우리는 아노펠레스의 염색체 준비와 물고기의 도움을 세르게이 Demin와 타티아나 Karamysheva 감사드립니다. 우리는 또한 텍스트를 편집 우리에게 Aedes과 Culex 게놈 DNA BAC 클론과 멜리사 웨이드를 제공하기 위해 데이비드 Severson 감사드립니다. 1R21 AI88035-01 마리아 V. Sharakhova와 1R21 AI094289-01에 이고르 V. Sharakhov :이 작품은 국립 보건원에서 두 보조금에 의해 지원되었다.

<ol>
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관심 없음 충돌이 선언 없습니다.

모기 mitotic 염색체에 원위치 하이브리드 화에 Nonfluorescent는 A. 쿠마르와 K. 라이 17에 의해 1990 년에 처음으로 공연되었다. 이 연구에서, 18S와 28S ribosomal DNA의 유전자는 플라스미드 하나에 함께 복제 된 모기 중 20 종의 염색체에 배치되었습니다. DNA 프로브는 radioactively 표시 및 뇌 신경절에서 염색체에 hybridized되었다. 세 모기 장군 중에서 물고기 기술은 애의 세포 라인에서 mitotic 염색체을 위해 개발되었습니다. aegypti 10,18,19하며 라이브 모기에서 mitotic 염색체에서 수행 된 적이있다. 최근, 우리는 4 일 instar의 애벌레 9의 ID에서 높은 품질의 염색체 준비를 얻기위한 간단하고 강력한 기술을 개발했다. 이 방법은 염색체의 높은 번호가 하나의 슬라이드에서 얻을 수 할 수 있으며, 보편적 모기의 모든 종에 사용될 수 있습니다. 만 애벌레의, pupal 또는 성인되지 역을 사용하는 필요성모기 릿지는 슬라이드 준비에 아마 방법의 한계입니다. 표준 생선 방법 12 Aedes, Culex, 그리고 아노펠레스의 mitotic 염색체에 대한 프로브로 게놈 BAC 클론과 IGS rDNA를 사용에 최적화되었습니다. 이러한 특정 응용 프로그램뿐만 아니라, 여기에 설명 된 생선 프로토콜은 다른 목적을 위해 사용할 수 있습니다. 불특정 하이브리드을 차단을위한 C 0t의 DNA 분수를 활용하여 고급 생선 프로토콜은 또한 BAC 클론 또는 아노펠레스의 heterochromatic 지역의 다른 큰 DNA 조각의 하이브리드에 적용 할 수 있습니다. Heterochromatic 지역은 transposable 요소와 다른 반복과 풍부한,이 지역에서 프로브는 일반적으로 염색체 3 강력한 배경을 생산하고 있습니다. 라벨이없는 C 0t의 DNA 분수를 사용하면 염색체에 프로브의 불특정 하이브리드을 줄이기 위해 도움이 될 것입니다. 물고기 PR의 간단한 버전otocol 어떤 rDNA 또는 모기와 다른 곤충의 mitotic 염색체에 반복적 인 DNA 프로브에 사용할 수 있습니다. 또한, 그것은 또한 아노펠레스 또는 Drosophila 등의 euchromatic 지역에서 낮은 반복적 인 DNA 콘텐츠가 포함 된 종의 BAC 클론의 DNA의 하이브리드에 적용 할 수 있습니다. 여기에 제안 된 프로토콜은 모기에 대한 높은 완성 된 염색체 기반의 게놈 어셈블리를 얻을하는 데 도움이되며, 널리 곤충의 다른 그룹에서 다양한 cytogenetic 응용 프로그램에 사용할 수 있습니다.

<table border="1"><tbody><tr><td> 시약 / 장비의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 </td></tr><tr><td> MZ6 Leica의 stereomicroscope </td><td> Leica </td><td> VA-OM-E194-354 </td><td> 다른 stereomicroscope를 사용할 수 있습니다 </td></tr><tr><td> 올림푸스 CX41 위상 현미경 </td><td> 하늘 </td><td> CX41 </td><td> 다른 위상 현미경을 사용할 수 있습니다 </td></tr><tr><td> 올림푸스 BX61 형광 현미경 </td><td> 하늘 </td><td> BX61 </td><td> 다른 형광 현미경을 사용 할 수 있습니다 </td></tr><tr><td> ThermoBrite 슬라이드 변성 / 하이브리드 시스템 </td><td> 애보트 분자 </td><td> 30-144110 </td><td> 가열 블록 및 습윤 챔버 역할을 </td></tr><tr><td> 해부 바늘 </td><td> 좋은 ScienceTools </td><td> 10130-10</td><td></td></tr><tr><td> 니들 홀더 </td><td> 정밀 과학 도구 </td><td> 26018-17 </td><td></td></tr><tr><td> 해부 가위 </td><td> 정밀 과학 도구 </td><td> 15000-03 </td><td></td></tr><tr><td> 75x25 더블 불투명 마이크로 슬라이드 </td><td> 코닝 </td><td> 2949-75x25 </td><td></td></tr><tr><td> 22x22 mm 현미경 coverslips </td><td> 피셔 과학 </td><td> 12-544-10 </td><td></td></tr><tr><td> Parafilm </td><td> 피셔 과학 </td><td> 13-374-10 </td><td></td></tr><tr><td> 고무 시멘트 </td><td> 피셔 과학 </td><td> 50-949-105 </td><td></td></tr><tr><td> 아세트산 </td><td> 피셔 과학 </td><td> A491-212 </td><td></td></tr><tr><td> 알코올 200 증명 </td><td> Decon 연구소 </td><td> 2701 </td><td></td></tr><tr><td> 프로피온산 </td><td> 시그마 Aldrich </td><td> 402907 </td><td></td></tr><tr><td> 염산 </td><td> 피셔 과학 </td><td> A144-500 </td><td></td></tr><tr><td> 나트륨 시트르산 이수화 </td><td> 피셔 과학 </td><td> S279-500 </td><td></td></tr><tr><td> 나트륨 아세테이트 trihydrate </td><td> 피셔 과학 </td><td> BP334-500 </td><td></td></tr><tr><td> 염화칼륨 </td><td> 피셔 과학 </td><td> BP366-500 </td><td></td></tr><tr><td> EDTA (에틸렌 다이아 민 테트라 초산) </td><td> 피셔 과학 </td><td> S311-500 </td><td></td></tr><tr><td> 트리스베이스 </td><td> 피셔 과학 </td><td> BP152-1 </td><td></td></tr><tr><td> 10X PBS </td><td> Invitrogen </td><td> P5493 </td><td></td></tr><tr><td> 10% NBF (중성는 포르말린 버퍼) </td><td> 시그마 - 알드리치 </td><td> HT501128 </td><td></td></tr><tr><td> 99 % 포름 아미드 </td><td> 피셔 Scientific </td><td> BP227500 </td><td></td></tr><tr><td> Dextran 황산 나트륨 소금 </td><td> 시그마 - 알드리치 </td><td> D8906 </td><td></td></tr><tr><td> 20x SSC 버퍼 </td><td> Invitrogen </td><td> AM9765 </td><td></td></tr><tr><td> 1 ㎜ 유유-1 요오드화물 (509분의 491) 솔루션 </td><td> Invitrogen </td><td> Y3601 </td><td></td></tr><tr><td> 골드 시약을 연장 Antifade </td><td> Invitrogen </td><td> <a href="http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen?cmd=catProductDetail&amp;productID=P36930" target="_blank">P36930</a> </td><td></td></tr><tr><td> dATP, dCTP, dGTP, dTTP </td><td> Fermentas </td><td> R0141, R0151, R0161, R0171 </td><td></td></tr><tr><td> Cy3-dUTP, Cy5-dUTP </td><td> GE 헬스 케어 </td><td> PA53022, PA55022 </td><td></td></tr><tr><td> BSA </td><td> 시그마 - 알드리치 </td><td> A3294 </td><td></td></tr><tr><td> DNA의 중합 효소 I </td><td> Fermentas </td><td> EP0041 </td><td></td></tr><tr><td> DNase I </td><td> Fermentas </td><td> EN0521 </td><td></td></tr><tr><td> S1 Nuclease </td><td> Fermentas </td><td> EN0321 </td><td></td></tr><tr><td> DNA 형성 촉매의 DNA 중합 효소의 </td><td> Invitrogen </td><td> 18038-042 </td><td></td></tr><tr><td> RNase </td><td> 시그마 - 알드리치 </td><td> 9001-99-4 </td><td></td></tr><tr><td> 펩신 </td><td> USB </td><td> 9001-75-6 </td><td></td></tr><tr><td> 연어 정자 DNA </td><td> 시그마 - 알드리치 </td><td> D7656 </td><td></td></tr><tr><td> Nonidet-P40 (NP40) </td><td> 미국 생물 </td><td> NC9375914 </td><td></td></tr><tr><td> Qiagen의 혈액과 세포 배양 Maxikit </td><td> Qiagen </td><td> 13,362 </td><td></td></tr><tr><td> Qiagen 대형 건립 키트 </td><td> Qiagen </td><td> 12,462 </tD&gt; <td></td></tr></tbody></table>

fluorescent in situ hybridization on mitotic chromosomes of mosquitoes

현장 하이브리드 화 (물고기)의 형광은 정기적으로 DNA와 RNA 프로브의 염색체 위치를 결정하기 위해 여러 실험실에서 사용하는 기술입니다. 이 방법의 중요한 응용 프로그램은 다양한 생물의 게놈 어셈블리를 개선하기위한 유용한 고품질의 물리적지도의 개발입니다. polytene와 mitotic 염색체의 자연 구간 패턴은 게놈 supercontigs의 정확한 순서와 방향에 대한 지침을 제공합니다. 즉 세 모기 장군, 아노펠레스, Aedes, 그리고 Culex, 중 잘 알려진 염색체 기반의 매핑 기술은 누구의 회원 읽을 수 polytene의 염색체 1을을 가지고 아노펠레스에 대한 개발되었습니다. 게놈 매핑 노력의 88 %의 결과로. gambiae 게놈은 정확한 염색체 위치 2,3에 배치되었습니다. 두 개의 다른 모기 장군, Aedes과 Culex는 제대로 염색체 B를 polytenized 한자신의 genomes 4, 5, 6 transposable 요소의 중요한 overrepresentation의 ecause. 게놈 supercontings 만 31, 9 %는 애의 염색체 명령 또는 방향없이 지정되었습니다. 각각 aegypti 7 CX. quinquefasciatus 8. 이 두 종에 대한 Mitotic의 염색체 준비는 이전에 뇌 신경절과 셀 라인으로 제한 하였다. 그러나, 모기의 뇌 신경절에서 준비 염색체 슬라이드는 일반적으로 metaphase 판 9 낮은 숫자가 포함되어 있습니다. 또한, 물고기 기술이 애의 셀 줄에서 mitotic 염색체을 위해 개발되었습니다 있지만. aegypti 10, 셀 라인 염색체 11에있는 여러 염색체 rearrangements의 축적 게놈지도에 그들을 쓸모합니다. 여기 4 일 instar의 애벌레의 imaginal 디스크 (IDS)에서 높은 품질의 mitotic의 염색체 준비를 얻기위한 간단하고 강력한 기술을 설명하는 C이 모기의 세 장군을 위해 사용될 수 없습니다. 표준 생선 프로토콜 12 애의 mitotic 염색체에 프로브로 게놈 DNA의 BAC 클론의 사용에 최적화되어 있습니다. aegypti 및 CX. quinquefasciatus와의 프로브로 intergenic 스페이서 (IGS) ribosomal DNA의 지역 (rDNA)를 이용하십시오. gambiae 염색체가. 물리적 매핑뿐만 아니라, 개발 된 기술은 인구 cytogenetics와 염색체 분류 / 모기와 다른 곤충 그룹의 systematics에 적용 할 수 있습니다.

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Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

즉 세 모기 장군, 중<em&gt; 아노펠레스,</em&gt;<em&gt; Aedes</em&gt; 및<em&gt; Culex</em&gt;, 물리적 게놈 매핑 기술은 전용 설립되었다<em&gt; 아노펠레스</em&gt;, 누구의 회원 읽을 수 polytene 염색체를 가지고 있습니다. 의 장군에 대한<em&gt; Aedes</em&gt;와<em&gt; Culex</em&gt; 그러나, cytogenetic 매핑 때문에 polytene 염색체의 품질에 도전 남아 있습니다. 여기 고품질의 mitotic 염색체의 준비와 모기의 세 장군을위한 최적화 된 생선 프로토콜을 얻기위한 범용 프로토콜을 제시한다.

형광성의<em&gt; 현장에서</em모기의 Mitotic의 염색체에&gt; 하이브리드

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

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JoVE Journal

Immunology and Infection

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

25.6K 조회수.  Virginia Tech. 즉 세 모기 장군, 중 아노펠레스, Aedes 및 Culex, 물리적 게놈 매핑 기술은 전용 설립되었다 아노펠레스, 누구의 회원 읽을 수 polytene 염색체를 가지고 있습니다. 의 장군에 대한 Aedes와 Culex 그러나, cytogenetic 매핑 때문에 polytene 염색체의 품질에 도전 남아 있습니다. 여기 고품질의 mitotic 염색체의 ...

비디오: Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

접착제 테이프 기반의 샘플링 및 형광의 조합 현장에서</em의 신속한 감지를 위해> 하이브 리다이 제이션 살모넬라균 신선한 농산물에 대한

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

연구

면역학 및 감염병학

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

옥내 핵산 흐름 Cytometry - 형광 현장에서 하이브 리다이 제이션 (LNA 흐름 - 물고기) : 세균의 소형 RNA 감지하는 방법

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

이종 교잡<em&gt; 현장에서</em&gt; 침샘, 난소, 그리고 벡터 모기의 태아의

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

시각화 및 정량하기 위해 형광 단백질을 사용하여<em&gt; 클라미디아</em&gt; 액포 성장 역학 살아있는 세포에서

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

2D and 3D Chromosome Painting in Malaria Mosquitoes

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.

Chromosome painting is a useful method for studying organization of the cell nucleus and evolution of the karyotype. Here, we demonstrate an approach to isolate and amplify specific regions of interest from single polytene chromosomes that are subsequently used for two- and three-dimensional fluorescent in situ hybridization (FISH).

말라리아 모기의 2D 및 3D 염색체 페인팅

Fluorescent in situ hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting ...

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

KSHV 감염 세포에서 특정 유전자 제품의 시투 혼성화 (FISH) 및 면역 형광 (IF)의 정량형 형광

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

시투 혼성화에서 형광을 사용하여 뮤린 위장에서 칸디다 알비칸스의 시각화

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine the rate of infection, disseminated infection, and potential transmission of the virus in the mosquito population in question. These procedures are widely utilized with various modifications in vector competence evaluations globally. They are important in determining the potential role that a given mosquito (i.e., species, population, individual) may play in the transmission of a given agent.

Vector Competence Analyses on Aedes aegypti Mosquitoes using Zika Virus

The presented protocol can determine the vector competence of Aedes aegypti mosquito populations for a given virus, such as Zika, in a containment setting.

지카 바이러스를 사용하여 Aedes aegypti 모기에 벡터 역량 분석

The procedures presented describe a generalized methodology to infect Aedes aegypti mosquitoes with Zika virus under laboratory conditions to determine ...

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of disease cases each year. E. aedis causes mortality and reduced reproductive fitness in the mosquito vector and has been explored for its potential as a biocontrol agent. The protocol we present for culturing E. aedis is based on its natural infection cycle, which involves both horizontal and vertical transmission at different life stages of the mosquito host. Ae. aegypti mosquitoes are exposed to spores in the larval stage. These infected larvae then mature into adults and transmit the parasite vertically to their offspring. Infected offspring are then used as a source of spores for future horizontal transmission. Culturing E. aedis can be challenging to the uninitiated given the complexities of the parasite&#8217;s life cycle, and this protocol provides detailed guidance and visual aids for clarification.

Propagation of the Microsporidian Parasite Edhazardia aedis in Aedes aegypti Mosquitoes

A protocol to culture the microsporidian parasite Edhazardia aedis. The parasite is passaged from one generation of Aedes aegypti mosquitoes to the next via horizontal transfer at the larval stage followed by vertical transmission at the adult stage. Live sporoplasms survive long-term in infected eggs.

Aedes aegypti 모기에서 현미경 포리디안 기생충 에드hazardia aedis의 전파

Edhazardia aedis is a microsporidian parasite of Aedes aegypti mosquitoes, a disease vector that transmits multiple arboviruses which cause millions of ...

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line&#160;and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

시투 혼성화에서 면역 RNA-형광을 이용한 SARS-CoV-2의 시각화

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...