이 작품은 생명 공학 및 생물 과학 연구위원회 (부여 31/C15982), 건강 연구의 캐나다 연구소, 혁신, 브리티시 컬럼비아 기술 개발 기금에 대한 캐나다 재단 CJR로 건강 연구에 대한 마이클 스미스 재단 (부여에 의해 지원되었다 로웬), 그리고 시력을 위해 싸웠습니다.

재료 및 방법 : <ol><li> 효모 종자 : <ul><li> Acp1 - GFP (GFP : : 그의) : Acp1의 C - 말단 GFP - 태그는 플라스미드 pKT128을 (GFP 및 HIS5를 포함)를 사용하여 PCR - 중재 상동 재조합에 의해 생성되었다. 긍정적인 transformants은 공촛점 현미경과 식민지 PCR에 의해 확인되었다. 분 연구소 (통과 분, 2006)에서 : 스트레인 배경 BY7043 (STE2pr - lue2 lyp1Δ his3Δ1 leu2Δ0 ura3Δ0 met15Δ0 MAT 알파 can1Δ)되었습니다. </li><li> 삭제 돌연변이 어레이 (DMA) (XXX : KanR) : 그것은 중요하지 않은 유전자의 약 5000 삭제 돌연변이의 배열입니다. 이러한 불필요한 유전자의 모든 G418에 기절했다. 배열의 변형 배경은 BY4741 (마타 his3_1 leu2_0 met15_0 ura3_0)입니다. 우리 DMA 원래 분이 연구실에서했다. </li></ul></li><li> DMA에 Acp1 - GFP를 통합 : GFP 배열을 만들기위한 다음과 같은 프로토콜은 수정과 합성 유전자 분석 (SGA) 기술 (통과 분, 2006)에서 적응됩니다. 복제 달아 단계의 모든 로봇 시스템을 arraying 싱어 로터 고밀도를 사용하여 수행할 수 있습니다. 또는, 수동 달아 또는 액체 기반의 고밀도 arraying 로봇이 사용할 수 있습니다. 약어 : YPD, 효모 추출물 - 펩톤 - 덱스 트로 오스, SD, 합성 중퇴, LRK, 루 / ARG /리스 중퇴 미디어, LHRK, 루 / 그 / ARG /리스 중퇴 미디어 준비 <ol><li> YPD 접시에 하룻밤 Acp1 - GFP 문화의 5 ML를 따르고하여 Acp1 - GFP 세포의 잔디를 성장. 접시 불꽃 또는 생물 학적 퓸 후드에서 충분히 건조하도록 허용합니다. </li><li> 30 ˚ C 하룻밤에 번호판을 품어. </li><li> 판 당 1,536 식민지로 로봇, 배열 Acp1 - GFP의 변형을 사용합니다. 동시에, 복제 YPD + G418 판을 달아하여 DMA의 새로운 사본을 준비합니다. </li><li> 30 ˚ C 하룻밤에 번호판을 품어. 번식 및 diploid 선택 </li><li> 복제 달아 및 YPD 접시에 식민지를 통해 부설하여 DMA와 Acp1 - GFP의 변형을 친구. </li><li> 30 ˚ C 하룻밤에 번호판을 품어. </li><li> diploid 세포를 (마타 / MATalpha)을 선택 그의 + G418 플레이트 - SD에 복제 판 식민지. </li><li> 30 ˚ C 하룻밤에 번호판을 품어. Sporulationg 및 발아 </li><li> meiotic 포자의 형성을 유도하기 위해 탄소와 질소 원의 감소 수준을 포함하고 향상된 sporulation 미디어에 복제 판 diploid 식민지. </li><li> 25 ˚ C 오일 최소 번호판을 품어. </li><li> LRK 미디어에 복제 - 도금 식민지로 마타 meiotic progenies를 발아하고 ˚ C 이틀 동안 30 번호판을 품어. </li><li> 이 미디어는 포자에서 haploid 세포의 발아를 유도합니다. LUE2 여는 독서 프레임 (ORF)가 마타 특정 STE2 프로 모터에 통합되어 있기 때문에, germinated 모든 haploids는 마타 것입니다. 두 대립 유전자의 선택 </li><li> 이제 GFP의 allele과 단일 돌연변이 allele을 모두 선택하는 것이 필요합니다. 이것을 달성하기 위해, 마타 전지은 복제 도금 유전자 삭제를 (XXX : kanR) 휴대 haploid 세포에 대한 선택 LRK + G418 미디어로. </li><li> 30 ˚ C 하룻밤에 번호판을 품어. </li><li> 마지막으로, 마타 meiotic progenies은 복제 도금 단일 돌연변이의 성장 (XXX : kanR :)에 대한 선택 LHRK + G418 미디어에 또한 Acp1 - GFP 은닉 (GFP를 : 그). 이 번호판은 ˚ C 최대 3 개월까지 4 저장할 수 있습니다. </li></ol> GFP 배열을 만들기위한 다음과 같은 프로토콜은 수정과 합성 유전자 분석 (SGA) 기술 (통과 분, 2006)에서 적응됩니다. 복제 달아 단계의 모든 로봇 시스템을 arraying 싱어 로터 고밀도를 사용하여 수행할 수 있습니다. 또는, 수동 달아 또는 액체 기반의 고밀도 arraying 로봇이 사용할 수 있습니다. 약어 : YPD, 효모 추출물 - 펩톤 - 덱스 트로 오스, SD, 합성 중퇴, LRK, 루 / ARG /리스 중퇴 미디어, LHRK, 루 / 그 / ARG /리스 중퇴 미디어 준비 </li><li> 현미경 : <ol><li> 판에서 직접 Acp1 - GFP를 표현 원하는 돌연변이 (들)을 선택하십시오. </li><li> 30 세포를 성장 ° YPD 또는 SD의 C - 초기 로그 단계에 그의 미디어. </li><li> GFP의 filterset를 사용 공촛점 현미경으로 시각화. 예상 결과의 예를 그림 1에 표시됩니다. </li></ol></li></ol> SGA 미디어 : 이 프로토콜에 사용되는 성장 미디어 (YPD)와 선택 미디어 (SD)는 정기적으로 효모 분자 생물학에서 사용됩니다. 자세한 설명을 위해 (앰버그 외., 2005) 효모의 방법을 참조하십시오. LRK, LHRK 미디어 (400 ML의 총) <table border="0"><tbody><tr><td> 순서에 추가 </td><td> 양 </td></tr><tr><td> 암모늄 황산과 효모 질소베이스 </td><td> 2.7 g </td></tr><tr><td> 아미노산혼합 </td><td> 0.8 g </td></tr><tr><td> 한천 </td><td> 8g </td></tr><tr><td> DH 2 O </td><td> 360 ML </td></tr><tr><td colspan="2"> 압력솥 </td></tr><tr><td> 20 % 덱스 트로 오스 </td><td> 40 ML </td></tr><tr><td colspan="2"> 65 쿨 ˚ C </td></tr><tr><td> 100 MG / ML canavanine </td><td> 0.2 ML </td></tr><tr><td> 100 MG / ML thialysine </td><td> 0.2 ML </td></tr><tr><td colspan="2"> 믹스, 플레이트 50 ML을 따르다 </td></tr></tbody></table> LRK, LHRK + G418 미디어 (400 ML의 총) <table border="0"><tbody><tr><td> 순서에 추가 </td><td> 양 </td></tr><tr><td> 암모늄 황산없이 효모 질소베이스 </td><td> 0.7 g </td></tr><tr><td> 아미노산 믹스 </td><td> 0.8 g </td></tr><tr><td> Monosodium 글루 탐 산염 </td><td> 0.4 </td></tr><tr><td> 한천 </td><td> 8g </td></tr><tr><td> DH 2 O </td><td> 360 ML </td></tr><tr><td colspan="2"> 압력솥 </td></tr><tr><td> 20 % 덱스 트로 오스 </td><td> 40 ML </td></tr><tr><td colspan="2"> 65 쿨 ˚ C </td></tr><tr><td> 100 MG / ML canavanine </td><td> 0.2 ML </td></tr><tr><td> 100 MG / ML thialysine </td><td> 0.2 ML </td></tr><tr><td> 200 MG / ML G418 </td><td> 0.4 ML </td></tr><tr><td colspan="2"> 믹스, 플레이트 50 ML을 따르다 </td></tr></tbody></table> 강화 sporulation 미디어 (400 ML의 총) <table border="0"><tbody><tr><td> 순서에 추가 </td><td> 양 </td></tr><tr><td> 칼륨 아세테이트 </td><td> 4g </td></tr><tr><td> 효모 추출물 </td><td> 0.4 g </td></tr><tr><td> 우선당 </td><td> 0.2 g </td></tr><tr><td> Sporulation 아미노산 믹스 </td><td> 0.04 g </td></tr><tr><td> 한천 </td><td> 8g </td></tr><tr><td> DH 2 O </td><td> 360 ML </td></tr><tr><td colspan="2"> 압력솥 </td></tr><tr><td colspan="2"> 65 쿨 ˚ C </td></tr><tr><td> 200 MG / ML G418 </td><td> 0.4 ML </td></tr><tr><td colspan="2"> 믹스, 플레이트 50 ML을 따르다 </td></tr></tbody></table>

<ol>
	<li>Amberg, D. C., Burke, D., Strathern, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in yeast genetics : a Cold Spring Harbor Laboratory course manual. , (2005).</li><li>Tong, A. H., Boone, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+genetic+array+analysis+in+Saccharomyces+cerevisiae.">Synthetic genetic array analysis in Saccharomyces cerevisiae.</a> Methods Mol Biol. 313, 171-192 (2006).</li></ol>

이 방법은 여러 가지 돌연변이 배경에 mitochondrial 마커, Acp1 - GFP를 통합할 효율적으로 도움이 될 수 있습니다. 그것은 로봇 시스템의 사용에 의존하고, 쉽게 어떤 로봇 시스템과 함께 사용하기 위해 채택 수 있습니다. 이 절차는 마커의 다른 유형을 통합 사용할 수 있습니다. 예를 들어, ER을 시각화하기 위해, 우리는 정기적으로 마커 Erg11 - GFP를 사용합니다. 우리의 대표 이미지, 돌연변이 및 Acp1 - GF...

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<td>The growth media (YPD) and synthetic dropout media (SD) including LRK and LHRK, and enriched sporulation media used in this protocol is routinely used in yeast molecular biology. Please refer to Methods in Yeast (Amberg et al., 2005) for detailed descriptions.</td>
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a high-throughput method to globally study the organelle morphology in s. cerevisiae

단백질 지방화 또는 organelle의 형태를 검사하기 위해 높은 처리량 방법은 단백질 상호 작용을 공부를위한 효과적인 도구이며 분자 경로의 포괄적인 이해를 얻을 수 있습니다. Saccharomyces의 cerevisiae에서 중요하지 않은 유전자 삭제 배열의 발달과 함께, 우리는 전세계 endoplasmic reticulum (ER)과 mitochondria 사용하여 GFP (또는 변형) - 마커 서로 다른 유전자 배경처럼 다른 organelles의 형태를 공부할 수 있습니다. 그러나, 각 단일 돌연변이에 결합 GFP 표식이 개별적으로 노동 집약적인 과정입니다. 여기, 우리는 정기적으로 저희 연구실에서 사용하는 절차를 설명합니다. 고밀도 효모 배열과 약물 선택 기법을 처리하는 로봇 시스템을 사용함으로써, 우리는 상당히 시간이 필요 단축 및 재현성을 향상시킬 수 있습니다. 간단한, 우리는 로봇 복제 달아하여 4,672 불필요한 유전자 삭제 돌연변이의 고밀도 배열 GFP - 태그 mitochondrial 마커 (Apc1 - GFP)을 교차. diploid 선택, sporulation, 발아 및 듀얼 마커 선택을 통해, 우리는 두 대립 유전자를 복구합니다. 결과적으로, 각 haploid 단일 돌연변이의 게놈 로커스에서 통합 Apc1 - GFP가 포함되어 있습니다. 이제, 우리는 모두 중요하지 않은 돌연변이 배경으로 mitochondria의 형태를 공부할 수 있습니다. 이 높은 처리량 접근 방식을 사용, 우리는 편리하게 연구와 게놈 전체 설정에서 상속 및 organelles의 형성에 관련된 경로와 유전자를 윤곽을 그리다 수 있습니다.

Watch this Scientific Journal Video about A high-throughput method to globally study the organelle morphology in S. cerevisiae at JoVE.com

A high-throughput method to globally study the organelle morphology in S. cerevisiae

GFP - 퓨전 단백질은 널리 공촛점 현미경에 의해 organelles을 시각화하는 데 사용됩니다. 그러나, organelles의 형태에 영향을 변이에 대한 검사는 일반적으로 개별적인 돌연변이 유발이 필요하며 시간이 소요됩니다. 여기, 우리는 동시에 효모 거의 5000이 아닌 필수적인 유전자에 organelle - GFP 표식을 통합하는 방법을 보여줍니다.

세계 S. cerevisiae의에서 organelle 형태를 연구하기 위해 높은 처리량 방법

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help ...

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10.3K 조회수.  University of British Columbia - UBC. GFP - 퓨전 단백질은 널리 공촛점 현미경에 의해 organelles을 시각화하는 데 사용됩니다. 그러나, organelles의 형태에 영향을 변이에 대한 검사는 일반적으로 개별적인 돌연변이 유발이 필요하며 시간이 소요됩니다. 여기, 우리는 동시에 효모 거의 5000이 아닌 필수적인 유전자에 organelle - GFP 표식을 통합하는 방법을 보여줍니다.

비디오: A high-throughput method to globally study the organelle morphology in S. cerevisiae

Assay for Adhesion and Agar Invasion in S. cerevisiae

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, biofilms can reduce industrial productivity, destroy structures, and threaten human life. 1-3 On the other hand, harnessing the power of biofilms can help clean the environment and generate sustainable energy. 4-8
The ability of S. cerevisiae to colonize surfaces and participate in complex biofilms was mostly ignored until the rediscovery of the differentiation programs triggered by various signaling pathways and environmental cues in this organism. 9, 10 The continuing interest in using S. cerevisiae as a model organism to understand the interaction and convergence of signaling pathways, such as the Ras-PKA, Kss1 MAPK, and Hog1 osmolarity pathways, quickly placed S. cerevisiae in the junction of biofilm biology and signal transduction research. 11-20 To this end, differentiation of yeast cells into long, adhesive, pseudohyphal filaments became a convenient readout for the activation of signal transduction pathways upon various environmental changes. However, filamentation is a complex collection of phenotypes, which makes assaying for it as if it were a simple phenotype misleading. In the past decade, several assays were successfully adopted from bacterial biofilm studies to yeast research, such as MAT formation assays to measure colony spread on soft agar and crystal violet staining to quantitatively measure cell-surface adherence. 12, 21 However, there has been some confusion in assays developed to qualitatively assess the adhesive and invasive phenotypes of yeast in agar.
Here, we present a simple and reliable method for assessing the adhesive and invasive quality of yeast strains with easy-to-understand steps to isolate the adhesion assessment from invasion assessment. Our method, adopted from previous studies, 10, 16 involves growing cells in liquid media and plating on differential nutrient conditions for growth of large spots, which we then wash with water to assess adhesion and rub cells completely off the agar surface to assess invasion into the agar. We eliminate the need for streaking cells onto agar, which affects the invasion of cells into the agar. In general, we observed that haploid strains that invade agar are always adhesive, yet not all adhesive strains can invade agar medium. Our approach can be used in conjunction with other assays to carefully dissect the differentiation steps and requirements of yeast signal transduction, differentiation, quorum sensing, and biofilm formation.

We describe a qualitative assay for yeast adhesion and agar invasion as a measure of invasive and pseudohyphal differentiation. This simple assay can be used to assess the invasive phenotype of various mutants as well as the effects environmental cues and signaling pathways on yeast differentiation.

S. cerevisiae의의 접착 및 아가르 침략을위한 분석

Yeasts are found in natural biofilms, where many microorganisms colonize surfaces. In artificial environments, such as surfaces of man-made objects, ...

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생물학

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

도마뱀의 운동의 속도와 방향에 시각 민감도 테스트

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

S. cerevisiae의의 양적 proteomics와 단백질 단지의 식별

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to the original protocol that both streamline the procedure and increase sample uniformity.  First, we normalize all sperm volumes using freezing media that does not contain the cryoprotectant. Second, cryopreserved sperm are stored in cryovials instead of capillary tubes.  The rates of sperm freezing and thawing (&delta;&deg;C/time) are probably the two most critical variables to control in this procedure.  For this reason, do not substitute different tubes for those specified.  Working in teams of 2 it is possible to freeze the sperm of 100 males per team in ~2 hrs.  Sperm cryopreserved using this protocol has an average of 25% fertility (measured as the number of viable embryos generated in an in vitro fertilization divided by the total number of eggs fertilized) and this percent fertility is stable over many years.

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a high-throughput sperm cryopreservation protocol for zebrafish. Sperm cryopreserved using this protocol has an average of 25% fertility in subsequent vitro fertilization and is stable over many years.

높은 처리량 Zebrafish의 정자 Cryopreservation에 대한 방법과 체외에서 수정

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

안녕하세요 - C : Genomes의 3 차원 구조를 공부하는 방법.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male urethra, which relies on androgenic signals and epithelial-mesenchymal interactions1,2. Understanding the molecular mechanisms responsible for prostate development may reveal growth mechanisms that are inappropriately reawakened later in life to give rise to prostate diseases such as benign prostatic hyperplasia and prostate cancer.
The developing LUT is anatomically complex. By the time prostatic budding begins on 16.5 days post conception (dpc), numerous cell types are present. Vasculature, nerves and smooth muscle reside within the mesenchymal stroma3. This stroma surrounds a multilayered epithelium and gives rise to the fetal prostate through androgen receptor-dependent paracrine signals4. The identity of the stromal androgen receptor-responsive genes required for prostate development and the mechanism by which prostate ductal epithelium forms in response to these genes is not fully understood. The ability to precisely identify cell types and localize expression of specific factors within them is imperative to further understand prostate development. In situ hybridization (ISH) allows for localization of mRNAs within a tissue. Thus, this method can be used to identify pattern and timing of expression of signaling molecules and their receptors, thereby elucidating potential prostate developmental regulators.
Here, we describe a high throughput ISH technique to identify mRNA expression patterns in the fetal mouse LUT using vibrating microtome-cut sections. This method offers several advantages over other ISH protocols. Performing ISH on thin sections adhered to a slide is technically difficult; cryosections frequently have poor structural quality while both cryosections and paraffin sections often result in weak signal resolution. Performing ISH on whole mount tissues can result in probe trapping. In contrast, our high throughput technique utilizes thick-cut sections that reveal detailed tissue architecture. Modified microfuge tubes allow easy handling of sections during the ISH procedure. A maximum of 4 mRNA transcripts can be screened from a single 17.5dpc LUT with up to 24 mRNA transcripts detected in a single run, thereby reducing cost and maximizing efficiency. This method allows multiple treatment groups to be processed identically and as a single unit, thereby removing any bias for interpreting data. Most pertinently for prostate researchers, this method provides a spatial and temporal location of low and high abundance mRNA transcripts in the fetal mouse urethra that gives rise to the prostate ductal network.

A High Throughput in situ Hybridization Method to Characterize mRNA Expression Patterns in the Fetal Mouse Lower Urogenital Tract

Here, we describe an efficient high throughput in situ hybridization (ISH) method for visualizing patterns of mRNA expression in developing fetal mouse prostate tissue sections. The method can be easily adapted to visualize mRNA expression patterns in other mouse tissues or in tissues from other species.

높은 처리량 현장에서</em태아 마우스 로우어 Urogenital 트랙트의 mRNA의 표현 패턴을 특성화> 하이브리드화 방법

Development of the lower urogenital tract (LUT) is an intricate process. This complexity is evidenced during formation of the prostate from the fetal male ...

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. The simplicity and power of the yeast aging model can also be explored to study DNA damage and genome maintenance as well as their contributions to diseases during aging. Here, we describe a system to study age-dependent DNA mutations, including base substitutions, frame-shift mutations, gross chromosomal rearrangements, and homologous/homeologous recombination, as well as nuclear DNA repair activity by combining the yeast chronological life span with simple DNA damage and mutation assays. The methods described here should facilitate the identification of genes/pathways that regulate genomic instability and the mechanisms that underlie age-dependent DNA mutations and cancer in mammals.

Studying Age-dependent Genomic Instability using the S. cerevisiae Chronological Lifespan Model

Here we describe a set of DNA mutation assays that can be combined with the yeast chronological life span model to study the genes/pathways that regulate or contribute to genomic DNA instability during aging.

를 사용하여 연령에 의존 게놈의 불안정성 유학 S. cerevisiae 연대기 수명 모델

Studies using the Saccharomyces cerevisiae aging model have uncovered life span regulatory pathways that are partially conserved in higher eukaryotes1-2. ...

Flow Cytometry-based Purification of S. cerevisiae Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae zygotes result from the fusion of haploid cells of distinct mating type (MATa, MATalpha) and give rise to corresponding stable diploids that successively generate as many as 20 diploid progeny as a result of their strikingly asymmetric mitotic divisions 2. Zygote formation is orchestrated by a complex sequence of events: In this process, soluble mating factors bind to cognate receptors, triggering receptor-mediated signaling cascades that facilitate interruption of the cell cycle and culminate in cell-cell fusion. Zygotes may be considered a model for progenitor or stem cell function.
Although much has been learned about the formation of zygotes and although zygotes have been used to investigate cell-molecular questions of general significance, almost all studies have made use of mating mixtures in which zygotes are intermixed with a majority population of haploid cells 3-8. Many aspects of the biochemistry of zygote formation and the continuing life of the zygote therefore remain uninvestigated.
Reports of purification of yeast zygotes describe protocols based on their sedimentation properties 9; however, this sedimentation-based procedure did not yield nearly 90% purity in our hands. Moreover, it has the disadvantage that cells are exposed to hypertonic sorbitol. We therefore have developed a versatile purification procedure. For this purpose, pairs of haploid cells expressing red or green fluorescent proteins were co-incubated to allow zygote formation, harvested at various times, and the resulting zygotes were purified using a flow cytometry-based sorting protocol. This technique provides a convenient visual assessment of purity and maturation. The average purity of the fraction is approximately 90%. According to the timing of harvest, zygotes of varying degrees of maturity can be recovered. The purified samples provide a convenient point of departure for "-omic" studies, for recovery of initial progeny, and for systematic investigation of this progenitor cell.

Flow Cytometry-based Purification of S. cerevisiae Zygotes

To purify zygotes of S. cerevisiae, haploid cells of opposite mating type were engineered to express red or green fluorescent proteins, co-incubated to allow zygote formation, and fractionated using a flow cytometry-based protocol. The highly-enriched fraction enables subsequent "-omic" studies, recovery of initial progeny, and systematic investigation of zygote morphogenesis.

의 흐름 세포 계측법 기반 정화<em&gt; S. cerevisiae</em&gt; Zygotes

Zygotes are essential intermediates between haploid and diploid states in the life cycle of many organisms, including yeast (Figure 1) 1. S. cerevisiae ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

이분자 형광 보완의 유세포 분석 : 단백질 - 단백질 상호 작용을 연구하는 높은 처리량 정량 방법

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically and Accurately

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to large-scale screening formats in every step of a drug screen, including large-scale image analysis. Currently there is no ready-to-use and free image analysis software to meet this large-scale format. Most existing methods involve manually drawing the length and width of the imaged 3D spheroids, which is a tedious and time-consuming process. This study presents a high-throughput image analysis software application &#8211; SpheroidSizer, which measures the major and minor axial length of the imaged 3D tumor spheroids automatically and accurately; calculates the volume of each individual 3D tumor spheroid; then outputs the results in two different forms in spreadsheets for easy manipulations in the subsequent data analysis. The main advantage of this software is its powerful image analysis application that is adapted for large numbers of images. It provides high-throughput computation and quality-control workflow. The estimated time to process 1,000 images is about 15 min&#160;on a minimally configured laptop, or around 1 min&#160;on a multi-core performance workstation. The graphical user interface (GUI) is also designed for easy quality control, and users can manually override the computer results. The key method used in this software is adapted from the active contour algorithm, also known as Snakes, which is especially suitable for images with uneven illumination and noisy background that often plagues automated imaging processing in high-throughput screens. The complimentary &#8220;Manual Initialize&#8221; and &#8220;Hand Draw&#8221; tools provide the flexibility to SpheroidSizer in dealing with various types of spheroids and diverse quality images. This high-throughput image analysis software remarkably reduces labor and speeds up the analysis process. Implementing this software is beneficial for 3D tumor spheroids to become a routine in vitro model for drug screens in industry and academia.

We present a high-throughput image analysis software application to measure the size of three-dimensional tumor spheroids imaged with bright-field microscopy. This application provides a fast and effective way to examine the effects of therapeutic drugs on spheroids, which is beneficial for researchers who wish to use spheroids in drug screens.

종양 상체의 높은 처리량의 이미지 분석 : 자동으로 정확하게 상체의 크기를 측정하는 사​​용자 친화적 인 소프트웨어 응용 프로그램

The increasing number of applications of three-dimensional (3D) tumor spheroids as an in vitro model for drug discovery requires their adaptation to ...