍼 (100 MM KH 2 PO 4 10 MM MgCl 2, 산도 6.0). </li><li> 1 시간 동안 화학 퓸 후드 아래에있는 한천 블록을 커버하기 위해 1% 오소 4의 같은 볼륨 [2 ML 오소 4 (2 %) + 2 ML 2X OS 버퍼] 튜브로 (얼음) 추가 후 같은 양의 두 번 씻어 십분 각 1X OS 버퍼. </li><li> 4 튜브가 하룻밤 사이에 남겨 ° 1X 운영 체제 버퍼에 C. </li><li> 정착액이 단계의 모든 세척은 유해 폐기물로 폐기됩니다. 오소 네를 포함한 모든 pipettes은 식물성 기름과 함께 배 씻어서되었습니다. </li></ol> 3. 씻고 탈수 <ol><li> 십분 파스퇴르 피펫을 사용하여 각각에 대해 차가운​​ 물 위의 동일한 볼륨 두 번 얼음 다음날 아침 와시 한천 차단합니다. </li><li> 25 % 동일한 볼륨, 50 %, 75 % 10 분 10 분 100 % 에탄올 2 세척하여 다음 각 95 % 에탄올로 순차적으로 씻으십시오. 4 박 남겨 ° C 100 % 에탄올 인치 </li><li> 이 단계의 모든 세척은 유해 폐기물로 폐기됩니다. </li></ol> 4. 스퍼의 준비 <ol><li> 다음날 아침 (최대한 빨리)은 스퍼의 시약을 준비 퓸 후드 아래 일회용 플라스틱 비커에 다음 솔루션을 (EM 과학)의 무게. 이 시약을 사용하는 모든 후속 작업 들어, 퓸 후드를 사용하여 계속 진행합니다. (ERL 4,221 솔루션 5g) (DER 736 솔루션 4g) (NSA 솔루션 13g) </li><li> 저어 막대를 사용하여 모자에 20 분 이러한 솔루션 (천천히) 믹스 </li><li> DMAE 솔루션에게 0.15 그램을 추가하고 위의 20 분 저어. </li><li> 모자에 1-2 시간에 대한 데가스 상기 혼합물. </li></ol> 5. 스퍼의 침투 <ol><li> 스퍼의 위의 십분 각각에 대해 100 % 객실 임시 에탄올의 동일 볼륨 한천 블록에게 5 번 씻어 만든 마자. 마지막으로 에탄올 세척 후 남아있는 에탄올 그냥 한천 블록을 커버하므로 파스퇴르 피펫을 사용하여 에탄올을 제거합니다. </li><li> 스퍼의 시약 (약 0.5 ML)의 약 동일한 금액을 추가하고 30 분 동안 서있게 다음 실온에서 15 분 동안 바퀴에 튜브를 회전합니다. 30 분 완전히 파스퇴르 피펫을 사용하여 솔루션을 제거한 후에 추가 스퍼는 한천 블록 (약 1.5 ML)를 커버하는이야, 실온에서 15 분 동안 바퀴에 튜브를 회전하고 30 분 동안 서있게합니다. 스퍼의 교체, 회전, 그리고 부화 3 번 이상 반복합니다. </li><li> 마지막 30 분 후에, 스퍼의 시약을 제거하고 블록을 충당하기 위해 새로운 스퍼의를 추가합니다. 한천 블록 4 시간에 대한 온도는 실온에 서 수 있습니다. </li><li> 스퍼의를 교체하고 하룻밤 회전. </li><li> 아침에 스퍼의를 교체하고 다음 날까지 회전 둡니다. </li><li> 다음날 아침 플레이스 스퍼는 다음 금형의 하단 커버 금형에 한천 블록을 추가하는 번호의 실리콘 금형 (피셔 과학)에 있어요. 4 시간 동안 60 ° C에서 알을 품다 </li><li> 더 많은 스퍼이고, 부화 60 ° C 하룻밤에있는 금형에서 맨 후 4 시간,. </li><li> 금형에서 블록을 제거 후 블록 아직도 약간 유연 경우, 금형으로 돌아가 추가 이일 품어 </li></ol> 6. Sectioning <ol><li> Leica Ultracut S 마이크로톰는 가벼운 현미경과 0.1 μ 전송 EM을위한 얇은 섹션 0.5 μ 두꺼운 부분을 절단하는 데 사용되었다. </li><li> 섹션 컷 것처럼 가벼운 현미경 들어, 그들은 DH 2 O의 드롭에 게재되었고, 52 ° C 열 블록에 건조. 말린 섹션은 다음 1.0 % toluidine 파랑, 5~15초 1 %의 나트륨 Borate의 드롭 물들일되었습니다. 얼룩 후 섹션은 즉시 물의 흐름에 따라 씻어 건조, 설치 미디어 (KPL)에서 설명하고 커버 용지는 샘플을 통해 봉인했다. </li></ol> 효모 및 기타 미생물의 패턴 형성을 측정하는 광범위한 관련는 이전에 다음 ID로 한 검토되었습니다. 특히주의의 분열 사회에 대한 상대 조직 미생물 식민지의 증가 기능입니다. 설명한 방법은 큰 전문 식민지 2 삽입하는 방법의 변경이며, 우리의 수정에 대한 간단한 설명 3 게시되었습니다. 야생 S.의 식민지의 중심을 통해 섹션의 가벼운 현미경의 예 cerevisiae 효모 콜로니는 그림 1에 표시됩니다. Asci, pseudohyphae 및 난형 효모 쉽게 구별할 수 있으며, 기본 한천을 침해 식민지의 지역도 분명 있습니다. 효모의 실험실 스트레인 (W303 배경)의 전자 현미경의 예제는 그림 2에 표시됩니다. 이미지 sporulated 세포의 높은 주파수를 포함하고있는 식민지의 영역에서이다. <img src="/files/ftp_upload/2510/2510fig1.jpg" alt="그림 1" /> 그림 1. 야생 효모 식민지의 빛 현미경. 이 효모 (YPS133)는 나무 exudates 4 최근에 고립되었다. 식민지 전에 sectioning에 YNA 중간 5 6 일간 incubated했다. 오픈 화살표가 가득한 화살촉은 길쭉한 세포의 체인 (pseudohyphae)와 asci를 나타내는, 채워진 화살표 대표 난형의 식물 세포를 나타냅니다, 대표 asci를 나타냅니다. <img src="/files/ftp_upload/2510/2510fig2.jpg" alt="그림 2" /> 그림 2. 실험실 효모 스트레인의 전자 현미경. SH1020 (W303 배경)의 식민지는 3 sectioning하기 전에 YNA 매체에서 6 일간 incubated했다. 이미지 asci 높은 주파수 부분의 영역을 보여줍니다. 화살표는 포자 벽의 이중 계층 구조를 나타냅니다. <img src="/files/ftp_upload/2510/2510fig3.jpg" alt="그림 3" /> 그림 3. 식민지 sporulated 세포의 배포판의 부량 : A)는 자신의 긴 쪽 함께 스택 15 인접한 사각형을 포함하는 격자는 식민지 섹션의 이미지의 중심 지역에 겹쳐있다. 의 비율이 지속적으로 유지하면서 그리드는 단지 식민지의 상단과 하단을 포함하기 위해, 크기를 조정합니다. (B &amp; C)는 직사각형의 전체 세포에 상대적으로 각 사각형의 sporulated 세포의 분율은 4 일 이전 (B)과 8 일 이전 (C) 식민지에서 이미지의 육안 검사에 의해 결정됩니다. 예를 들어, 식민지의 상단에있는 직사각형은 ( &quot;1&quot;표시) 그래프의 왼쪽에 해당합니다. 4 식민지의 뜻이 나타납니다, 오차 막대는 표준을 표시합니다. 오류가 발생했습니다.

<ol>
	<li>Kimmel, A. R., Firtel, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+symmetries:+regulation+of+Dictyostelium+development+through+chemoattractant+and+morphogen+signal-response.">Breaking symmetries: regulation of Dictyostelium development through chemoattractant and morphogen signal-response.</a> Curr Opin Genet Dev. 14, 540-540 (2004).</li><li>Palkova, Z., Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Life+within+a+community:+benefit+to+yeast+long-term+survival.">Life within a community: benefit to yeast long-term survival.</a> FEMS Microbiol Rev. 30, 806-806 (2006).</li><li>Shapiro, J., Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significances+of+bacterial+colony+patterns.">The significances of bacterial colony patterns.</a> Bioessays. 17, 597-597 (1995).</li><li>Shapiro, J., Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thinking+about+bacterial+populations+as+multicellular+organisms.">Thinking about bacterial populations as multicellular organisms.</a> Annu Rev Microbiol. 52, 81-81 (1998).</li><li>Engelberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicellular+stalk-like+structures+in+Saccharomyces+cerevisiae.">Multicellular stalk-like structures in Saccharomyces cerevisiae.</a> J Bacteriol. 180, 3992-3992 (1998).</li><li>Scherz, R., Shinder, V., Engelberg, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+analysis+of+Saccharomyces+cerevisiae+stalk-like+structures+reveals+spatial+organization+and+cell+specialization.">Anatomical analysis of Saccharomyces cerevisiae stalk-like structures reveals spatial organization and cell specialization.</a> J Bacteriol. 183, 5402-5402 (2001).</li><li>Piccirillo, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Rim101p/PacC+pathway+and+alkaline+pH+regulate+pattern+formation+in+yeast+colonies.">The Rim101p/PacC pathway and alkaline pH regulate pattern formation in yeast colonies.</a> Genetics. 184, 707-707 (2010).</li><li>Sniegowski, P. D., Dombrowski, P. G., Fingerman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Saccharomyces+cerevisiae+and+Saccharomyces+paradoxus+coexist+in+a+natural+woodland+site+in+North+America+and+display+different+levels+of+reproductive+isolation+from+European+conspecifics.">Saccharomyces cerevisiae and Saccharomyces paradoxus coexist in a natural woodland site in North America and display different levels of reproductive isolation from European conspecifics.</a> FEMS Yeast Res. 1, 299-299 (2002).</li><li>Piccirillo, S., Honigberg, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sporulation+patterning+and+invasive+growth+in+wild+and+domesticated+yeast+colonies.">Sporulation patterning and invasive growth in wild and domesticated yeast colonies.</a> Res Microbiol. 161, 390-390 (2002).</li><li>Vachova, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+developing+multicellular+yeast+colony:+spatio-temporal+expression+of+Ato1p+ammonium+exporter.">Architecture of developing multicellular yeast colony: spatio-temporal expression of Ato1p ammonium exporter.</a> Environ Microbiol. , (2009).</li></ol>

관심 없음 충돌 선언하지 않습니다.

제출 방법은 식민지의 내부 구조를 보여준다. 방법은 S.의 범위의 셀 유형의 패턴을 결정하는 효과가 있기 때문에 다른 콜로니 morphologies로 cerevisiae의 변종, 또한 관련 종 S. paradoxus 5, 방법은 곰팡이와 다른 미생물의 다양한에서 작동 가능성이 높습니다. 방법의 성공을 위해 한 중요한 단계는 식민지의 상단을 포함한 전체 식민지는, 프로토콜에 걸쳐 한천...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>Osmium tetroxide</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 19152</td>
<td>&nbsp;</td>
<tr>
<td>Silicone embedding molds</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>NC 9975029</td>
<td>&nbsp;</td>
<tr>
<td>Cycloaliphatic epoxide resin</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 15004</td>
<td>ERL 4221</td>
</tr>
<tr>
<td>Epoxy resin</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 13000</td>
<td>DER 736</td>
</tr>
<tr>
<td>Nonenyl Succinic Anhydride</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 19050</td>
<td>NSA</td>
</tr>
<tr>
<td>2-Dimethyl aminoethanol	</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>RT 13300</td>
<td>DMAE</td>
</tr>
<tr>
<td>Mounting media</td>
<td>&nbsp;</td>
<td>KPL</td>
<td>71-00-16</td>
<td>&nbsp;</td>
<tr>
<td>Rotating wheel</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>Pelco 1055</td>
<td>&nbsp;</td>
<tr>
<td>Microtome</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>Ultracut S</td>
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yeast colony embedding method

배아 다른 세포 유형의 Patterning는 metazoan 개발에 중요한 메커니즘입니다. 이러한 식민지와 biofilms과 같은 미생물의 커뮤니티는 세포 유형의 패턴을 표시합니다. 예를 들어, 효모의 S. cerevisiae는 sporulated 세포 pseudohyphal 세포 균일 식민지에서 배포되지 않습니다. patterning 이러한 패턴을 기초 분자 메커니즘의 기능 중요성은 아직도 제대로 이해하고 있습니다. 곰팡이 식민지의 세포 유형의 패턴을 조사에 관한 한 과제는 metazoan 조직과는 달리, 식민지의 세포가 상대적으로 약하게 서로 연결된 것입니다. 특히, 곰팡이 식민지는 대부분의 조직에서 발견 세포외 기질 같은 광범위한 수준을 포함​​하지 않습니다. 여기이 식민지에서 세포 유형의 내부 패턴을 보여줍니다 효모 식민지를 포함하고 sectioning하는 방법에 대한 리포트입니다. 방법은 가벼운 현미경 및 전송 전자 현미경에 적합한 얇은 섹션 (0.1 μ)에 유용 (0.5 μ) 두꺼운 부분을 준비하는 데 사용할 수 있습니다. 이러한 세포의 내부 구조가 EM으로 시각 수 있지만 Asci과 pseudohyphal 세포는 쉽게, 가벼운 현미경으로 난형의 효모 세포를 구분할 수 있습니다. 방법은 한천과 식민지를 둘러싼 스퍼의 중간 그들을 침투하고, 다음 sectioning을 기반으로합니다. 1~2mm의 범위에있는 직경이 식민지는이 프로토콜에 적합합니다. 식민지의 내부를 시각화뿐만 아니라, 방법은 기본 한천을 침공 식민지 지역의 시각화 수 있습니다.

Watch this Scientific Journal Video about Yeast Colony Embedding Method at JoVE.com

Yeast Colony Embedding Method

빛과 전자 현미경에 대한 sectioning 수 있도록 효모 식민지를 포함하는 방법. 이 프로토콜은 곰팡이 지역 사회 내에서 세포 유형의 조직을 이해하는 방향으로 새로운 도구를 제공하는 식민지 이내 sporulated 세포 pseudohyphal 세포의 분포의 결정 수 있습니다.

효모 콜로니 임베딩 방법

Patterning of  different cell types in embryos is a key mechanism in metazoan development. Communities of microorganisms, such as colonies and biofilms ...

Research

JoVE Journal

Biology

11.4K 조회수.  University of Missouri - Kansas City. 빛과 전자 현미경에 대한 sectioning 수 있도록 효모 식민지를 포함하는 방법. 이 프로토콜은 곰팡이 지역 사회 내에서 세포 유형의 조직을 이해하는 방향으로 새로운 도구를 제공하는 식민지 이내 sporulated 세포 pseudohyphal 세포의 분포의 결정 수 있습니다.

비디오: Yeast Colony Embedding Method

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

버딩 효모에 Replicative 수명 측정

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

연구

생물학

Purification of Mitochondria from Yeast Cells

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

효모 세포 Mitochondria의 정화

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

비디오 생물 정보학 인간의 배아 줄기 세포 콜로니 성장 분석

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

높은 처리량 효모 플라스미드 Overexpression 화면

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

Barcoded 효모 게놈 라이브러리의 경쟁력 화면

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay.
To overcome these limitations, a semi-solid media based colony assay for pancreatic progenitors has been developed and is presented in this report. This method takes advantage of an existing concept from the hematopoietic colony assay, in which methylcellulose is used to provide viscosity to the media, allowing the progenitor cells to stay in three-dimensional space as they undergo proliferation as well as differentiation. To enrich insulin-expressing colony-forming progenitors from a heterogeneous population, we utilized cells that express neurogenin (Ngn) 3, a pancreatic endocrine progenitor cell marker. Murine embryonic stem (ES) cell-derived Ngn3 expressing cells tagged with the enhanced green fluorescent protein reporter were sorted and as many as 25,000 cells per well were plated into low-attachment 24-well culture dishes. Each well contained 500 &mu;L of semi-solid media with the following major components: methylcellulose, Matrigel, nicotinamide, exendin-4, activin &beta;B, and conditioned media collected from murine ES cell-derived pancreatic-like cells. After 8 to 12 days of culture, insulin-expressing colonies with distinctive morphology were formed and could be further analyzed for pancreatic gene expression using quantitative RT-PCR and immunoflourescent staining to determine the lineage composition of each colony.
In summary, our colony assay allows easy detection and quantification of functional progenitors within a heterogeneous population of cells. In addition, the semi-solid media format allows uniform presentation of extracellular matrix components and growth factors to cells, enabling progenitors to proliferate and differentiate in vitro. This colony assay provides unique opportunities for mechanistic studies of pancreatic progenitor cells at the single cell level.

A three-dimensional clonogenic assay that allows pancreatic-like progenitors to differentiate into insulin-expressing colonies is described. This method takes advantage of semi-solid media containing methylcellulose, Matrigel and growth factors, in which single progenitors proliferate and differentiate in vitro, permitting quantification of the number of functional progenitors in a population.

인슐린을 표현 콜로니를 형성 Progenitors에 대한 정량 분석

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the ...

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

높은 활성 효모 변이의 색층 정화

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

The Soft Agar Colony Formation Assay

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

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

소프트 한천 콜로니 형성 분석

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

Surface Spreading and Immunostaining of Yeast Chromosomes

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution of chromatin-bound proteins. Surface spreading of yeast nuclei results in expansion of chromatin without loss of bound proteins. A method for surface spreading balances fixation of DNA bound proteins with detergent treatment. The method demonstrated is slightly modified from that described by Josef Loidl and Franz Klein1,2. The method has been used to characterize the localization of many chromatin-bound proteins at various stages of the mitotic cell cycle, but is especially useful for the study of meiotic chromosome structures such as meiotic recombinosomes and the synaptonemal complex. We also describe a modification that does not require use of Lipsol, a proprietary detergent, which was called for in the original procedure, but no longer commercially available. An immunostaining protocol that is compatible with the chromosome spreading method is also described.

A method for surface-spreading chromosomes from budding yeast is presented. This method is derived from a method previously described by Loidl and Klein. In addition, we demonstrate a procedure for immunostaining of spread chromosomes.

표면 확산과 효모 염색체의 면역 염색

The small size of nuclei of the budding yeast Saccharomyces cerevisiae limits the utility of light microscopy for analysis of the subnuclear distribution ...

Microscopy of Fission Yeast Sexual Lifecycle

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the mechanics of cell division and cell polarity. Furthermore, classical experiments on its sexual reproduction have yielded results pivotal to current understanding of DNA recombination and meiosis. More recent analysis of fission yeast mating has raised interesting questions on extrinsic stimuli response mechanisms, polarized cell growth and cell-cell fusion. To study these topics in detail we have developed a simple protocol for microscopy of the entire sexual lifecycle. The method described here is easily adjusted to study specific mating stages. Briefly, after being grown to exponential phase in a nitrogen-rich medium, cell cultures are shifted to a nitrogen-deprived medium for periods of time suited to the stage of the sexual lifecycle that will be explored. Cells are then mounted on custom, easily built agarose pad chambers for imaging. This approach allows cells to be monitored from the onset of mating to the final formation of spores.

We provide a reproducible basic method for the long-term microscopy of the fission yeast sexual lifecycle. With minor adjustments described, the presented protocol allows research focus on different steps of the reproductive process.

분열 효모 성적 라이프 사이클의 현미경

The fission yeast Schizosaccharomyces pombe has been an invaluable model system in studying the regulation of the mitotic cell cycle progression, the ...