وأيد هذا البحث من قبل المعاهد الوطنية للصحة منح GM055360 إلى SLB وU54HD068157 لRGM. وأيد JMB من التدريب غرانت T32 علم الوراثة في جامعة ولاية بنسلفانيا (GM008216).

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يعلن الكتاب أنه ليس لديهم مصالح مالية المتنافسة. هذا البروتوكول يتم شرحها ينطوي على استخدام الفئران المختبرية وأعدم في الامتثال لجميع المبادئ التوجيهية واللوائح والوكالات التنظيمية ذات الصلة. تتم المحافظة على الحيوانات المستخدمة في هذا البروتوكول بتوجيه وموافقة من جامعة جنة رعاية الحيوان واستخدام المؤسسية ولاية بنسلفانيا (IACUC بروتوكول # 804284).

أولئك الذين يدرسون الحيوانات المنوية الاعتماد على النماذج الحيوانية لعينات الخلايا المنوية، كنظام ثقافة الخلية موثوق بها لا وجود لها حتى الآن طريقة لتوليد كل أنواع الخلايا المنوية 3. على الرغم من أن الخلايا المنوية يتم جمعها بسهولة من الخصيتين كله، النتائج فق...

الحيوانات المنوية لدى الثدييات هو عملية التمايز المعقدة التي تحدث في مراحل عدة في الأنابيب المنوية من الخصيتين 1. لفترة وجيزة، وقف مثل أمهات المني الموجودة بالقرب من ظهارة نبيب الفجوة المنوية وتفرق في الخلايا المنوية، التي تخضع ثم الانقسامات الانتصافي. بعد الانقسام الاختزالي كاملة، وخلايا فرداني الناتجة عن ذلك، أو طلائع منوية الجولة، الخضوع لتكون النطاف، وهي عملية التمايز التي تنطوي على سفك السيتوبلازم والضغط من النواة. طلائع منوية تتطور تدريجيا السوط والخضوع استطالة والتكثيف من نواة، وإنتاج التمطيط ثم التكثيف طلائع منوية، على التوالي. المنتجات النهائية هي الحيوانات المنوية، والتي تم إصدارها في تجويف أنبوب صغير المنوية ومنه إلى البربخ حيث تنضج أخرى. لأن عملية تكوين الحيوانات المنوية يعتمد على الظروف الهرمونية والجزيئية الخاصة فيالخصيتين، لم يتم حتى الآن وضع نظام موثوق الثقافة في المختبر لكامل عملية تكوين الحيوانات المنوية 2،3. وقد تم تطوير أساليب لخلق ثقافة &quot;الجرثومية البدائية خلايا تشبه الخلايا&quot; و &quot;الخلايا مثل أرومة النطفة الجولة&quot; فرداني، من الخلايا الجذعية، ولكن هذه الأساليب ليست قادرة على توليد أعداد كبيرة من هذه الخلايا وتفشل في إنتاج الخلايا المنوية في وقت لاحق بعد أنواع 4،5. لحسن الحظ، وأنواع الخلايا المنوية تختلف اختلافا كبيرا في الحجم، والذي يسمح لنظام التعليق وحيدة الخلية التي تم الحصول عليها من الخصيتين كله أن تكون مفصولة مع التدرج السائل. طريقة STA-PUT، تظاهر هنا، يستخدم الانحدار الخطي BSA والترسيب بسيطة لفصل الخلايا المنوية على أساس الحجم والكتلة 6-9. طريقة STA-PUT ديها العديد من المزايا أكثر من غيرها من الأساليب الأكثر استخداما على نطاق واسع لفصل أنواع الخلايا المنوية: نظام مراقبة الأصول الميدانية وتصويل 10-13. جهاز STA-PUT يتطلب قلذ عدة قطع من الأواني الزجاجية المتخصصة تجميعها في غرفة باردة أو ثلاجة كبيرة. وبالتالي، فمن أقل تكلفة من استخدام فارز الخلية أو elutriator. طريقة STA-PUT ينتج كميات أكبر من الخلايا لكل نوع من الخلايا والخصية مما يمكن الفرز حسب نظام مراقبة الأصول الميدانية في إطار زمني مشابه، على الرغم من نقاء كل السكان الخلية ليست عالية مثل تلك التي حصلت مع نظام مراقبة الأصول الميدانية 11. فرز الخلايا باستخدام الخرز المغناطيسي (المغناطيسية الفرز الخلية تفعيلها، MACS) مؤخرا استخدمت بنجاح لتخصيب أمهات المني من السكان الخلية الخصية مختلطة، لكنه غير مناسب حاليا لفصل الخلايا المنوية أو طلائع منوية بسبب الافتقار إلى المعرفة السطحية المناسبة علامات 14. ميزة إضافية للأسلوب STA-PUT على نظام مراقبة الأصول الميدانية أو MACS هو القدرة على عزل خلايا قابلة للحياة مناسبة لاحقة لأن الثقافة، وعلى النقيض من معظم بروتوكولات نظام مراقبة الأصول الميدانية، أنها لا تتطلب أي الحمض النووي أو أنواع أخرى من تلطيخ. للدراسات التي تتطلب لغلة الدو من أنواع الخلايا المنوية في ~ 90٪ النقاء، وSTA-PUT هو الأسلوب الأمثل.

<table border="0" cellpadding="0" cellspacing="0" width="856">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">BSA</td>
			<td style="width:109px;">Affymetrix/USB</td>
			<td style="width:255px;">10857 100MG</td>
			<td style="width:183px;">Alternate can be used.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:311px;">0.5%, 2%, and 4% BSA solutions</td>
			<td colspan="2" style="width:364px;">Dissolve 0.25 g, 11 g, and 22 g (respectively) in total volumes of 50 ml, 550 ml, and 550 ml of 1x KREBS (respectively).</td>
			<td style="width:183px;">To be made day of the STA-PUT procedure. Filter sterilize.</td>
		</tr>
		<tr height="91">
			<td height="91" style="height:91px;width:311px;">KREBS (10x)</td>
			<td colspan="2" style="width:364px;">3.26 g KH2PO4 + 139.5 g NaCl +5.89 g MgSO4/7H20 + 40 g Dextrose + 3.78 g CaCl2/2H20 + 7.12 g KCl, bring to 2 L with ddH20</td>
			<td style="width:183px;">Autoclave/filter and store at 4 &#176;C for several months.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:311px;">KREBS (1x)</td>
			<td colspan="2" style="width:364px;">Dissolve 4.24 g NaHCO3 in 100 ml ddH20. Add 200 ml 10x KREBS. Bring to 2 L with ddH20.</td>
			<td style="width:183px;">To be made day of the STA-PUT procedure. Filter sterilize.</td>
		</tr>
		<tr height="17">
			<td height="35" rowspan="2" style="height:35px;width:311px;">Collagenase</td>
			<td rowspan="2" style="width:109px;">Sigma</td>
			<td rowspan="2" style="width:255px;">C9891-1G</td>
			<td rowspan="2" style="width:183px;"> </td>
		</tr>
		<tr height="18">
		</tr>
		<tr height="17">
			<td height="35" rowspan="2" style="height:35px;width:311px;">DNAse</td>
			<td rowspan="2" style="width:109px;">Sigma</td>
			<td rowspan="2" style="width:255px;">DNEP-5MG</td>
			<td rowspan="2" style="width:183px;"> </td>
		</tr>
		<tr height="18">
		</tr>
		<tr height="17">
			<td height="35" rowspan="2" style="height:35px;width:311px;">Trypsin</td>
			<td rowspan="2" style="width:109px;">Sigma</td>
			<td rowspan="2" style="width:255px;">T9201-1G</td>
			<td rowspan="2" style="width:183px;"> </td>
		</tr>
		<tr height="18">
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">DAPI</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Triton-X100</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Formaldehyde (~37%)</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"> </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:311px;">TABLE 2: EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;"> </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Equipment</td>
			<td style="width:109px;">Company</td>
			<td style="width:255px;">Catalog #</td>
			<td style="width:183px;">Comments</td>
		</tr>
		<tr height="169">
			<td height="169" style="height:169px;width:311px;">Complete STA-PUT apparatus</td>
			<td style="width:109px;">ProScience Glass Shop</td>
			<td style="width:255px;">STA-PUT (this procedure uses the standard sedimentation chamber: Cat. No. 56700-500)</td>
			<td style="width:183px;">Includes all glassware and equipment for the apparatus. Alternate glassware is available for performing STA-PUT with smaller cell numbers.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">10 &#956;m mesh filter</td>
			<td style="width:109px;">Fisherbrand</td>
			<td style="width:255px;">22363549</td>
			<td style="width:183px;"> </td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Mouse dissection tools</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;width:311px;">14 ml round bottom fraction collection tubes with caps</td>
			<td colspan="3" style="width:547px;">Preference of researcher </td>
		</tr>
		<tr height="22">
			<td colspan="3" height="22" style="height:22px;width:547px;">Need approximately 100 tubes</td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;width:311px;">Fraction collector</td>
			<td style="width:109px;">GE Healthcare Life Sciences</td>
			<td style="width:255px;">Model: Frac-920, Product code: 18-1177-40</td>
			<td style="width:183px;">Alternate can be used.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Microscope slides, cover glass</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:311px;">Light/Fluorescent Microscope</td>
			<td colspan="3" style="width:547px;">Preference of researcher</td>
		</tr>
	</tbody>
</table>

separation of spermatogenic cell types using sta-put velocity sedimentation

الحيوانات المنوية لدى الثدييات هو عملية التمايز المعقدة التي تحدث في مراحل عدة في الأنابيب المنوية من الخصيتين. حاليا، لا يوجد نظام موثوق زراعة الخلايا مما يسمح لتمايز المنوية في المختبر، وتتطلب معظم الدراسات البيولوجية للخلايا المنوية الحصاد الأنسجة من النماذج الحيوانية مثل الماوس والفئران. لأن الخصية يحتوي على العديد من أنواع الخلايا - على حد سواء غير المنوية (ايديغ، سيرتولي، النخاعي، والخلايا الظهارية) والمنوية (أمهات المني، الخلايا المنوية، طلائع منوية الجولة، التكثيف طلائع منوية والحيوانات المنوية) - دراسات الآليات البيولوجية المعنية في الحيوانات المنوية تتطلب العزلة وإثراء هذه أنواع مختلفة من الخلايا. يسمح أسلوب STA-PUT لفصل السكان غير متجانسة من الخلايا - في هذه الحالة، من الخصيتين - من خلال BSA الانحدار الخطي. أنواع الخلايا الفردية الرواسب مع مختلف سرعة الترسيب وفقا لمحجم ليرة لبنانية، والكسور المخصب لأنواع مختلفة من الخلايا التي يمكن جمعها والاستفادة منها في إجراء المزيد من التحليلات. في حين أن طريقة STA-PUT لا يؤدي إلى كسور نقية للغاية من أنواع الخلايا، على سبيل المثال كما يمكن الحصول مع بعض أساليب الخلية والفرز، وأنها لا توفر عائدات أعلى بكثير من مجموع الخلايا في كل جزء (~ 1 × 10 8 خلايا / نوع من الخلايا المنوية من السكان بدءا من 7-8 × 10 8 خلايا). يتطلب هذا الأسلوب عالية الغلة الأواني الزجاجية المتخصصة فقط ويمكن القيام بها في أي غرفة باردة أو ثلاجة كبيرة، مما يجعلها وسيلة مثالية للمختبرات التي لديها محدودية فرص الحصول على المعدات المتخصصة مثل مضان تنشيط فارز الخلية (نظام مراقبة الأصول الميدانية) أو elutriator.

نتيجة مثالية من الإجراء STA-PUT هو فصل ملحوظ نسبيا من الخلايا من الخصيتين يعتمد على حجم الخلية وكثافة. في حين أن الخلايا المعزولة من الخصيتين والمترسبة من خلال التدرج BSA، يمكن ملاحظة عدة نطاقات مميزة من الخلايا. أي كتل من الخلايا تميل إلى تنزل الى قاع الانحدار ولن تلوث ال...

Watch this Scientific Journal Video about Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation at JoVE.com

Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

يسمح أسلوب STA-PUT للفصل بين مجموعات مختلفة من الخلايا المنوية على أساس الحجم والكثافة.

الفصل بين أنواع الخلايا المنوية عن طريق STA-PUT سرعة الترسيب

Mammalian spermatogenesis is a complex  differentiation process that occurs in several stages in the seminiferous  tubules of the testes. Currently, there ...

separation-spermatogenic-cell-types-using-sta-put-velocity

Research

JoVE Journal

Biology

25.5K Views.  University of Pennsylvania.  يسمح أسلوب STA-PUT للفصل بين مجموعات مختلفة من الخلايا المنوية على أساس الحجم والكثافة. 

Video: Separation of Spermatogenic Cell Types Using STA-PUT Velocity Sedimentation

Measuring Exocytosis in Neurons Using FM Labeling

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time imaging of vesicles labeled with FM dye to monitor the rate of presynaptic vesicle release.  FM4-64 is a red fluorescent amphiphilic styryl dye that embeds into the membranes of synaptic vesicles as endocytosis is stimulated.  Lipophilic interactions cause the dye to greatly increase in fluorescence, thus emitting a bright signal when associated with vesicles and a nominal one when in the extracellular fluid. After a wash step is used to help remove external dye within the plasma membrane, the remaining FM is concentrated within the vesicles and is then expelled when exocytosis is induced by another round of electrical stimulation. The rate of vesicles release is measured from the resulting decrease in fluorescence.  Since FM dye can be applied external and transiently, it is a useful tool for determining rates of exocytosis in neuronal cultures, especially when comparing the rates between transfected synapses and neighboring control boutons.



The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission. Here we used real-time imaging of vesicles labeled with the red fluorescent dye FM 4-64 to measure the rate of presynaptic vesicle release in hippocampal neuronal cultures.

قياس إيماس في الخلايا العصبية عن طريق وصفها وزير الخارجية

The ability to measure the kinetics of vesicle release can help provide insight into some of the basics of neurotransmission.  Here we used real-time ...

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

أكتين المشارك الترسيب الفحص ، وبالنسبة للتحليل البروتين ملزمة لF - أكتين

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

Electrophoretic Separation of Proteins

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to investigate subunit compositions, and to verify homogeneity of protein samples. It can also serve to purify proteins for use in further applications. In polyacrylamide gel electrophoresis, proteins migrate in response to an electrical field through pores in a polyacrylamide gel matrix; pore size decreases with increasing acrylamide concentration. The combination of pore size and protein charge, size, and shape determines the migration rate of the protein. In this unit, the standard Laemmli method is described for discontinuous gel electrophoresis under denaturing conditions, i.e., in the presence of sodium dodecyl sulfate (SDS). 

In this video, we demonstrate a method for electrophoretic separation of proteins using poly-acrylimide gel electrophoresis (PAGE).

الكهربي فصل البروتينات

Electrophoresis is used to separate complex mixtures of proteins (e.g., from cells, subcellular fractions, column fractions, or immunoprecipitates), to ...

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and genetics because of its simple life cycle, ease of growth and manipulation for genetic analysis, genomic resources, and transformability of the nucleus and both organelles. Mating strains is a common practice when genetic approaches are used in Chlamydomonass, to create vegetative diploids for analysis of dominance, or following tetrad dissection to ascertain nuclear vs. organellar inheritance, to test allelism, to analyze epistasy, or to generate populations for the purpose of map-based cloning. Additionally, genetic crosses are routinely used to combine organellar genotypes with particular nuclear genotypes. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad separation. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas. Key parameters and trouble spots are explained. Finally, resources for further information and alternative methods are provided.

Mating and Tetrad Separation of Chlamydomonas reinhardtii for Genetic Analysis

Mating and tetrad separation are required for genetic analysis in Chlamydomonas reinhardtii. Here we demonstrate standard methods for gametogenesis, mating, zygote germination and tetrad dissection. This protocol consists of an easy-to-follow series of steps that will make genetic approaches amenable to scientists who are less familiar with Chlamydomonas.

وفصل التزاوج من الرباعيات المتلحفة reinhardtii عن التحليل الوراثي

The unicellular green alga Chlamydomonas reinhardtii (Chlamydomonas) has become a popular organism for research in diverse areas of cell biology and ...

Two Types of Assays for Detecting Frog Sperm Chemoattraction

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension and microscopically visualize sperm accumulation around the pipette1. Sperm chemoattraction in vertebrates such as frogs, rodents and humans is more difficult to detect and requires quantitative assays. Such assays are of two major types - assays that quantitate sperm movement to a source of chemoattractant, so-called sperm accumulation assays, and assays that actually track the swimming trajectories of individual sperm.
Sperm accumulation assays are relatively rapid allowing tens or hundreds of assays to be done in a single day, thereby allowing dose response curves and time courses to be carried out relatively rapidly. These types of assays have been used extensively to characterize many well established chemoattraction systems - for example, neutrophil chemotaxis to bacterial peptides and sperm chemotaxis to follicular fluid. Sperm tracking assays can be more labor intensive but offer additional data on how chemoattractancts actually alter the swimming paths that sperm take. This type of assay is needed to demonstrate the orientation of sperm movement relative to the chemoattrractant gradient axis and to visualize characteristic turns or changes in orientation that bring the sperm closer to the egg.
Here we describe methods used for each of these two types of assays. The sperm accumulation assay utilized is called a "two-chamber" assay. Amphibian sperm are placed in a tissue culture plate insert with a polycarbonate filter floor having 12 &mu;m diameter pores. Inserts with sperm are placed into tissue culture plate wells containing buffer and a chemoatttractant carefully pipetted into the bottom well where the floor meets the wall (see Fig. 1). After incubation, the top insert containing the sperm reservoir is carefully removed, and sperm in the bottom chamber that have passed through the membrane are removed, pelleted and then counted by hemocytometer or flow cytometer.
The sperm tracking assay utilizes a Zigmond chamber originally developed for observing neutrophil chemotaxis and modified for observation of sperm by Giojalas and coworkers2,3. The chamber consists of a thick glass slide into which two vertical troughs have been machined. These are separated by a 1 mm wide observation platform. After application of a cover glass, sperm are loaded into one trough, the chemoattractant agent into the other and movement of individual sperm visualized by video microscopy. Video footage is then analyzed using software to identify two-dimensional cell movements in the x-y plane as a function of time (xyt data sets) that form the trajectory of each sperm.

Eggs and the extracellular coatings around eggs frequently release peptides, proteins and small molecules that communicate with sperm to guide them to the egg thereby promoting fertilization. Using frog sperm we describe and compare two classes of assays used to detect sperm chemoattraction &#x2013; sperm accumulation assays and sperm tracking assays.

نوعان من فحوصات الكشف عن الضفدع Chemoattraction الحيوانات المنوية

Sperm chemoattraction in invertebrates can be sufficiently robust that one can place a pipette containing the attractive peptide into a sperm suspension ...

Agarose Gel Electrophoresis for the Separation of DNA Fragments

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from the seaweed genera Gelidium and Gracilaria, and consists of repeated agarobiose (L- and D-galactose) subunits2. During gelation, agarose polymers associate non-covalently and form a network of bundles whose pore sizes determine a gel&#39;s molecular sieving properties. The use of agarose gel electrophoresis revolutionized the separation of DNA. Prior to the adoption of agarose gels, DNA was primarily separated using sucrose density gradient centrifugation, which only provided an approximation of size. To separate DNA using agarose gel electrophoresis, the DNA is loaded into pre-cast wells in the gel and a current applied. The phosphate backbone of the DNA (and RNA) molecule is negatively charged, therefore when placed in an electric field, DNA fragments will migrate to the positively charged anode. Because DNA has a uniform mass/charge ratio, DNA molecules are separated by size within an agarose gel in a pattern such that the distance traveled is inversely proportional to the log of its molecular weight3. The leading model for DNA movement through an agarose gel is &#34;biased reptation&#34;, whereby the leading edge moves forward and pulls the rest of the molecule along4. The rate of migration of a DNA molecule through a gel is determined by the following: 1) size of DNA molecule; 2) agarose concentration; 3) DNA conformation5; 4) voltage applied, 5) presence of ethidium bromide, 6) type of agarose and 7) electrophoresis buffer. After separation, the DNA molecules can be visualized under uv light after staining with an appropriate dye. By following this protocol, students should be able to: 
 
1. Understand the mechanism by which DNA fragments are separated within a gel matrix 
2. Understand how conformation of the DNA molecule will determine its mobility through a gel matrix 
3. Identify an agarose solution of appropriate concentration for their needs 
4. Prepare an agarose gel for electrophoresis of DNA samples 
5. Set up the gel electrophoresis apparatus and power supply 
6. Select an appropriate voltage for the separation of DNA fragments 
7. Understand the mechanism by which ethidium bromide allows for the visualization of DNA bands 
8. Determine the sizes of separated DNA fragments 
&#160;

A basic protocol for the separation of DNA fragments using agarose gel electrophoresis is described.

الاغاروز الكهربائي للهلام لفصل أجزاء الحمض النووي

Agarose gel electrophoresis is the most effective way of separating DNA fragments of varying sizes ranging from 100 bp to 25 kb1. Agarose is isolated from ...

A Novel Bayesian Change-point Algorithm for Genome-wide Analysis of Diverse ChIPseq Data Types

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of protein-bound DNA and aligning the short reads to a reference genome. Enriched regions are revealed as peaks, which often differ dramatically in shape, depending on the target protein1. For example, transcription factors often bind in a site- and sequence-specific manner and tend to produce punctate peaks, while histone modifications are more pervasive and are characterized by broad, diffuse islands of enrichment2. Reliably identifying these regions was the focus of our work.
Algorithms for analyzing ChIPseq data have employed various methodologies, from heuristics3-5 to more rigorous statistical models, e.g. Hidden Markov Models (HMMs)6-8. We sought a solution that minimized the necessity for difficult-to-define, ad hoc parameters that often compromise resolution and lessen the intuitive usability of the tool. With respect to HMM-based methods, we aimed to curtail parameter estimation procedures and simple, finite state classifications that are often utilized.
Additionally, conventional ChIPseq data analysis involves categorization of the expected read density profiles as either punctate or diffuse followed by subsequent application of the appropriate tool. We further aimed to replace the need for these two distinct models with a single, more versatile model, which can capably address the entire spectrum of data types.
To meet these objectives, we first constructed a statistical framework that naturally modeled ChIPseq data structures using a cutting edge advance in HMMs9, which utilizes only explicit formulas-an innovation crucial to its performance advantages. More sophisticated then heuristic models, our HMM accommodates infinite hidden states through a Bayesian model. We applied it to identifying reasonable change points in read density, which further define segments of enrichment. Our analysis revealed how our Bayesian Change Point (BCP) algorithm had a reduced computational complexity-evidenced by an abridged run time and memory footprint. The BCP algorithm was successfully applied to both punctate peak and diffuse island identification with robust accuracy and limited user-defined parameters. This illustrated both its versatility and ease of use. Consequently, we believe it can be implemented readily across broad ranges of data types and end users in a manner that is easily compared and contrasted, making it a great tool for ChIPseq data analysis that can aid in collaboration and corroboration between research groups. Here, we demonstrate the application of BCP to existing transcription factor10,11 and epigenetic data12 to illustrate its usefulness.

Our Bayesian Change Point (BCP) algorithm builds on state-of-the-art advances in modeling change-points via Hidden Markov Models and applies them to chromatin immunoprecipitation sequencing (ChIPseq) data analysis. BCP performs well in both broad and punctate data types, but excels in accurately identifying robust, reproducible islands of diffuse histone enrichment.

رواية النظرية الافتراضية تغيير نقطة حسابي لتحليل الجينوم-اسعة من أنواع البيانات المتنوعة ChIPseq

ChIPseq is a widely used technique for investigating protein-DNA interactions. Read density profiles are generated by using next-sequencing of ...

Separation of Mouse Embryonic Facial Ectoderm and Mesenchyme

Orofacial clefts are the most frequent craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal facial development3. In human and mouse, initial growth and patterning of the face relies on several small buds of tissue, the facial prominences4,5. The face is derived from six main prominences: paired frontal nasal processes (FNP), maxillary prominences (MxP) and mandibular prominences (MdP). These prominences consist of swellings of mesenchyme that are encased in an overlying epithelium. Studies in multiple species have shown that signaling crosstalk between facial ectoderm and mesenchyme is critical for shaping the face6. Yet, mechanistic details concerning the genes involved in these signaling relays are lacking. One way to gain a comprehensive understanding of gene expression, transcription factor binding, and chromatin marks associated with the developing facial ectoderm and mesenchyme is to isolate and characterize the separated tissue compartments.
Here we present a method for separating facial ectoderm and mesenchyme at embryonic day (E) 10.5, a critical developmental stage in mouse facial formation that precedes fusion of the prominences. Our method is adapted from the approach we have previously used for dissecting facial prominences7. In this earlier study we had employed inbred C57BL/6 mice as this strain has become a standard for genetics, genomics and facial morphology8. Here, though, due to the more limited quantities of tissue available, we have utilized the outbred CD-1 strain that is cheaper to purchase, more robust for husbandry, and tending to produce more embryos (12-18) per litter than any inbred mouse strain8. Following embryo isolation, neutral protease Dispase II was used to treat the whole embryo. Then, the facial prominences were dissected out, and the facial ectoderm was separated from the mesenchyme. This method keeps both the facial ectoderm and mesenchyme intact. The samples obtained using this methodology can be used for techniques including protein detection, chromatin immunoprecipitation (ChIP) assay, microarray studies, and RNA-seq.

A protocol for separation of embryo facial ectoderm and mesenchyme is described. We use Dispase II to treat whole embryos first, dissect whole facial prominences out, and then separate the facial ectoderm and mesenchyme.

فصل الأدمة الجنينية الماوس الوجه واللحمة المتوسطة

 Orofacial clefts are the most frequent  craniofacial defects, which affect 1.5 in 1,000 newborns worldwide1,2. Orofacial clefting is caused by abnormal ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

بمساعدة الليزر تسليخ مجهري (لام) كأداة للالترانسكربتي التنميط من أنواع الخلايا الفردية

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

Comparison of Tobacco Host Cell Protein Removal Methods by Blanching Intact Plants or by Heat Treatment of Extracts

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical proteins, such as antibodies, vaccine candidates and enzymes. These must be purified from the plant biomass but chromatography steps are hindered by the high concentrations of host cell proteins (HCPs) in plant extracts. However, most HCPs irreversibly aggregate at temperatures above 60 &#176;C facilitating subsequent purification of the target protein. Here, three methods are presented to achieve the heat precipitation of tobacco HCPs in either intact leaves or extracts. The blanching of intact leaves can easily be incorporated into existing processes but may have a negative impact on subsequent filtration steps. The opposite is true for heat precipitation of leaf extracts in a stirred vessel, which can improve the performance of downstream operations albeit with major changes in process equipment design, such as homogenizer geometry. Finally, a heat exchanger setup is well characterized in terms of heat transfer conditions and easy to scale, but cleaning can be difficult and there may be a negative impact on filter capacity. The design-of-experiments approach can be used to identify the most relevant process parameters affecting HCP removal and product recovery. This facilitates the application of each method in other expression platforms and the identification of the most suitable method for a given purification strategy.

Three heat precipitation methods are presented that effectively remove more than 90% of host cell proteins (HCPs) from tobacco extracts prior to any other purification step. The plant HCPs irreversibly aggregate at temperatures above 60 &#176;C.

مقارنة التبغ طرق إزالة المضيف بروتين الخلية من قبل السلق النباتات سليمة أو عن طريق المعالجة الحرارية من مقتطفات

Plants not only provide food, feed and raw materials for humans, but have also been developed as an economical production system for biopharmaceutical ...