그리고 어린이 병원 보스턴에서 심장 전도 기금에 기부,이 작품은 연구 국립 보건원에서 보조금 (HL088206 HL068915)에 의해 지원됩니다.

1 부 : 전신 마취의 유도 <ol><li> 200-250g 루이스 쥐 50 MG / kg 케타민을 및 intraperitoneal 캐비티에 30 G 바늘로 주입 4 MG / kg Xylazine과 anesthetized 있습니다. 상단 복부 오른쪽 가슴은 머리를 철저하게 70 %의 에탄올 다음 povidone - 요오드 국소 살균 솔루션 청소 절개 사이트를 제거 꺾을 수 있습니다. </li><li> 동물은 endotracheally 작은 동물 인공 호흡기 (하버드 장치)에 연결된 16 G 정맥 카테터를 사용하여 intubated 있습니다. 인공 호흡기가 전신 마취를 유지하기 위해 100 % 산소와 혼합 Isoflurane 1.0 % (박스터)에 0.5을 공급하는 기화기에 연결되어 있습니다. 호흡 속도와 갯벌 볼륨이 쥐의 체중에 따라 조정됩니다. 동물은 수술하는 동안 체온을 유지하기 위해 가열 패드 (후드 온습 패드)에 배치됩니다. </li><li> 수술 pulsoximeter 장치 (Nellcor)을 통해 심장 박동과 산소 포화도를 모니터링하려면 것은 다시 사지 중 하나에 잘립니다. </li></ol> 2 부 : Thoracotomy 및 엔지니어링 조직 주입 <ol><li> 외과 사이트 타월 클램프와 장소에서 개최되는 불임 수술 draping와 준비가되어 있습니다. </li><li> 절개는 단 5 일 늑간 공간 위의 안테 - 측면에서 오른쪽 가슴의 측면 벽에 이루어집니다. 가슴의 내부는 5 일 늑간 공간을 통해 액세스할 수 있으며, 작은 견인기 (Roboz)은 전망을 최대화하기 위해 배치됩니다. </li><li> 심낭은 단지 마음의 꼭대기쪽으로 기지에서 오른쪽 아트리움과 우심실의 부록 위의 컷입니다. 그 후 유명 홈이 식별되고 aorto - atrio - 심실 삼각형에 인접한 epicardial 계층은 부드럽게 떨어져 조롱이다. </li><li> 7-0 Prolene 봉합사 (Ethicon)은 우심실의 기초 어깨를 통해 배치됩니다. A 2 X 2 X 2mm 기사가 설계 조직 구조의 해부 및 유명 홈 내에 위치하고 부드럽게 Prolene 봉합사로 묶어입니다. </li><li> 수술 부위는 출혈에 대한 검사 두 4-0 Vicryl 봉합 (Ethicon)은 리브 - 케이지의 폐쇄에 대한 상위 및 하위 늑간 공간 사이에 위치하고 있습니다. </li><li> 모든 명백한 pneumothorax은 환풍기의 방법으로 폐를 확대하여 발굴이다. </li><li> 가슴 벽 그런 다음 레이어에서 닫힙니다. </li></ol> 파트 3 : 복구 <ol><li> 동물은 수술에서 회복하는 동안, 절개 사이트 5 ML 0.25 % Bupivacaine [2]로 가득 차 있습니다. </li><li> 동물은 인공 호흡기의 이유 다음 extubated이며 완전히 깨어과 흉골되면 새로운 침구와 케이지로 반환됩니다. 쥐는 이후 3 일간 0.01 밀리그램 / kg Buprenorphine 진통제 subcutaneously 매 12 시간을 관리합니다. </li></ol> 4 부 : 대표 결과 제대로 수행한 경우, 동물보다 90 % 이상 복구에 (그림 1) 심장의 AV - 홈 결과의 설계 조직의 이식 수술을 설명했다. <img src="/files/ftp_upload/1139/Figure1.jpg" alt="그림 1" /> 그림 1. 설계 조직의 이식의 사이트를 보여주는 노출 마음의 사진.

<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>

동물 실험은 어린이의 병원 보스톤에있는 기관 애니멀 케어 및 사용위원회에 의해 명시된 지침과 규정에 따라 수행되었다.

수술에 유리한 결과가 들면, 그것은 제대로 intubate 및 invasively 동물을 환기하는 것이 중요합니다. 쥐가 단순히 진정하고 저절로 숨을 쉴 수있다면 흉부가 설치류의 일반적인 늑막에 의한 개설하는 동안, 모두 폐 필연적으로 붕괴됩니다. 가슴을 닫기 전에 모든 pneumothorax 완전히 제거되어야하며 폐가 완전히 무기폐을 줄이기 위해 확장. 그렇지 않으면, 인공 호흡기 및 복구에서 이유하는 것은 실패?...

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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>
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<td>Inspira ASV Ventilator</td>
<td>Equipment</td>
<td>Harvard Apparatus, Holliston, MA 01746 USA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Handheld Pulse Oximeter</td>
<td>Equipment</td>
<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>
<td>RS-6514</td>
<td>&nbsp;</td>
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<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>
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<td>Xylazine (AnaSed)</td>
<td>Reagent</td>
<td>Lloyd</td>
<td>LB15705A</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>
<td>&nbsp;</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

설치류 수술은 종종 엔지니어링 조직의 유틸리티를 평가하는 중요한 구성 요소입니다. 수술 절차의 다양한는 일반적인 실험실 쥐 또는 마우스로 진행 이러한 꽤 자주 벤치 - 상단 실험 및 대형 동물 실험이나 인체에 실험 사이의 중간 단계 역할을 수 있습니다. 설치류는 비계의 재료와 세포의 새로운 조합을 테스트에 설립, 비용 효율적인, 그리고 생리학 - 관련 모델 시스템을 제공 감안할 때, 그들은 심장 혈관 조직 공학 연구에 적합 특히있다. 현재, 우리는 방실에 myogenic 전구 세포를 포함하는 설계 조직 (AV) 쥐 심장의 홈을 이식하는 심장 절개 수술을 설명합니다. 이 이식은 전체 심장 블록 [1]과 소아 환자에서 기존의 맥박 조정기 주입에 대한 대안 치료를 제공하는 궁극적인 목표로 오른쪽 아트리움과 우심실 사이의 전선관을 만들 것입니다. 설계 조직은 thoracotomy를 통해 AV - 홈에 이식합니다. 우리 목적을 위해서도, 루이스 쥐가 anesthetized 및 invasively 멸균 수술 도중에 긍정적인기도 압력을 유지하기 위해 통풍이 있습니다. 심장에 접근은 5 안테 - 가로 절개를 통해 권리 thoracotomy에 의해 수행됩니다<sup&gt; 일</sup&gt; 늑간 공간. 조직 구조는 하나의 7-0 Prolene의 봉합사를 사용하여 AV 홈에 고정하고 마음의 복부 부분에서 우심실과 아트리움 사이에 위치합니다. epicardium는 부분적으로받는 심근 세포 및 엔지니어링 조직에 포함된 사람 간의 직접 접촉을 허용하는 제거됩니다. 이식 다음, 가슴 벽이 레이어에 폐쇄 모든 pneumothorax은 대피하고, 동물은 extubated와 진통제로 치료합니다.

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

여기, 우리는 방실 (AV) - 홈 성인 루이스 쥐에서 설계 조직을 이식하는 심장 수술을 설명합니다.

랫 중심에 엔지니어링된 조직의 주입

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 조회수.  Children's Hospital Boston and Harvard Medical School. 여기, 우리는 방실 (AV) - 홈 성인 루이스 쥐에서 설계 조직을 이식하는 심장 수술을 설명합니다.

비디오: 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].

Myogenic 엔지니어링된 조직 구조의 제작

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

연구

생물학

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.

성인 쥐 심장 Myocytes의 차 문화

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.

에 잔 심장을 떠올리 Drosophila</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.

의 전자 현미경을위한 배아의 준비 Drosophila 태아의 심장 관

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.

Ionising 방사선에 대한 응답으로 γH2AX Foci의 부량

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.

조직 샘플에서 γH2AX Foci의 Quantitation

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.

Mitochondria 높은 결합 랫 마음의 준비

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.

이온화 방사선 다음 γH2AX의 공간적 분포 평가

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.

Clonogenic 분석 : 자기편 셀

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.

전 · Cryo 키트를 사용하여 마우스 정자 Cryopreservation 및 복구

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