ويدعم هذا العمل عن طريق المنح البحثية من المعاهد الوطنية للصحة (HL068915 ؛ HL088206) والمساهمات للصندوق التوصيل القلب في مستشفى بوسطن للأطفال.

الجزء 1 : تحريض التخدير العام <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> A 200-250 ز الفئران لويس هو تخدير مع الكيتامين ملغم / كغم من 50 و 4 ملغم / كغم زيلازين حقنه بإبرة G 30 في تجويف داخل الصفاق. يتم قص الجزء العلوي من البطن والصدر الحق في إزالة الشعر ، وموقع شق تنظيفها مع الايثانول 70 ٪ وبعد ذلك ، بوفيدون اليود محلول مطهر موضعي. </li><li style=";text-align:right;direction:rtl"> هو intubated الحيوان endotracheally باستخدام قسطرة الوريد 16 G متصلا بجهاز التنفس الحيوانات الصغيرة (جهاز هارفارد). يتم توصيل جهاز التنفس الاصطناعي لالمرذاذ لتوريد 0،5-1،0 ٪ Isoflurane (باكستر) مختلطة مع الأكسجين 100 ٪ للحفاظ على التخدير العام. يتم ضبط معدل التنفس وحجم المد والجزر وفقا لوزن الجسم من الفئران. يتم وضع الحيوان على وسادة تدفئة (هود الحرارية لوحة) للحفاظ على درجة حرارة الجسم أثناء العملية الجراحية. </li><li style=";text-align:right;direction:rtl"> لرصد معدل ضربات القلب وتشبع الأوكسجين في جميع أنحاء العملية الجراحية جهاز pulsoximeter (Nellcor) يتم قص على واحد من أطرافه الخلفية. </li></ol> الجزء 2 : بضع الصدر وزرع الأنسجة المهندسة <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> مستعدة للموقع الجراحي مع اللف الجراحية العقيمة التي تعقد في مكان مع منشفة المشابك. </li><li style=";text-align:right;direction:rtl"> يتم إجراء شق من الاطراف انتيرو على الجدار الجانبي من الصدر حق ، فوق مساحة 5 الوربية ال. يتم الوصول إلى المناطق الداخلية من الصدر عبر الفضاء في 5 الوربية عشر ويتوضع a مبعاد الصغيرة (Roboz) لزيادة العرض. </li><li style=";text-align:right;direction:rtl"> هو قطع تأمور فقط فوق ملحق الأذين الأيمن والبطين الأيمن من القاعدة باتجاه قمة القلب. بعد ذلك ، يتم التعرف على الأخدود AV ومازحت برفق طبقة epicardial المتاخمة للمثلث aorto - الأذين البطين إربا. </li><li style=";text-align:right;direction:rtl"> يتم وضع 7-0 Prolene خياطة (Ethicon) من خلال الكتف القاعدية من البطين الأيمن. A 2 × 2 × 2 قطعة يتم تشريح مم من بناء الأنسجة المهندسة وضعه داخل الأخدود ، وتعادل AV برفق إلى أسفل مع خياطة Prolene. </li><li style=";text-align:right;direction:rtl"> يتم فحص الميداني للجراحة لوقف نزيف وضعت اثنين الغرز Vicryl 4-0 (Ethicon) بين العلوي والسفلي الوربية الفضاء لإغلاق القفص الصدري. </li><li style=";text-align:right;direction:rtl"> غير المحفورة أي استرواح الواضح من خلال توسيع الرئتين عن طريق جهاز التنفس. </li><li style=";text-align:right;direction:rtl"> ثم يتم إغلاق جدار الصدر في الطبقات. </li></ol> الجزء 3 : الاسترداد <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> في حين أن الحيوان للشفاء من عملية جراحية ، وتسللوا الى موقع شق مع 5 مل من 0.25 ٪ [2] بوبيفاكايين. </li><li style=";text-align:right;direction:rtl"> هو extubated الحيوان التالية الفطام من التنفس الاصطناعي ويتم إرجاعها إلى قفص مع الفراش مرة جديدة أنها مستيقظا تماما والقصية. ويدير لاحقا الفئران 0.01 ملغم / كغم البوبرينورفين تسكين تحت الجلد كل 12 ساعة لمدة 3 أيام. </li></ol> الجزء 4 : نتائج الممثل إذا أجريت بشكل صحيح ، ووصف العمليات الجراحية زرع الأنسجة المهندسة في الأخدود - AV من القلب (الشكل 1) النتائج في استرداد أكثر من 90 ٪ من الحيوانات. <img src="/files/ftp_upload/1139/Figure1.jpg" alt="الشكل 1" /> الشكل 1. صورة لقلب يتعرض تبين موقع زرع أنسجة هندسيا.

<ol>
	<li>Choi, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+conduction+through+engineered+tissue.">Cardiac conduction through engineered tissue.</a> Am J Pathol. 169, 72-85 (2006).</li><li>Shin, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low-dose+systemic+bupivacaine+prevents+the+development+of+allodynia+after+thoracotomy+in+rats.+Anesth+Analg.">Low-dose systemic bupivacaine prevents the development of allodynia after thoracotomy in rats. Anesth Analg.</a> 107, 1587-1591 (2008).</li><li>Horstick, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surgical+procedure+affects+physiological+parameters+in+rat+myocardial+ischemia:+need+for+mechanical+ventilation.">Surgical procedure affects physiological parameters in rat myocardial ischemia: need for mechanical ventilation.</a> Am J Physiol. 276, H472-H479 (1999).</li></ol>

وقد أجريت تجارب على الحيوانات وفقا للمبادئ التوجيهية واللوائح التي وضعتها المؤسسات ورعاية الحيوان اللجنة استخدم في مستشفى بوسطن للأطفال ق.

لتحقيق نتيجة إيجابية لعملية جراحية ، من الأهمية بمكان intubate صحيح وتهوية جراحية الحيوان. إذا كان مخدرا ببساطة الفئران ، وسمحت للتنفس من تلقاء أنفسهم ، وسوف تنهار حتما كلا الرئتين ، بينما يتم فتح القفص الصدري بسبب الجنبة شيوعا في القوارض. قبل أن يغلق في الصدر ، أي استروا...

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<tr>
<td>7x4 Thermo Pad 4/box</td>
<td>Equipment</td>
<td>Hood Thermo-Pad, Summerland, BC Canada</td>
<td>MODEL 704R</td>
<td>&nbsp;</td>
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<td>Inspira ASV Ventilator</td>
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<td>Harvard Apparatus, Holliston, MA 01746 USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Handheld Pulse Oximeter</td>
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<td>Nellcor, Boulder, CO 80301 USA </td>
<td>N20PA</td>
<td>&nbsp;</td>
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<td>Retractor</td>
<td>Equipment</td>
<td>Roboz, Gaithersburg, MD 20898 USA</td>
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<td>Isoflurane (Forane)</td>
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<td>Baxter</td>
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<td>Ketamine HCl</td>
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<td>HOSPIRA Inc., Lake Forest, IL 60045</td>
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<td>Lloyd</td>
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<td>Buprenorphine HCL Inject</td>
<td>Reagent</td>
<td>Bedford Labs, Bedford, OH 44146 USA</td>
<td>Div-BUP-P00</td>
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<td>Bupivacaine 0.25%</td>
<td>Reagent</td>
<td>HOSPIRA Inc., Lake Forest, IL 60045</td>
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implantation of engineered tissue in the rat heart

جراحة القوارض غالبا ما يشكل عنصرا هاما في تقييم جدوى الأنسجة هندسيا. يمكن تنفيذ مجموعة واسعة من العمليات الجراحية في الفئران المختبرية المشتركة أو الفئران ، وهذه كثيرا ما يكون بمثابة خطوة وسيطة بين التجارب بين كبار مقاعد البدلاء والكبيرة التجارب على الحيوانات أو التجارب على الانسان. بالنظر إلى أن القوارض توفير نظام النموذج المعمول بها ، فعالة من حيث التكلفة ، وذات الصلة من الناحية الفسيولوجية ، والتي لاختبار تركيبات جديدة من المواد السقالات والخلايا ، فهي مناسبة تماما وخاصة القلب والأوعية الدموية للدراسات هندسة الأنسجة. في الوقت الحاضر ، ونحن تصف إجراء جراحة القلب المفتوح لزراعة الأنسجة المهندسة التي تحتوي على خلايا مولدة للعضل السلف في الأذينية البطينية (AV) تلم قلب الفئران. وتهدف هذه الغرسات لإنشاء قناة الكهربائية بين الأذين الأيمن والبطين الأيمن مع الهدف النهائي المتمثل في توفير العلاج البديل لزرع جهاز تنظيم ضربات القلب التقليدية في طب الأطفال المرضى مع كتلة القلب الكامل [1]. يتم زرع الأنسجة المهندسة في الأخدود - AV عن طريق بضع الصدر. لأغراضنا ، لويس لتخدير الفئران والتهوية جراحية للحفاظ على الضغط الهوائي الإيجابي أثناء إجراء العمليات الجراحية ومعقمة. يتم تنفيذ هذا النهج إلى القلب من قبل بضع الصدر الحق من خلال شق انتيرو - الجانبية في ال 5<sup&gt; عشر</sup&gt; الوربية الفضاء. يتم إصلاح بناء الأنسجة في الأخدود AV باستخدام واحدة 7-0 وخياطة Prolene المتمركزة بين البطين الأيمن والأذين في الجزء البطني للقلب. تتم إزالة النخاب جزئيا للسماح للاتصال مباشر بين خلايا عضلة القلب المتلقي وتلك الواردة في النسيج هندسيا. بعد زرع ، يتم إغلاق جدار الصدر في الطبقات ، هو إجلاء أي استرواح الصدر ، وextubated الحيوان ومعاملته مسكن.

Watch this Scientific Journal Video about Implantation of Engineered Tissue in the Rat Heart at JoVE.com

Implantation of Engineered Tissue in the Rat Heart

هنا ، نحن تصف إجراء جراحة لزرع القلب الأنسجة المهندسة في الأذينية البطينية (AV) الاخدود من الفئران لويس الكبار.

زرع الأنسجة المهندسة في قلب فأر

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in ...

implantation-of-engineered-tissue-in-the-rat-heart

Research

JoVE Journal

Biology

10.2K Views.  Children's Hospital Boston and Harvard Medical School. هنا ، نحن تصف إجراء جراحة لزرع القلب الأنسجة المهندسة في الأذينية البطينية (AV) الاخدود من الفئران لويس الكبار.

Video: Implantation of Engineered Tissue in the Rat Heart

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

تلفيق عضلي يبني الأنسجة المهندسة

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

Primary Culture of Adult Rat Heart Myocytes

Cultured primary adult rodent heart cells are an important model system for cardiovascular research. Nevertheless, establishment of robust, viable cultured adult myocytes can be a technically challenging, rate-limiting step for many researchers. Here we described a protocol to obtain a high yield of adult rat heart myocytes that remain viable in culture for several days. The heart is isolated and perfused with collagenase and protease under low Ca2+ conditions to recover single myocytes. Ca2+-tolerant cells are obtained by stepwise increases in extracellular Ca2+ concentration in three subsequent wash steps. Cells are filtered, resuspended in culture medium, and plated on laminin coated slips. Cultured myocytes obtained using this protocol are viable for up to four days and are suitable for most experiments including electrophysiology, biochemistry, imaging and molecular biology.

In this paper, we described a typical way to isolate and culture adult rat heart myocytes. Collagenase and protease are used to digest and isolate single myocytes. Myocytes cultured follow this protocol meet most experiment requirements.

الابتدائية ثقافة الكبار Myocytes قلب فأر

Cultured primary adult rodent heart cells are an important model system for cardiovascular research.  Nevertheless, establishment of robust, viable ...

Visualizing the Beating Heart in Drosophila

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in myogenic hearts. A key step in examining heart function in the fly is finding a way to access the heart in a manner that preserves its myogenic function while still allowing the beating heart organ to be observed and recorded. Two different methods for observing and recording the beating heart in both larva and adult Drosophila are described here. Our semi-intact preparation using adult flies allows clear visualization of the abdominal heart without interference from the pigmented cuticle and overlying fat bodies. To record larval heart beats it is necessary to immobilize the larva, which minimizes body wall movements thereby reducing heart movements that are not associated with myocardial contractions. Our methodologies produce stable adult and larval heart preparations that can beat for hours at rates of 1-3 Hz.

Visualizing the Beating Heart in Drosophila

Technique required for visualizing the beating heart in larval and adult Drosophila are presented. Each life stage requires a different methodology.

تصور القلب النابض في ذبابة الفاكهة</em

The Drosophila heart has recently emerged as a good model system for examining the genetic, cellular, and molecular mechanisms underlying function in ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

إعداد الأجنة المجهري للإلكترون من ذبابة الفاكهة أنبوب القلب الجنينية

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

الكمي بؤر γH2AX في الاستجابة للإشعاع المؤين

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Quantitation of &gamma;H2AX Foci in Tissue Samples

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) processes or by exogenous factors, particularly ionizing radiation and radiomimetic compounds. Phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX, is an early response to DNA double-strand breaks1. This phosphorylation event is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Overall, DSB induction results in the formation of discrete nuclear &gamma;H2AX foci which can be easily detected and quantitated by immunofluorescence microscopy2. Given the unique specificity and sensitivity of this marker, analysis of &gamma;H2AX foci has led to a wide range of applications in biomedical research, particularly in radiation biology and nuclear medicine. The quantitation of &gamma;H2AX foci has been most widely investigated in cell culture systems in the context of ionizing radiation-induced DSBs. Apart from cellular radiosensitivity, immunofluorescence based assays have also been used to evaluate the efficacy of radiation-modifying compounds. In addition, &gamma;H2AX has been used as a molecular marker to examine the efficacy of various DSB-inducing compounds and is recently being heralded as important marker of ageing and disease, particularly cancer3. Further, immunofluorescence-based methods have been adapted to suit detection and quantitation of &gamma;H2AX foci ex vivo and in vivo4,5. Here, we demonstrate a typical immunofluorescence method for detection and quantitation of &gamma;H2AX foci in mouse tissues.

Quantitation of &#947;H2AX Foci in Tissue Samples

Quantitation of DNA double-strand breaks on the basis of &gamma;H2AX foci has become an invaluable tool, particularly in radiation biology, for the evaluation of tissue radiosensitivity and effects of radiation modifying compounds. Here we demonstrate the use of an immunofluorescence assay for quantitation of &gamma;H2AX foci in tissue samples.

الكميات بؤر γH2AX في عينات الأنسجة

DNA double-strand breaks (DSBs) are particularly lethal and genotoxic lesions, that can arise either by endogenous (physiological or pathological) ...

Preparation of Highly Coupled Rat Heart Mitochondria

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there have been significant advances in our understanding of the functions of mitochondria other than the generation of energy. These include their role in apoptosis, acting as signalling organelles, mammalian development and ageing as well as their contribution to the coordination between cell metabolism and cell proliferation. Our understanding of biological processes modulated by mitochondria is based on robust methods for isolation and handling of intact mitochondria from tissues of the laboratory animals. Mitochondria from rat heart is one of the most common preparations for past and current studies of cellular metabolism including studies on knock-out animals.
Here we describe a detailed rapid method for isolation of intact mitochondria with a high degree of coupling. Such preparation of rat heart mitochondria is an excellent object for functional and structural research on cellular bioenergetics, transport of biomolecules, proteomic studies and analysis of mitochondrial DNA, proteins and lipids.

We describe &#1072; protocol for isolation of pure, highly coupled rat heart mitochondria for functional or structural studies of cellular bioenergetics, biophysical measurements, proteomics or mitochondrial DNA and lipids analysis.

إعداد قلب فأر اقترن الارتحال الميتوكوندريا

The function of mitochondria in generation of cellular ATP in the process of oxidative phosphorylation is widely recognised. During the past decades there ...

Evaluation of the Spatial Distribution of &gamma;H2AX following Ionizing Radiation

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone H2AX1,2. This phosphorylation of H2AX is mediated by the phosphatidyl-inosito 3-kinase (PI3K) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)3. The phosphorylated form of H2AX, referred to as &gamma;H2AX, spreads to adjacent regions of chromatin from the site of the DSB, forming discrete foci, which are easily visualized by immunofluorecence microscopy3. Analysis and quantitation of &gamma;H2AX foci has been widely used to evaluate DSB formation and repair, particularly in response to ionizing radiation and for evaluating the efficacy of various radiation modifying compounds and cytotoxic compounds4.
Given the exquisite specificity and sensitivity of this de novo marker of DSBs, it has provided new insights into the processes of DNA damage and repair in the context of chromatin. For example, in radiation biology the central paradigm is that the nuclear DNA is the critical target with respect to radiation sensitivity. Indeed, the general consensus in the field has largely been to view chromatin as a homogeneous template for DNA damage and repair. However, with the use of &gamma;H2AX as molecular marker of DSBs, a disparity in &gamma;-irradiation-induced &gamma;H2AX foci formation in euchromatin and heterochromatin has been observed5-7. Recently, we used a panel of antibodies to either mono-, di- or tri- methylated histone H3 at lysine 9 (H3K9me1, H3K9me2, H3K9me3) which are epigenetic imprints of constitutive heterochromatin and transcriptional silencing and lysine 4 (H3K4me1, H3K4me2, H3K4me3), which are tightly correlated actively transcribing euchromatic regions, to investigate the spatial distribution of &gamma;H2AX following ionizing radiation8. In accordance with the prevailing ideas regarding chromatin biology, our findings indicated a close correlation between &gamma;H2AX formation and active transcription9. Here we demonstrate our immunofluorescence method for detection and quantitation of &gamma;H2AX foci in non-adherent cells, with a particular focus on co-localization with other epigenetic markers, image analysis and 3D-modeling.

Evaluation of the Spatial Distribution of &#947;H2AX following Ionizing Radiation

Microscopic analysis of &gamma;H2AX foci, which form following the phosphorylation of H2AX at Ser-139 in response to DNA double-strand breaks, has become an invaluable tool in radiation biology. Here we used an antibody to mono-methylated histone H3 at lysine 4 as an epigenetic marker of actively transcribing euchromatin, to evaluate the spatial distribution of radiation-induced &gamma;H2AX formation within the nucleus.

تقييم التوزيع المكاني للγH2AX التالية الإشعاعات المؤينة

An early molecular response to DNA double-strand breaks (DSBs) is phosphorylation of the Ser-139 residue within the terminal SQEY motif of the histone ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

مولد الرمع الفحص : خلايا منتسب

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Mouse Sperm Cryopreservation and Recovery using the I&middot;Cryo Kit

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies. Many of these valuable animals exist only as live animals, with no backup plan in case of emergency. Cryopreservation of embryos can provide this backup, but is costly, can be a lengthy procedure, and generally requires a large number of animals for success. Since the discovery that mouse sperm can be successfully cryopreserved with a basic cryoprotective agent (CPA) consisting of 18&#37; raffinose and 3&#37; skim milk, sperm cryopreservation has become an acceptable and cost-effective procedure for archiving, distributing and recovery of these valuable strains.
 Here we demonstrate a newly developed I&bull;Cryo kit for mouse sperm cryopreservation. Sperm from five commonly-used strains of inbred mice were frozen using this kit and then recovered. Higher protection ratios of sperm motility (&gt; 60&#37;) and rapid progressive motility (&gt; 45&#37;) compared to the control (basic CPA) were seen for sperm frozen with this kit in 5 inbred mouse strains. Two cell stage embryo development after IVF with the recovered sperm was improved consistently in all 5 mouse strains examined. Over a 1.5 year period, 49 GM mouse lines were archived by sperm cryopreservation with the I&bull;Cryo kit and later recovered by IVF.

Here we demonstrate the newly developed I&#x2022;Cryo kit for mouse sperm cryopreservation. Two-cell stage embryo development with frozen-thawed sperm was improved consistently in 5 mouse strains with the use of this kit. Over a 1.5 year period, 49 genetically modified mouse lines were archived by sperm cryopreservation with the I&#x2022;Cryo kit and later successfully recovered by IVF.

الماوس حفظ بالبرودة والانتعاش الحيوانات المنوية باستخدام · لقد كرو كيت

Thousands of new genetically modified (GM) strains of mice have been created since the advent of transgenesis and knockout technologies.  Many of these ...