עבודה זו נתמכת על ידי מענקי מחקר מן המכון הלאומי לבריאות (HL068915; HL088206) ותרומות לקרן הולכה לב בבית החולים לילדים בבוסטון.

חלק 1: אינדוקציה של הרדמה כללית <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> חולדה 200-250 גרם לואיס הוא הרדים עם קטמין מ&quot;ג / ק&quot;ג 50 ו -4 מ&quot;ג / ק&quot;ג Xylazine מוזרק עם מחט 30 G לחלל intraperitoneal. הבטן העליונה והחזה הנכונים מקוטע להסיר שיער באתר חתך לנקות ביסודיות עם אתנול 70% ולאחר מכן פתרון povidone-יוד חיטוי מקומי. </li><li style=";text-align:right;direction:rtl"> בעל חיים הוא מחובר לצינורות endotracheally באמצעות קטטר תוך ורידי 16 G מחובר למכונת הנשמה חיה קטנה (Apparatus הרווארד). הנשמה מחוברת מאדה לספק 0.5% עד 1.0 Isoflurane (Baxter) מעורבב עם חמצן 100% כדי לשמור על הרדמה כללית. קצב הנשימה ונפח גאות מותאם על פי משקל הגוף של החולדה. חיה ממוקם על כרית חימום (הוד Thermo-Pad), כדי לשמור על חום הגוף במהלך ההליך הכירורגי. </li><li style=";text-align:right;direction:rtl"> כדי לעקוב אחר קצב הלב ואת הרוויה חמצן בכל ההליך הכירורגי מכשיר pulsoximeter (Nellcor) נחתך על אחת הגפיים בחזרה. </li></ol> חלק 2: פתיחת בית החזה ואת השתלת רקמות מהונדסות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> האתר כירורגית מוכן עם כורכת כירורגי סטרילי כי הוא החזיק במקום מלחציים עם מגבת. </li><li style=";text-align:right;direction:rtl"> מבצעים חתך מן antero-לרוחב הקיר לרוחב של החזה הימני, ממש מעל המרחב צלעי ה 5. החלל הפנימי של החזה הוא לגשת בחלל צלעי 5 וה מפשק קטן (Roboz) ממוקמת על מנת למקסם את התצוגה. </li><li style=";text-align:right;direction:rtl"> קרום הלב היא לחתוך ממש מעל התוספתן של אטריום הנכון החדר הימני מהבסיס לכיוון הקודקוד של הלב. לאחר מכן, חריץ ה-AV מזוהה ואת השכבה epicardial סמוך למשולש aorto-atrio-חדרית היא הקניטה בעדינות מזה. </li><li style=";text-align:right;direction:rtl"> 7-0 Prolene תפר (Ethicon) ממוקם דרך הכתף הבסיס של החדר הימני. 2 x 2 x 2 מ&quot;מ הוא חתיכת גזור מן לבנות את רקמות מהונדסות וממוקמים בתוך חריץ ה-AV וקשר מעדנות עם תפר Prolene. </li><li style=";text-align:right;direction:rtl"> בתחום כירורגית לוודא שהדימום ושני תפרים Vicryl 4-0 (Ethicon) ממוקמים בין העליון מרחב צלעי נמוך סגירת כלוב הצלעות. </li><li style=";text-align:right;direction:rtl"> Pneumothorax נראית לעין הוא נחפר על ידי הרחבת הריאות בדרך של מכונת ההנשמה. </li><li style=";text-align:right;direction:rtl"> קיר החזה סגור אז בשכבות. </li></ol> חלק 3: שחזור <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בעוד החיה מתאושש מהניתוח, האתר הוא חתך שחדר עם 5 מ&quot;ל 0.25% Bupivacaine [2]. </li><li style=";text-align:right;direction:rtl"> החיה היא extubated הבאים הגמילה ממכונת ההנשמה והוא חזר כלוב עם מצע טרי פעם אחת הוא ער לחלוטין sternal. עכברוש ניתנת לאחר מכן 0.01 מ&quot;ג / ק&quot;ג עצירות כאבים תת עורי כל 12 שעות במשך 3 ימים. </li></ol> חלק 4: תוצאות נציג אם מבוצעת כראוי, תיאר כירורגי של השתלת רקמות מהונדסות ב AV-חריץ של הלב (איור 1) תוצאות התאוששות יותר מ 90% של בעלי החיים. <img src="/files/ftp_upload/1139/Figure1.jpg" alt="איור 1" /> באיור 1. תצלום של הלב החשוף מראה את האתר של השתלת רקמות מהונדסות.

<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>

ניסויים בבעלי חיים בוצעו בהתאם להנחיות והתקנות שנקבעו על ידי טיפול בבעלי חיים מוסדיים ועדת השתמש ב מבית החולים לילדים בבוסטון זה.

לקבלת תוצאה חיובית כדי ההליך הכירורגי, חשוב שצריך לצנרר ו פולשני לאוורר את החיה. אם הוא עכברוש מסומם בפשטות מותר לנשום באופן ספונטני, הריאות הן בהכרח התמוטטות בעוד החזה נפתח עקב הצדר נפוץ במכרסמים. לפני שסגר את החזה, pneumothorax כל צריך לחסל לחלוטין הריאות מלא מורחבת להפחי...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<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>
<tr>
<td>Inspira ASV Ventilator</td>
<td>Equipment</td>
<td>Harvard Apparatus, Holliston, MA 01746 USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Handheld Pulse Oximeter</td>
<td>Equipment</td>
<td>Nellcor, Boulder, CO 80301 USA </td>
<td>N20PA</td>
<td>&nbsp;</td>
<tr>
<td>Retractor</td>
<td>Equipment</td>
<td>Roboz, Gaithersburg, MD 20898 USA</td>
<td>RS-6514</td>
<td>&nbsp;</td>
<tr>
<td>Isoflurane (Forane)</td>
<td>Reagent</td>
<td>Baxter</td>
<td>1001936040</td>
<td>&nbsp;</td>
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<td>Ketamine HCl</td>
<td>Reagent</td>
<td>HOSPIRA Inc., Lake Forest, IL 60045</td>
<td>RL-0065</td>
<td>&nbsp;</td>
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<td>Xylazine (AnaSed)</td>
<td>Reagent</td>
<td>Lloyd</td>
<td>LB15705A</td>
<td>&nbsp;</td>
<tr>
<td>Buprenorphine HCL Inject</td>
<td>Reagent</td>
<td>Bedford Labs, Bedford, OH 44146 USA</td>
<td>Div-BUP-P00</td>
<td>&nbsp;</td>
<tr>
<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

ניתוח מכרסמים לעתים קרובות מרכיב חשוב בהערכת התועלת של רקמות מהונדסות. מגוון רחב של ניתוחים ניתן לבצע חולדות מעבדה או עכברים משותפים אלה לעתים קרובות לשמש שלב ביניים בין העליונה הספסל ניסויים ניסויים בבעלי חיים גדולים או בניסויים בבני אדם. בהתחשב בכך מכרסמים לספק מערכת מודל הוקמה, העלות האפקטיבית, ו-פיזיולוגית רלוונטי אשר לבדוק שילובים הרומן של חומרים פיגומים התאים, הם מתאימים במיוחד ללימודי הנדסה רקמות וכלי דם. נכון להיום, אנו מתארים הליך ניתוחי לב פתוח להשתיל רקמות מהונדסות המכיל ובתאים myogenic בתוך החדרים והעליות (AV) חריץ של לב חולדה. שתלים אלו נועדו ליצור צינור חשמל בין אטריום הנכון החדר הימני עם המטרה הסופית של מתן טיפול חלופי השתלת קוצב לב קונבנציונאלי מטופלים ילדים עם לחסום לב שלם [1]. רקמות מהונדסות הוא מושתל ה-AV-Groove באמצעות פתיחת בית החזה. לענייננו, חולדות לואיס מורדמים ומאוורר פולשני כדי לשמור על לחץ חיובי בדרכי הנשימה במהלך ההליך הכירורגי סטרילית. הגישה אל הלב מבוצע על ידי פתיחת בית החזה זכות דרך חתך לרוחב antero-ב 5<sup&gt; ה</sup&gt; צלעי בחלל. לבנות רקמת קבוע בחריץ ה-AV באמצעות תפר אחד 7-0 Prolene וממוקמים בין החדר אטריום ימין על החלק הגחוני של הלב. Epicardium יוסר חלקית כדי לאפשר מגע ישיר בין התאים הנמען שריר הלב לבין אלה הכלולים רקמות מהונדסות. לאחר ההשתלה, בית החזה נסגר בשכבות, pneumothorax כל הוא פונה, והוא בעל החיים extubated ומטופלים עם משכך כאבים.

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

כאן אנו מתארים הליך ניתוחי לב להשתיל רקמה מהונדסת העליות והחדרים (AV), חריץ של עכברוש לואיס מבוגר.

השתלת רקמות מהונדסות בלב החולדה

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. כאן אנו מתארים הליך ניתוחי לב להשתיל רקמה מהונדסת העליות והחדרים (AV), חריץ של עכברוש לואיס מבוגר.

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].

המצאה של Myogenic בונה רקמות מהונדסות

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.

תרבות ראשונית של הלב Myocytes למבוגרים עכברוש

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.

לדמיין את הלב הפועם תסיסנית</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.

הכנת עוברים אלקטרונים מיקרוסקופית של תסיסנית לב עוברי צינור

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 &amp;#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.

כימות γH2AX מוקדים בתגובה קרינה Ionising

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 &amp;#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.

Quantitation של γH2AX מוקדים של דגימות רקמה

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.

הכנה של הלב עכברוש מצמידים מאוד המיטוכונדריה

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 &amp;#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.

הערכת התפלגות מרחבית של γH2AX הבאים בקרינה מייננת

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.

Assay Clonogenic: תאים חסיד

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.

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

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.

הזרע עכבר cryopreservation ושחזור באמצעות · אני 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 ...