وأيد هذا العمل من قبل مكتب البحوث البحرية عبر بحوث البحرية الامريكية الأموال الأساسية مختبر.

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Probes. 17, 165-169 (2003).</li><li>Obika, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+2'-O,+4'-C-methyleneuridine+and+-cytidine.+Novel+bicyclic+nucleosides+having+a+fixed+C3'-endo+sugar+puckering.">Synthesis of 2'-O, 4'-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3'-endo sugar puckering.</a> Tetrahedron. Lett. 38, 8735-8738 (1997).</li><li>Koshkin, A. 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الكتاب ليس لديهم ما يكشف.

وقد استخدم تدفق الأسماك LNA الأسلوب المعروضة هنا للكشف عن التعبير عن الحمض الريبي النووي الذواب من سلبية الغرام البحرية بكتيريا الضمة campbellii التعبير الذي سبق أن أكد عبر التنميط القائم على التعبير ميكروأري وعكس رد فعل البلمرة النسخ 16. حتى الآن ، وقد استخدمنا هذا الأسلوب لمراقبة التعبير عن مجموعة متنوعة من RNAs (مثلا عبر الحمض الريبي النووي الذواب المشفرة ، التي تعدل الحمض الريبي النووي الذواب نشاط البروتين ، riboswitches ، ومرنا). على هذا النحو ، ونحن واثقون من أن هذه الطريقة قابلة للتكيف ، ويمكن استخدامها للكشف عن أي هدف أو الحمض الريبي النووي الذواب مرنا ويمكن تعديلها لاستخدامها في أي نوع من الأنواع البكتيرية أو نوع من الخلايا. إذا كان تعديل هذا البروتوكول هو ضروري لأنواع الخلايا الأخرى ، والمتغير الأهم للنظر في التلاعب هو الخطوة permeabilization. على سبيل المثال ، عند تعديل شروط permeabilization الموصوفة هنا ، يجب اختبار التغيرات في تركيزات بروتين الليزوزيم وكاف. وجدنا أن تضاعف أوثلاثة أضعاف كمية بروتين الليزوزيم وكاف أدى إلى تغييرات كبيرة في النتائج تدفق الأسماك LNA. علاوة على ذلك ، عند اختبار الظروف permeabilization إضافية ، ينبغي تحليل الخلايا للتكتل ، وفقدان الخلية ، ويمكن الحصول على أفضل إشارة إلى نسبة الخلفية مضان. ويمكن اختبار مدى التثاقل عن الخلية و / أو المجهري التدفق الخلوي. من جانب التدفق الخلوي ، كتل الخلايا موجودة في ذيول في الأمام مقابل الجانب مؤامرة نقطة مبعثر والأسلوب الصحيح permeabilization سيقلل هذا التأثير المخلفات. هذا الأسلوب هو أيضا قابل للكشف عن الحمض الريبي النووي الذواب متعددة دون تعديلات رئيسية في البروتوكول كما هو موضح إضافية يمكن أن تضاف تحقيقات LNA المسمى مع haptens أخرى مثل digoxigenin خلال الخطوة التهجين والكشف عن الأجسام المضادة ثم مع مكافحة digoxigenin والثانوية للأجسام المضادة المتقارن fluorophore. حاليا ، والقيد الوحيد التي واجهناها مع هذا الأسلوب هو عدم قدرتها على توليد ما يكفي من الإشارات السابقة ضعيفةالحمض الريبي النووي الذواب أنواع الضغط والجهود جارية لتحديد عتبة الكشف (في عدد النسخ المطلق لكل خلية). عموما ، فإن تدفق الأسماك LNA الأسلوب يتيح الفرصة لقياس الحمض الريبي النووي الذواب البكتيرية أو التعبير مرنا على مستوى خلية واحدة بطريقة إنتاجية عالية لتقديم رؤى حول وجود وفرة نسبية لأنواع سرنا والدرجة التي يختلف التعبير عنها في عدد السكان.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم الكاشف </td><td> شركة </td><td> فهرس العدد </td><td> تعليقات </td></tr><tr><td> 10X الفوسفات مخزنة المالحة (PBS) ، ودرجة الحموضة 7.4 </td><td> تطبيق النظم البيولوجية / Ambion </td><td> AM9625 </td><td></td></tr><tr><td> Nuclease خالية من المياه </td><td> تطبيق النظم البيولوجية / Ambion </td><td> AM9932 </td><td> (لا DEPC المعالجة) </td></tr><tr><td> 32 ٪ بارافورمالدهيد </td><td> الإلكترون المجهري العلوم </td><td> RT 15714 </td><td> الإيثانول مجانا </td></tr><tr><td> حامض الخليك </td><td> العضوية Acros </td><td> 327290010 </td><td></td></tr><tr><td> إثيل pyrocarbonate (DEPC) </td><td> سيغما الدريتش </td><td> D5758 </td><tد&gt; احتفظ المجفف </td></tr><tr><td> الليزوزيم </td><td> سيغما الدريتش </td><td> L2879 </td><td></td></tr><tr><td> بروتين كاف (20 ملغ / مل) </td><td> تطبيق النظم البيولوجية / Ambion </td><td> AM2546 </td><td></td></tr><tr><td> Formamide </td><td> تطبيق النظم البيولوجية / Ambion </td><td> AM9342 </td><td> منزوع الأيونات ، وتجميد قسامة </td></tr><tr><td> ديكستران ميغاواط سلفات&gt; 500000 </td><td> سيغما الدريتش </td><td> D8906 </td><td> قسامة حل 60 ٪ وتجميد </td></tr><tr><td> التركيز على حل دينهارت 50X </td><td> سيغما الدريتش </td><td> D2532 </td><td> قسامة وتجميد </td></tr><tr><td> سيترات الصوديوم العازلة 20X </td><td> تطبيق النظم البيولوجية / Ambion </td><td> AM9770 </td><td></td></tr><tr> &lt;TD الحيوانات المنوية سالمون&gt; الحمض النووي (المنفصمة) 10 ملغ / مل </td><td> تطبيق النظم البيولوجية / Ambion </td><td> AM9680 </td><td> قسامة وتجميد </td></tr><tr><td> الخميرة الحمض الريبي النووي النقال (10 ملغ / مل) </td><td> تقنيات الحياة / Invitrogen </td><td> 15401-011 </td><td> قسامة وتجميد </td></tr><tr><td> توين - 20 </td><td> سيغما الدريتش </td><td> P9416 </td><td></td></tr><tr><td> 5X في محلول التهجين حجب الموقع </td><td> ناقلات مختبرات </td><td> MB - 1220 </td><td></td></tr><tr><td> DyLight 488 streptavidin </td><td> ناقلات مختبرات </td><td> SA - 5488-1 </td><td></td></tr><tr><td> Biotinylated مكافحة streptavidin </td><td> ناقلات مختبرات </td><td> BA - 0500 </td><td> قسامة وتجميد </td></tr></tbody></table>

locked nucleic acid flow cytometry-fluorescence in situ hybridization (lna flow-fish): a method for bacterial small rna detection

مضان التهجين في الموضع (FISH) هي تقنية قوية التي تستخدم لكشف وتوطين محددة تسلسل الحمض النووي في البيئة الخلوية. من أجل زيادة الإنتاجية ، يمكن الجمع بين سمك مع التدفق الخلوي (تدفق FISH) لتمكين الكشف عن تسلسل الحمض النووي المستهدف في الآلاف من الخلايا الفردية. نتيجة لذلك ، تدفق الأسماك يوفر ميزة واضحة على مدى lysate / الفرقة القائمة على الكشف عن الحمض النووي الأساليب لأن كل خلية يعامل بوصفه مراقبة مستقلة ، مما يسمح أقوى التحليلات الإحصائية والتباين. وقد دفعت هذه الصفات استخدام أساليب السمكية وتدفق الأسماك في عدد من التطبيقات المختلفة ، وكانت فائدة هذه الأساليب أثبتت بنجاح في تحديد هوية طول التيلومير ، 1،2 الخلوية والتعبير الجيني 3،4 ، ورصد تكاثر الفيروس في الخلايا المصابة 5 ، والبكتيرية تحليل المجتمع والتعداد6. تقليديا ، وقد تم الكشف عنها من خصوصية وسائل السمكية وتدفق الأسماك بواسطة المجسات قليل النوكليوتيد الحمض النووي. لكن في الآونة الأخيرة ، ازداد استبدال تحقيقات مع قليل النوكليوتيد النظير DNA الحمض النووي وتحقيقات السمكية وتدفق الأسماك كلا من حساسية وخصوصية كل تقنية نظرا لارتفاع درجات الحرارة في ذوبان (T م) من هذه الأحماض النووية لالنظير الطبيعي 7،8 . الحمض النووي مؤمن (LNA) تحقيقات هي نوع من التناظرية التي تحتوي على الحمض النووي النيوكليوتيدات LNA ارتفعت طوال تسلسل الحمض النووي الريبي أو 9،10. عندما يقترن مع تدفق الأسماك ، تحقيقات LNA سبق أظهرت التحقيقات أن يتفوق الحمض النووي التقليدية 7،11 واستخدمت بنجاح لكشف حقيقية النواة مرنا 12 و RNA فيروسي في الخلايا الثديية 5. هنا نوسع هذه القدرة ، ووصف تدفق الأسماك LNA الأسلوب الذي يسمح للكشف محددة من الحمض النووي الريبي في الخلية البكتيريةق (الشكل 1). على وجه التحديد ، ونحن مهتمون في الكشف الصغيرة غير الترميز التنظيمية الحمض النووي الريبي (الحمض الريبي النووي الذواب) والتي استقطبت اهتماما كبيرا في السنوات القليلة الماضية حيث تم العثور عليها لتكون بمثابة العناصر التنظيمية الرئيسية في العديد من العمليات الخلوية الحرجة 13. ومع ذلك ، هناك أدوات محدودة لدراسة sRNAs والتحديات للكشف عن الحمض الريبي النووي الذواب في الخلايا البكتيرية ويرجع ذلك جزئيا إلى الحجم الصغير نسبيا (عادة 5-30 النيوكليوتيدات في الطول) وفرة منخفضة من جزيئات الحمض الريبي النووي الذواب فضلا عن صعوبة في العمل العام مع أصغر الخلايا البيولوجية بدرجات الأغشية الخلوية. في هذه الطريقة ، ونحن تصف التثبيت وpermeabilzation الشروط التي تحافظ على بنية الخلايا البكتيرية والسماح للتغلغل تحقيقات LNA فضلا عن الخطوات التي تمكن من تضخيم إشارة الكشف محددة من الحمض الريبي النووي الذواب فرة منخفضة (الشكل 2).

Watch this Scientific Journal Video about Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection at JoVE.com

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

وصفت رواية عالية الإنتاجية الأسلوب الذي يتيح اكتشاف والكميات النسبية للRNA الصغيرة والتعبير مرنا من الخلايا البكتيرية واحد باستخدام مسابر مؤمن الحمض النووي وتدفق الخلوي ومضان<em&gt; في الموقع</em&gt; التهجين.

تأمين تدفق الأحماض النووية الخلوي ومضان في الموقع التهجين (LNA تدفق FISH) : طريقة لاكتشاف الحمض النووي الريبي البكتيرية الصغيرة

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

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Immunology and Infection

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

25.2K Views.  Naval Research Laboratory. وصفت رواية عالية الإنتاجية الأسلوب الذي يتيح اكتشاف والكميات النسبية للRNA الصغيرة والتعبير مرنا من الخلايا البكتيرية واحد باستخدام مسابر مؤمن الحمض النووي وتدفق الخلوي ومضان في الموقع التهجين.

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

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).

مزيج من الشريط اللاصق على أساس أخذ العينات والإسفار في الموقع تهجين للكشف السريع عن السالمونيلا على المنتجات الطازجة

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

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new infections each year. Despite decades of research, there is still no vaccine against malaria, highlighting the need for novel control strategies. One innovative approach is the use of genetically modified mosquitoes to effectively control malaria parasite transmission. Deliberate alterations of cell signaling pathways in the mosquito, via targeted mutagenesis, have been found to regulate parasite development 1. From these studies, we can begin to identify potential gene targets for transformation. Targeted mutagenesis has traditionally relied upon the homologous recombination between a target gene and a large DNA molecule. However, the construction and use of such complex DNA molecules for generation of stably transformed cell lines is costly, time consuming and often inefficient. Therefore, a strategy using locked nucleic acid-modified oligonucleotides (LNA-ONs) provides a useful alternative for introducing artificial single nucleotide substitutions into episomal and chromosomal DNA gene targets (reviewed in 2). LNA-ON-mediated targeted mutagenesis has been used to introduce point mutations into genes of interest in cultured cells of both yeast and mice 3,4. We show here that LNA-ONs can be used to introduce a single nucleotide change in a transfected episomal target that results in a switch from blue fluorescent protein (BFP) expression to green fluorescent protein (GFP) expression in both Anopheles gambiae and Anopheles stephensi cells. This conversion demonstrates for the first time that effective mutagenesis of target genes in mosquito cells can be mediated by LNA-ONs and suggests that this technique may be applicable to mutagenesis of chromosomal targets in vitro and in vivo.

Oligonucleotides can be used to site specifically substitute a single nucleotide of transfected target genes in both Anopheles gambiae and Anopheles stephensi cells.

ترنسفكأيشن والطفرات في الجينات المستهدفة في الخلايا عن طريق البعوض مغلق [أليغنوكليوتيد] حمض تعديل الأحماض النووية

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new ...

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

&#39;Bioluminescent&#39; بالعاثية مراسل للكشف عن مسببات الأمراض البكتيرية الفئة أ

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

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.

تهجين<em&gt; في الموقع</em&gt; من الغدد اللعابية والمبيض والأجنة من البعوض الحامل للمرض

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

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

فلوري<em&gt; في الموقع</em&gt; التهجين على الكروموسومات الميتوزى من البعوض

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

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

بكتيريا الاصطناعية الكروموسومات: أداة علم الجينوم وظيفية لدراسة الفيروسات RNA إيجابي حبلا

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

استخراج سريعة وآمنة وبسيطة دليل السرير الحمض النووي للكشف عن الفيروس في عينات الدم كله

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

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).

الفلورة الكمية في الموقع التهجين (FISH) والفلورة المناعية (IF) من منتجات جينية محددة في الخلايا المصابة KSHV

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

Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293T&#160;overexpressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines.&#160;HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.&#160;&#160;

This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and traditional hepatic cells (HepG2-NE, expressing human NTCP).

النمذجة التهاب الكبد الوبائي باء العدوى بفيروس في الخلايا غير الكبدية 293T-NE-3NRs

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV ...

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia coli and Salmonella Typhimurium, could repress the expression of outer membrane proteins. To further investigate the regulation function of micC in Salmonella Enteritidis, we cloned the micC gene in the Salmonella Enteritidis strain 50336, and then constructed the mutant 50336&#916;micC by the &#955; Red-based recombination system and the complemented mutant 50336&#916;micC/pmicC carrying recombinant plasmid pBR322 expressing micC. qRT-PCR results demonstrated that transcription of ompD in 50336&#916;micC was 1.3-fold higher than that in the wild type strain, while the transcription of ompA and ompC in 50336&#916;micC were 2.2-fold and 3-fold higher than those in the wild type strain. These indicated that micC represses the expression of ompA and ompC. In the following study, the pathogenicity of 50336&#916;micC was detected by both infecting 6-week-old Balb/c mice and 1-day-old chickens. The result showed that the LD50 of the wild type strain 50336, the mutants 50336&#916;micC and 50336&#916;micC/pmicC for 6-week-old Balb/c mice were 12.59 CFU, 5.01 CFU, and 19.95 CFU, respectively. The LD50 of the strains for 1-day-old chickens were 1.13 x 109 CFU, 1.55 x 108 CFU, and 2.54 x 108 CFU, respectively. It indicated that deletion of micC enhanced virulence of S. Enteritidis in mice and chickens by regulating expression of outer membrane proteins.

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

An &#955;-Red-mediated recombination system was used to create a deletion mutant of a small non-coding RNA micC.

يساهم ميكروفون الحمض النووي الريبي الصغير غير المشفر في الفوعة في بروتينات الغشاء الخارجي في السالمونيلا المعوية

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia ...