기증자 준비 및 심장 하베스트 : <ol><li style="text-align: left"> 마우스 수술 분야에 pentobarbital과 배치 부정사의 intraperitoneal 주사와 함께 anesthetized 있습니다. </li><li style="text-align: left"> 긴 정중선 복부 절개가 이루어집니다. 복부 내용은 복부 대동맥과 하부 대정 맥을 노출하기 위해 왼쪽에 거즈로 철회 있습니다. </li><li style="text-align: left"> 헤파린 (10u/ml) 1 ML은 heparinization에 대한 열등 베나의 정맥에 주입합니다. </li><li style="text-align: left"> thoracotomy이 이루어집니다 : 갈비뼈는 척추의 양쪽에 나누어 및 흉부 앞쪽에 벽은 심장을 노출 levered 최대집니다. </li><li style="text-align: left"> 하부 대정 맥, 우수한 베나 카바와 azygous 정맥은 6-0 실크 출혈도 잡았과 ligatures 우수 나누어집니다. </li><li style="text-align: left"> 오름차순 대동맥과 폐동맥이 멀리 distally 최대한 분리하고 나누어집니다. </li><li style="text-align: left"> 폐 정맥은 출혈도 잡았과 유대 관계에 말초 나누어집니다. </li><li style="text-align: left"> 마음 4 C O 생리 식염수로 perfused입니다. </li><li style="text-align: left"> 마음은 얼음 차가운 살린 솔루션에 저장됩니다. </li></ol> 수신자 준비 및 이식 <ol><li style="text-align: left"> 마우스 수술 분야에 pentobarbital과 배치 부정사의 intraperitoneal 주사와 함께 anesthetized 있습니다. </li><li style="text-align: left"> 긴 정중선 복부 절개가 이루어집니다. 복부 내용은 복부 대동맥과 하부 대정 맥을 노출 거즈와 복부 외부 철회 있습니다. </li><li style="text-align: left"> 복부 대동맥 및 하부 대정 맥의 지점은 노출과 10-0 봉합과 출혈도 잡았 있습니다. </li><li style="text-align: left"> 근위 및 말초 ligatures는 각각 대동맥 및 하부 베나의 정맥 주위에 배치됩니다. </li><li style="text-align: left"> venotomy는 30 게이지 바늘로받는 사람의 하부 대정 맥에서 이루어집니다. 오프닝은 다음 마이크로 가위로 기증자의 폐동맥 대동맥와 같은 길이로 연장됩니다. </li><li style="text-align: left"> aortotomy는 30 게이지 바늘로받는 사람의 복부 대동맥에서 이루어집니다. </li><li style="text-align: left"> 오픈 복부 대동맥 및 하부 대정 맥은 생리 식염수로 irrigated 있습니다. </li><li style="text-align: left"> 기증자의 심장은받는 사람의 복부의 왼쪽에 위치하고 거즈로 덮혀 있습니다. </li><li style="text-align: left"> 숙박 봉합은 기증자의 상승 대동맥과 복부 대동맥 수신자의 근위 및 말초 apexes에 게재됩니다. </li><li style="text-align: left"> 받는 사람의 복부 대동맥과 기증자의 오름차순 대동맥의 오른쪽의 문합은 계속 봉합과 함께 완료됩니다. </li><li style="text-align: left"> 두 사람은 남아의 봉합은 기증자의 폐동맥과받는 사람의 하부 대정 맥의 문합을위한 기증자의 폐동맥과 근위 아펙스과받는 사람의 하부 대정 맥의 말초 꼭대기에서 먼저입니다. </li><li style="text-align: left"> 하부 대정 맥과 기증자의 폐동맥의 왼쪽 벽면은 하부 대정 맥 내부의 지속적인 봉합과 함께 닫힙니다. </li><li style="text-align: left"> 근위 아펙스 숙박 봉합 하나의 매듭 후, 하부 대정 맥과 기증자의 폐동맥의 오른쪽 벽은 하부 베나 동굴 밖으로 지속적으로 봉합과 함께 폐쇄됩니다. </li><li style="text-align: left"> 기증자의 심장은받는 사람의 복부의 오른쪽으로 켜집니다. 받는 사람의 복부 대동맥과 기증자의 상승 대동맥의 문합의 오른쪽 벽은 계속 봉합과 함께 폐쇄됩니다. </li><li style="text-align: left"> 문합가 만든 후, 말초 묶인는 문합에서 출혈을 확인 제거됩니다. </li><li style="text-align: left"> 문합로부터 출혈이 거의가있는 경우, 근위 묶인가 제거됩니다. 기증자의 마음은 즉시 피로 채우고, 마음의 색깔은 빨간색되고 그것은 다시 때려 시작합니다. </li><li style="text-align: left"> 창자는 6-0로 연속 봉합 폐쇄 복부에 반환됩니다. </li><li style="text-align: left"> 받는 마우스가 따뜻한 지역에 위치합니다. 일시간 후받는 복구합니다. </li></ol>

<ol>
	<li>Corry, R. o. b. e. r. t. . J., Winn, H. e. n. r. y. . J., Russell, P. a. u. l. . S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primarily+vascularized+allografts+of+heart+in+mice.">Primarily vascularized allografts of heart in mice.</a> Transplantation. 16, 343-350 (1973).</li></ol>

검색 결과 우리는 성공적으로 생쥐에서 심장 이식 heterotopic 이상의 1000 명의 환자를 수행하고 야생 유형, 유전자 변형과 유전자 녹아웃 마우스를 포함한 마우스의 다양한 변종, 90 %의 성공률을 통해 달성했습니다. 토론 DRS 이후. 코리와 러셀은 1973 년 생쥐에서 heterotopic 심장 이식을 도입,이 모델은 면역 거부 반응을 공부하고 새로운 immunosuppressive 전략을 개발하기 위해 ?...

heterotopic heart transplantation in mice

그것이 DRS에 의해 도입된 이후 마우스 heterotopic 심장 이식이 널리 사용되었습니다. 1973 년 코리와 러셀. 이제 새로운 유전자 변형과 유전자 녹아웃 마우스가 사용 가능한지 거부하고 면역 반응을 공부에 특히 유용하며, immunologic의 시약의 다수가 개발되었습니다. 심장 이식 모델은 있지만, 기술적으로 많은 도전, 피부 이식 모델보다 엄격한 있습니다. 우리는 수정 기술을 개발하고 생쥐에서 심장 이식 heterotopic 1000 성공적으로 가지 경우를 통해 완료했습니다. 상승 대동맥과 복부 대동맥의 교합 (anastomosis)을 만들 때 두 숙박 봉합 그런 다음 계속 봉합과 대동맥의 두 측면에 문합에 대한 11-0 봉합사를 사용하여 기증자의 상승 대동맥과받는 복부 대동맥의 근위 및 말초 apexes에 게재됩니다. 숙박 봉합은 문합 쉽게 만들어 11-0는 출혈과 혈전증을 방지하기위한 이상적인 봉합의 크기입니다. 폐동맥과 하부 대정 맥의 교합 (anastomosis)을 만들 때 두 숙박 봉합은 기증자의 폐동맥과 함께 근위 아펙스과받는 사람의 하부 대정 맥의 말초 꼭대기에서 이루어집니다. , 근위 아펙스 체류 한 매듭이 하부 대정 맥의 오른쪽 벽과 기증자의 폐동맥을 봉합 후 하부 대정 맥과 기증자의 폐동맥의 왼쪽 벽면은 하부 대정 맥의 내부에서 계속 봉합과 함께 폐쇄 10-0 봉합과 하부 베나 동굴 밖으로 지속적으로 봉합과 함께 폐쇄됩니다. 문합 단지 하부 대정 맥과 10-0 봉합의 한쪽에 만들어되기 때문에이 방법을 수행하기 쉬운 것은 출혈과 혈전증을 피하기 위해 오른쪽 크기입니다. 이 문서에서는, 우리는 비디오를 보완하기 위해 기술의 세부 사항을 제공합니다.

Watch this Scientific Journal Video about Heterotopic Heart Transplantation in Mice at JoVE.com

Heterotopic Heart Transplantation in Mice

마우스 heterotopic 심장 이식 모델은 거부하고 면역 반응의 메커니즘을 연구를위한 중요한 방법으로 많은 수사관에 의해 입증되었습니다. 그러나, 관련 기술은 여전히​​ 도전하고 있습니다. 표준 기술을 수정하여 우리는 1000 개 이상의 이식 성공을 있었다.

마우스의 Heterotopic 심장 이식

The mouse heterotopic heart transplantation has been used widely since it was introduced by Drs. Corry and Russell in 1973. It is particularly valuable ...

heterotopic-heart-transplantation-in-mice

Research

JoVE Journal

Biology

29.4K 조회수.  University of California, San Francisco - UCSF. 마우스 heterotopic 심장 이식 모델은 거부하고 면역 반응의 메커니즘을 연구를위한 중요한 방법으로 많은 수사관에 의해 입증되었습니다. 그러나, 관련 기술은 여전히​​ 도전하고 있습니다. 표준 기술을 수정하여 우리는 1000 개 이상의 이식 성공을 있었다.

비디오: Heterotopic Heart Transplantation in Mice

Small Bowel Transplantation In Mice

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive medications, infection prophylaxis, and suitable patient selection helped improve actuarial graft and patient survival rates for all types of intestine transplantation. Patients with irreversible intestinal failure and complications of parenteral nutrition should now be routinely considered for small intestine transplantation. However, Survival rates for small intestinal transplantation have been slow to improve compares increasingly favorably with renal, liver, heart and lung. The small bowel transplantation is still unsatisfactory compared with other organs. Further progress may depend on better understanding of immunology and physiology of the graft and can be greatly facilitated by animal models. A wider use of mouse small bowel transplantation model is needed in the study of immunology and physiology of the transplantation gut as well as efficient methods in diagnosing early rejection. However, this model is limited to use because the techniques involved is an extremely technically challenging. We have developed a modified technique. When making anastomosis of portal vein and inferior vena cava, two stay sutures are made at the proximal apex and distal apex of the recipient s inferior vena cava with the donor s portal vein. The left wall of the inferior vena cava and donor s portal vein is closed with continuing sutures in the inside of the inferior vena cava after, after one knot with the proximal apex stay suture the right wall of the inferior vena cava and the donor s portal vein are closed with continuing sutures outside the inferior vena cave with 10-0 sutures. This method is easier to perform because anastomosis is made just on the one side of the inferior vena cava and 10-0 sutures is the right size to avoid bleeding and thrombosis.  In this article, we provide details of the technique to supplement the video.

The mouse small bowel transplantation model has been recognized as an important tool to study mechanismes of immune rejection and screen new immunosuppressive drugs. However, this model is limited to use because the techniques involved is an extremely technically challenge. Now we introduce the modified technique.

마우스에 작은 창자 이식

Since 1990, the development of tacrolimus-based immunosuppression and improved surgical techniques, the increased array of potent immunosuppressive ...

연구

생물학

Transplantation of Pancreatic Islets Into the Kidney Capsule of Diabetic Mice

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice. We found that it was easier to concentrate the islets or cells into pellets in the final delivery tubing (PE50) used to transplant the cells under the kidney capsule. This technique provides both speed and ease while reducing any undue stress to the cells or to the mouse.

Loading: Settled, hand picked, islets or pelleted cells are carefully aspirated off the bottom of a 1.5 mL microcentrifuge tube using a p200 pipetteman and a straight, thin-wall pipette tip. A length of PE50 tubing is attached to the pipette tip using a small silicone adapter tubing. Cells are allowed to settle, in the tip, and then are transferred to the PE50 tubing by slowly dialing the pipetteman. Once the cells are near the end of the PE50 tubing, a kink is made and the silicone adaptor tubing is placed over the kink. The PE50 tubing is transferred to a 15 mL conical containing a cut 5 mL pipet, and the PE50 tubing is taped over the side of the 5 mL pipet to prevent curling during centrifuging. Cells are allowed to reach 1,000 rpm and stopped.
Transplantation: Recipient mice are anesthetized, shaved, and cleaned. A small incision is made on the left flank of the mouse and the kidney is exposed. The kidney, fat, and tissue are kept moist with normal saline swab. The distal end of the PE50 is attached to a Hamilton screw drive syringe, containing a pipette tip, using the silicone adaptor tubing. A small nick is made on the right flank side of the kidney, not too large nor too deep. The beveled end of the PE50 tubing, nearest the cells, is carefully placed under the capsule, the tubing is moved around gently to make space while swabbing normal saline; a dry capsule can tear easily. A small air bubble is delivered under the capsule by slowly dialing the syringe screw drive. Islets are then slowly delivered behind the air bubble. Once the islets have been delivered kidney homeostasis is maintained and the knick is cauterized with low heat. The kidney is placed back into the cavity and the peritoneum and skin are sutured and stapled. Mice are immediately treated with Flunixin and Buprenorphine s.q. and placed in a cage on a heating pad.

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of mice. Cells are concentrated into pellets in the final tubing used for transplanting the cells under the kidney capsule. The ease of this technique reduces stress to the cells and the mouse.

당뇨병 마우스의 신장 캡슐으로 췌장 아일 레츠의 이식

Our protocol was developed to cleanly and easily deliver islets or cells under the kidney capsule of diabetic or normal mice.  We found that it was easier ...

Murine Skin Transplantation

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to MHC-disparate donor antigens. The aim of this video-article is to provide the viewer with a step-by-step visual demonstration of skin transplantation using the mouse model. The protocol is divided into 5 main components: 1) harvesting donor skin; 2) preparing recipient for transplant; 3) skin transplant; 4) bandage removal and monitoring graft rejection; 5) helpful hints. Once proficient, the procedure itself should take &lt;10 min to perform.

Allogeneic skin transplantation is a standard model to assay host T cell responses to MHC-disparate donor antigens. This video-article provides a visual tutorial of each step involved in performing a BALB/c--&gt;C57BL/6 skin transplant.

Murine 피부 이식

As one of the most stringent and least technically challenging models, skin transplantation is a standard method to assay host T cell responses to ...

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 ...

Heterotopic and Orthotopic Tracheal Transplantation in Mice used as Models to Study the Development of  Obliterative Airway Disease

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the pathophysiology, treatment and prevention of OAD, different animal models of tracheal transplantation in rodents have been developed (1-7). Here, we use two established models of trachea transplantation, the heterotopic and orthotopic model and demonstrate their advantages and limitations.
For the heterotopic model, the donor trachea is wrapped into the greater omentum of the recipient, whereas the donor trachea is anastomosed by end-to-end anastomosis in the orthotopic model.
In both models, the development of obliterative lesions histological similar to clinical OAD has been demonstrated (1-7).
This video shows how to perform both, the heterotopic as well as the orthotopic tracheal transplantation technique in mice, and compares the time course of OAD development in both models using histology.

This video shows and compares two experimental models to study the development of obliterative airway disease (OAD) in mice, the heterotopic and orthotopic tracheal transplantation model.

Obliterative 에어웨이 질환의 발전을 연구하는 모델로 사용 마우스의 Heterotopic 및 Orthotopic Tracheal 이식

Obliterative airway disease (OAD) is the major complication after lung transplantations that limits long term survival (1-7).
To study the ...

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 &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.

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 ...

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.

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. ...

Germ Cell Transplantation and Testis Tissue Xenografting in Mice

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies showed that microinjection of germ cells from fertile donor mice into the seminiferous tubules of infertile recipient mice results in donor-derived spermatogenesis and sperm production by the recipient animal2. The use of donor males carrying the bacterial &beta;-galactosidase gene allowed identification of donor-derived spermatogenesis and transmission of the donor haplotype to the offspring by recipient animals1. Surprisingly, after transplantation into the lumen of the seminiferous tubules, transplanted germ cells were able to move from the luminal compartment to the basement membrane where spermatogonia are located3. It is generally accepted that only SSCs are able to colonize the niche and re-establish spermatogenesis in the recipient testis. Therefore, germ cell transplantation provides a functional approach to study the stem cell niche in the testis and to characterize putative spermatogonial stem cells. To date, germ cell transplantation is used to elucidate basic stem cell biology, to produce transgenic animals through genetic manipulation of germ cells prior to transplantation4,5, to study Sertoli cell-germ cell interaction6,7, SSC homing and colonization3,8, as well as SSC self-renewal and differentiation9,10.
Germ cell transplantation is also feasible in large species11. In these, the main applications are preservation of fertility, dissemination of elite genetics in animal populations, and generation of transgenic animals as the study of spermatogenesis and SSC biology with this technique is logistically more difficult and expensive than in rodents. Transplantation of germ cells from large species into the seminiferous tubules of mice results in colonization of donor cells and spermatogonial expansion, but not in their full differentiation presumably due to incompatibility of the recipient somatic cell compartment with the germ cells from phylogenetically distant species12. An alternative approach is transplantation of germ cells from large species together with their surrounding somatic compartment. We first reported in 2002, that small fragments of testis tissue from immature males transplanted under the dorsal skin of immunodeficient mice are able to survive and undergo full development with the production of fertilization competent sperm13. Since then testis tissue xenografting has been shown to be successful in many species and emerged as a valuable alternative to study testis development and spermatogenesis of large animals in mice14.

Protocols for germ cell transplantation and testis tissue xenografting are described. Germ cell transplantation results in donor-derived spermatogenesis in recipient testes and represents a functional reconstitution assay for identification of spermatogonial stem cells (SSCs). Testis tissue xenografting reproduces testis development and spermatogenesis of various donor species in recipient mice.

배아 세포 이식 및 Testis 조직 마우스에 Xenografting

Germ cell transplantation was developed by Dr. Ralph Brinster and colleagues at the University of Pennsylvania in 19941,2. These ground-breaking studies ...