وأيد هذا العمل من خلال منحة من مركز للبحوث باكارد ALS في جامعة جونز هوبكنز (المدير العام المساعد) ، وجائزة من مدير المعاهد الوطنية للصحة ومبتكر جديد 1DP2OD004417 - 01 (المدير العام المساعد) ، NIH R01 NS065317 (المدير العام المساعد) ، وجائزة الباحث ألن ريتا المؤسسة. مساعد المدير العام لمركز بيو باحثة في العلوم الطبية الحيوية ، وبدعم من صناديق بيو الخيرية.

1. الاستعدادات للتحول الخميرة ويهدف هذا البروتوكول لمدة عشر لوحات 96 - جيدا ولكن يمكن تحجيمها صعودا أو هبوطا وفقا لذلك. لقد وجدنا أن هذا البروتوكول لا تعمل بشكل جيد لوحات 96 - جيدا أكثر من عشرين في جولة من التحول. فإن التحول الداخلي بأكمله (من الخطوة I.3) اتخاذ ما يقرب من ثماني ساعات. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قسامة 5 - 10μL من الحمض النووي البلازميد (100 نانوغرام / ميكرولتر) من مكتبة الخميرة ORF FLEXGene في كل بئر قاع الجولة - 96 - جيدا لوحة مع معالج RapidPlate Biorobot السائل. وكشف مكان لوحات 96 - جيدا في غطاء مقاعد البدلاء النظيفة الدخان حتى يجف بين عشية وضحاها. لقد تم العثور على كفاءات مماثلة مع التحول CEN والبلازميدات 2μ الخميرة. </li><li style=";text-align:right;direction:rtl"> تلقيح 200mL من ببتون الخميرة وسائل الاعلام (YPD) سكر العنب (10G / L خلاصة الخميرة ، ببتون 20G / L ، 20G / L الجلوكوز) مع سلالة الاستعلام في دورق مخروطي 500mL حيرة. يحضن بين عشية وضحاها على 30 درجة مئوية مع اهتزاز (200 دورة في الدقيقة). إذا لزم الأمر ، يمكن أيضا وسائل الإعلام الانتقائي تستعمل لهذه الثقافات بداية. </li><li style=";text-align:right;direction:rtl"> في صباح اليوم التالي ، يخفف من توتر الاستعلام لOD 600 = 0.1 في 2L من YPD. يهز لمدة 4-6 ساعات عند 30 درجة مئوية (200 دورة في الدقيقة) حتى تصل إلى ثقافة OD 600 = 0،8-1،0. لتحقيق النمو الأمثل ، ويكبر ثقافتين 1L منفصلة في قوارير L 2.8 حيرة. إذا لزم الأمر ، يمكن أيضا وسائل الإعلام الانتقائي استخدامها بدلا من YPD. </li></ol> 2. خميرة التحول <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> حصاد الخلايا بواسطة الطرد المركزي. ملء أربع أنابيب القابل للتصرف المخروطية 225mL مع الثقافة الخميرة وتدور في 3000rpm (~ 1800 x ج) لمدة 5 دقائق في جهاز الطرد المركزي منضدية (إيبندورف 5810R). تخلصي طاف وكرر حتى يتم حصاد جميع الخلايا. </li><li style=";text-align:right;direction:rtl"> غسل الخلايا في 200mL من تعقيمها المقطر O. 2 H Resuspend الخلية بيليه عن طريق هز أو vortexing. الجمع بين الخلايا المخروطية في أنبوب one 225mL. تدور في 3000rpm لمدة 5 دقائق. إزالة طاف. </li><li style=";text-align:right;direction:rtl"> يعد حل 0.1MLiOAc/1XTE (0.1M LiOAc ، 10MM تريس ، 1MM EDTA ، ودرجة الحموضة في الماء المقطر 8 تعقيمها). غسل الخلايا في 0.1MLiOAc/1XTE 100ML. تدور في 3000rpm لمدة 5 دقائق. إزالة طاف. </li><li style=";text-align:right;direction:rtl"> Resuspend الخلية بيليه في 70mL 0.1M LiOAc/1xTE. هزة و / أو دوامة لضمان معلق تماما بيليه. </li><li style=";text-align:right;direction:rtl"> احتضان مع اهتزاز عند 30 درجة مئوية لمدة 30 دقيقة (200 دورة في الدقيقة). </li><li style=";text-align:right;direction:rtl"> إضافة إلى β - المركابتويثانول 0.1M. احتضان مع اهتزاز عند 30 درجة مئوية لمدة 30 دقيقة (200 دورة في الدقيقة). ملاحظة : يمكن تجاهل هذه الخطوة إذا باستخدام سلالات الخميرة التي تعتبر حساسة لالمركابتويثانول - β. لقد وجدنا أن هذه الخطوة غير ضرورية لعملية التحول بنجاح ، بيد أن ذلك لا تحسين كفاءة التحويل. </li><li style=";text-align:right;direction:rtl"> تغلي 2mL من الحمض النووي sonicated السلمون الحيوانات المنوية (10mg/mL) لمدة 15 دقيقة ، ثم البرد على الجليد. </li><li style=";text-align:right;direction:rtl"> إضافة 2mL السلمون المسلوق من الحمض النووي الحيوانات المنوية إلى خليط الخلية. الحذر : قد تترسب على شكل إضافة السلمون الحمض النووي الحيوانات المنوية إلى خليط الخلية. استخدام غيض pipetman و 200 ميكرولتر ، إزالة بعناية أي يعجل أن الأشكال لأن ذلك قد تتداخل مع pipetting المصب. </li><li style=";text-align:right;direction:rtl"> 50μL قسامة من خليط الخلية إلى كل بئر من لوحة 96 - جيدا باستخدام Rapidplate BioRobot (يمكن أيضا استخدام متعدد القنوات ماصة). لا يختلطان. </li><li style=";text-align:right;direction:rtl"> يحضن في درجة حرارة الغرفة لمدة 30 دقيقة دون أن تهتز. </li><li style=";text-align:right;direction:rtl"> إعداد 200mL 40 ٪ PEG-3350/10 DMSO/0.1M LiOAc ٪ (50 ٪ 160mL PEG - 3350 ، DMSO 20mL ، LiOAc 20mL 1M). يعد حل مباشرة قبل الاستعمال. </li><li style=";text-align:right;direction:rtl"> إضافة 125μL من PEG / LioAc / DMSO حل كل بئر. مزيج من قبل الشفط والاستغناء تعليق خلية ثماني مرات مع RapidPlate. </li><li style=";text-align:right;direction:rtl"> يحضن في درجة حرارة الغرفة لمدة 30 دقيقة دون أن تهتز. </li><li style=";text-align:right;direction:rtl"> الصدمة الحرارية والخلايا في درجة حرارة 42 درجة مئوية لمدة 15 دقيقة في حاضنة الجافة. لا مكدس لوحات. ملاحظة : لقد وجدنا أن هذه الخطوة الصدمة الحرارية ليست ضرورية لتحقيق التحول الناجح. لسلالات الخميرة معينة (على سبيل المثال ، درجة الحرارة المسوخ الحساسة) ، الصدمة الحرارية هي ضارة. خفضنا سابقا الوقت صدمة الحرارة لمدة دقيقة واحدة وتمكنت من تحويل بنجاح سلالة الخميرة بايواء طفرة الحرارة ypt1 الحساسة باستخدام هذا البروتوكول. </li><li style=";text-align:right;direction:rtl"> لوحات تدور في 2500rpm (~ 1100 x ج) لمدة 5 دقائق ، وذلك باستخدام محولات أجهزة الطرد المركزي لاستيعاب 96 - جيدا لوحات. إزالة حل عن طريق الربط على عكس لوحات دلو النفايات. وصمة عار على طبق مقلوب منشفة ورقية لإزالة السائل المتبقية (الخلايا ستبقى على الجزء السفلي من الآبار). </li><li style=";text-align:right;direction:rtl"> شطف الخلايا من خلال إضافة الحد الأدنى من وسائل الإعلام 200μL (مثل SD / - أورا) إلى كل جيدا مع RapidPlate. </li><li style=";text-align:right;direction:rtl"> لوحات تدور في 2500rpm لمدة 5 دقائق وإزالة طاف بواسطة دلو النفايات على عكس والنشاف على مناشف ورقية. </li><li style=";text-align:right;direction:rtl"> إضافة 200μL الحد الأدنى من وسائل الإعلام (مثل SD / - أورا) إلى كل جيدا مع RapidPlate. </li><li style=";text-align:right;direction:rtl"> في احتضان 30 درجة مئوية لمدة 2 أيام دون أن تهتز. 2 بعد يوم ، وينبغي أن مستعمرات صغيرة من الخلايا تبدأ في النموذج في أسفل كل حولت بنجاح أيضا. </li></ol> 3. اكتشاف مقايسة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الاستغناء عن وسائل الإعلام 200μL رافينوز أدنى القائمة (مثل SRaf / - أورا) في كل بئرجديد مسطحة القاع لوحة 96 - جيدا. </li><li style=";text-align:right;direction:rtl"> مزيج من كل جانب لوحة التحول بواسطة الشفط والاستغناء تعليق خلية ثماني مرات مع Rapidplate. </li><li style=";text-align:right;direction:rtl"> تطعيم لوحات SRaf 5μL مع مزيج من خلية لوحة التحول. </li><li style=";text-align:right;direction:rtl"> في احتضان 30 درجة مئوية ليوم واحد. وينبغي أن يكون حاضرا مستعمرات صغيرة في الجزء السفلي من كل بئر بعد يوم واحد. </li><li style=";text-align:right;direction:rtl"> المستعمرات بقعة على أورا / - SD وSGal / - أورا لوحات في غطاء محرك السيارة. تعقيم 96 - الترباس المكرر (عبة Frogger) بواسطة المشتعلة. مزيج مع المستعمرات عبة Frogger قبل اكتشاف وسائل الاعلام على لوحات انتقائية. بعد اكتشاف ، والسماح لوحات ليجف في غطاء محرك السيارة لمدة 5-10 دقائق. </li><li style=";text-align:right;direction:rtl"> في احتضان 30 درجة مئوية لمدة 2-3 أيام. </li></ol> لوحات فوتوغرافية مع كاميرا رقمية ، وقارن بين بصريا نمو المستعمرات في SGal / - أورا لوحات لإيجاد مستعمرات في الاستعلام الذي تم تحسين السلالة سمية (أبطأ نمو / المستعمرات أقل كثافة) أو قمعها (أسرع نمو / المستعمرات أكثر كثافة). 4. ممثل النتائج : <img src="/files/ftp_upload/2836/2836fig1.jpg" alt="الشكل 1"/> الشكل 1. الشاشة الخميرة overexpression البلازميد لتحديد المكثفات ومحسنات من TDP 43 السمية. TDP - 43 هو بروتين الإنسان التي تورطت في التسبب في التصلب الوحشي الضموري (داء لو جيهريج). مجاميع من هيولي TDP - 43 تتراكم في الدماغ والخلايا العصبية في النخاع الشوكي لمرضى ALS 17. معربا عن TDP - 43 في نتائج التجميع في خلايا الخميرة والسمسة 18. وقد استخدمنا هذا النظام النموذجي لتحديد آليات سمية 19،20 TDP - 43. أظهرت أمثلة repesentative لوحات من الخميرة لدينا TDP - 43 معدل سمية الشاشة. هذه اللوحات تعرض المستعمرات مع البلازميد متكاملة TDP - 43 - الغالاكتوز محرض وحولت أيضا مع البلازميدات من التعبير FLEXGene مكتبة ORF. لوحة على اليسار يحتوي على الجلوكوز ، والتي تقمع التعبير عن TDP - 43 أو البلازميدات FLEXGene. لوحة على اليمين يحتوي على سكر اللبن ، والذي يحرض على التعبير عن TDP - 43 وORFs في البلازميدات FLEXGene. رأس السهم الأخضر يشير إلى وجود مستعمرة تحولت مع البلازميد الكابت للسمية TDP - 43. رأس السهم الأحمر يشير إلى وجود مستعمرة تحولت مع البلازميد يعزز سمية TDP - 43.

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الإعلان عن أي تضارب في المصالح.

هنا نطرح على بروتوكول لتنفيذ عالية الإنتاجية الشاشة overexpression البلازميد في الخميرة. هذا النهج يسمح للفحص السريع وغير منحازة عن المعدلات الوراثية للكثير من الظواهر الخلوية المختلفة. باستخدام هذا النهج ، يمكن للباحث الشاشة جزء كبير من الجينوم الخميرة في غضون أسابيع. هذ...

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high-throughput yeast plasmid overexpression screen

الخميرة في مهدها ، السكيراء البيرة ، هو نموذج النظام قوية لتحديد الآليات الأساسية لكثير من العمليات الخلوية الهامة ، بما فيها تلك التي لها صلة مباشرة الأمراض التي تصيب البشر. لأن من وقته قصير والجيل جيدا اتسم الجينوم ، ميزة كبيرة التجريبية لنظام الخميرة النموذج هو القدرة على أداء شاشات الجينية لتحديد الجينات والمسارات التي تشارك في عملية معينة. على مدى السنوات الثلاثين الماضية قد استخدمت مثل هذه الشاشات الجينية لاستجلاء دورة الخلية ، ومسار إفرازية ، والعديد من الجوانب الأكثر حفظا للغاية من بيولوجيا الخلايا حقيقية النواة 1-5. في السنوات القليلة الماضية ، تم إنشاء العديد من المكتبات genomewide سلالات الخميرة والبلازميدات 6-10. هذه المجموعات تسمح الآن للاستجواب المنهجي للحصول على وظائف الجينات باستخدام والخسارة من وظيفة نهج 11-16. هنا نقدم بروتوكول مفصلة لاستخدام بروتوكول خميرة التحول عالية الإنتاجية مع الروبوت مناولة السائل لأداء شاشة overexpression البلازميد ، وذلك باستخدام مكتبة المحتشدة من الخميرة البلازميدات 5500. لقد تم استخدام هذه الشاشات لتحديد معدلات السمية الوراثية المرتبطة تراكم تجميع البروتينات البشرية المعرضة للمرض الاعصاب. الأساليب المقدمة هنا هي قابلة للتكيف بسهولة لدراسة الظواهر الخلوية الأخرى ذات الاهتمام.

Watch this Scientific Journal Video about High-throughput Yeast Plasmid Overexpression Screen at JoVE.com

High-throughput Yeast Plasmid Overexpression Screen

نحن هنا وصف شاشة overexpression في البلازميد<em&gt; السكيراء الجعوية</em&gt; ، وذلك باستخدام مكتبة المحتشدة البلازميد والتحول خميرة بروتوكول عالية الإنتاجية مع الروبوت مناولة السائل.

شاشة عالية الإنتاجية الخميرة Overexpression البلازميد

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

high-throughput-yeast-plasmid-overexpression-screen

Research

JoVE Journal

Biology

16.3K Views.  University of Pennsylvania School of Medicine. نحن هنا وصف شاشة overexpression في البلازميد السكيراء الجعوية ، وذلك باستخدام مكتبة المحتشدة البلازميد والتحول خميرة بروتوكول عالية الإنتاجية مع الروبوت مناولة السائل.

Video: High-throughput Yeast Plasmid Overexpression Screen

A High Throughput Screen for Biomining Cellulase Activity from Metagenomic Libraries

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel production 1. Chemical methods to modify or degrade cellulose typically require strong acids and high temperatures. As such, enzymatic methods have become prominent in the bioconversion process. While the identification of active cellulases from bacterial and fungal isolates has been somewhat effective, the vast majority of microbes in nature resist laboratory cultivation. Environmental genomic, also known as metagenomic, screening approaches have great promise in bridging the cultivation gap in the search for novel bioconversion enzymes. Metagenomic screening approaches have successfully recovered novel cellulases from environments as varied as soils 2, buffalo rumen 3 and the termite hind-gut 4 using carboxymethylcellulose (CMC) agar plates stained with congo red dye (based on the method of Teather and Wood 5). However, the CMC method is limited in throughput, is not quantitative and manifests a low signal to noise ratio 6. Other methods have been reported 7,8 but each use an agar plate-based assay, which is undesirable for high-throughput screening of large insert genomic libraries. Here we present a solution-based screen for cellulase activity using a chromogenic dinitrophenol (DNP)-cellobioside substrate 9. Our library was cloned into the pCC1 copy control fosmid to increase assay sensitivity through copy number induction 10. The method uses one-pot chemistry in 384-well microplates with the final readout provided as an absorbance measurement. This readout is quantitative, sensitive and automated with a throughput of up to 100X 384-well plates per day using a liquid handler and plate reader with attached stacking system.

This protocol describes a high throughput screen for cellulolytic activity from a metagenomic library expressed in Escherichia coli. The screen is solution based and highly automated, and uses one-pot chemistry in 384 well microplates with the final readout as an absorbance measurement.

شاشة إنتاجية عالية للنشاط Biomining سلولاز من مكتبات Metagenomic

Cellulose, the most abundant source of organic carbon on the planet, has wide-ranging industrial applications with increasing emphasis on biofuel ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

الإنتاجية العالية للمواد الفحص تسكر Lignocellulosic

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

High-throughput Purification of Affinity-tagged Recombinant Proteins

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further biochemical analyses to obtain detailed insights into structure/function relationships. Advances in oligonucleotide- and gene synthesis technology make large-scale mutagenesis strategies increasingly feasible, including the substitution of target residues by all 19 other amino acids. Gain- or loss-of-function phenotypes then allow systematic conclusions to be drawn, such as the contribution of particular residues to catalytic activity, protein stability and/or protein-protein interaction specificity.
In order to attribute the different phenotypes to the nature of the mutation - rather than to fluctuating experimental conditions - it is vital to purify and analyse the proteins in a controlled and reproducible manner. High-throughput strategies and the automation of manual protocols on robotic liquid-handling platforms have created opportunities to perform such complex molecular biological procedures with little human intervention and minimal error rates1-5.
Here, we present a general method for the purification of His-tagged recombinant proteins in a high-throughput manner. In a recent study, we applied this method to a detailed structure-function investigation of TFIIB, a component of the basal transcription machinery. TFIIB is indispensable for promoter-directed transcription in vitro and is essential for the recruitment of RNA polymerase into a preinitiation complex6-8. TFIIB contains a flexible linker domain that penetrates the active site cleft of RNA polymerase9-11. This linker domain confers two biochemically quantifiable activities on TFIIB, namely (i) the stimulation of the catalytic activity during the 'abortive' stage of transcript initiation, and (ii) an additional contribution to the specific recruitment of RNA polymerase into the preinitiation complex4,5,12 . We exploited the high-throughput purification method to generate single, double and triple substitution and deletions mutations within the TFIIB linker and to subsequently analyse them in functional assays for their stimulation effect on the catalytic activity of RNA polymerase4. Altogether, we generated, purified and analysed 381 mutants - a task which would have been time-consuming and laborious to perform manually. We produced and assayed the proteins in multiplicates which allowed us to appreciate any experimental variations and gave us a clear idea of the reproducibility of our results.
This method serves as a generic protocol for the purification of His-tagged proteins and has been successfully used to purify other recombinant proteins. It is currently optimised for the purification of 24 proteins but can be adapted to purify up to 96 proteins.

We describe a method for the affinity-tagged purification of recombinant proteins using liquid-handling robotics. This method is generally applicable to the small-scale purification of soluble His-tagged proteins in a high-throughput format.

عالية الإنتاجية تنقية تقارب الموسومة البروتينات المؤتلف

X-ray crystallography is the method of choice for obtaining a detailed view of the structure of proteins. Such studies need to be complemented by further ...

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

RNA تنبؤ هيكل الثانوية باستخدام الشكل عالي الإنتاجية

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

عالية الإنتاجية إبر دقيقة جدا لقنفذ البحر بيضات ملقحة

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

عالية الإنتاجية المعايرة من luciferase المراسل، معربا عن الفيروسات المؤتلف

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

دليل جزيء صغير شاشة تقترب عالية الإنتاجية عن طريق الزرد الأجنة

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

التعافي من اسماك الزرد الكبار قلوب للتطبيقات عالية الإنتاجية

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

الإنتاجية العالية للكشف عن المغيرون الكيميائية من الجينات المشاركة ما بعد transcriptionally الخاضعة للرقابة

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

عالية الإنتاجية<em&gt; دانيو Rerio</em&gt; طاقة الإنفاق الفحص

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...