Donor Vorbereitung und Herz Harvest: <ol><li style="text-align: left"> Die Maus ist betäubt mit einer intraperitonealen Injektion von Pentobarbital und legte sich rücklings auf den operativen Bereich. </li><li style="text-align: left"> Eine lange Mittellinie Bauchschnitt gemacht wird. Bauch-Inhalte sind mit Gaze nach links zurückgezogen, um die Bauchaorta und die untere Hohlvene aussetzen. </li><li style="text-align: left"> 1 ml Heparin (10u/ml) wird in die untere Hohlvene für Heparinisierung injiziert. </li><li style="text-align: left"> Die Thorakotomie gemacht wird: die Rippen auf beiden Seiten der Wirbelsäule gespalten und der vorderen Brustwand ist gehebelt bis das Herz aussetzen. </li><li style="text-align: left"> Die untere Hohlvene, die Vena cava superior und azygous Vene sind mit 6-0 Seide unterbunden und durchtrennt überlegen die Ligaturen. </li><li style="text-align: left"> Die aufsteigende Aorta und Lungenarterie sind getrennt und so weit distal wie möglich aufgeteilt. </li><li style="text-align: left"> Die Pulmonalvenen sind ligiert und durchtrennt distal der Bande. </li><li style="text-align: left"> Das Herz ist mit 4 C o Kochsalzlösung perfundiert. </li><li style="text-align: left"> Das Herz ist in eiskalten Salzlösung gespeichert. </li></ol> Empfänger Vorbereitung und Transplantation <ol><li style="text-align: left"> Die Maus ist betäubt mit einer intraperitonealen Injektion von Pentobarbital und legte sich rücklings auf den operativen Bereich. </li><li style="text-align: left"> Eine lange Mittellinie Bauchschnitt gemacht wird. Bauch-Inhalte liegen außerhalb des Bauches mit Gaze, die Bauchaorta und die untere Hohlvene freizulegen. </li><li style="text-align: left"> Die Äste der abdominalen Aorta und der Vena cava inferior ausgesetzt sind und ligiert mit 10-0 Nähten. </li><li style="text-align: left"> Die proximalen und distalen Ligaturen sind rund um die Aorta und untere Hohlvene plaziert. </li><li style="text-align: left"> Die Venotomie ist in die untere Hohlvene des Empfängers mit einem 30-Gauge-Nadel gemacht. Die Eröffnung ist dann zu einer Länge von gleich des Spenders pulmonalen Aorta mit Mikro-Schere erweitert. </li><li style="text-align: left"> Die Aortotomie ist in der abdominalen Aorta des Empfängers mit einem 30-Gauge-Nadel gemacht. </li><li style="text-align: left"> Das eröffnet Bauchaorta und die untere Hohlvene mit Kochsalzlösung gespült. </li><li style="text-align: left"> Der Spender ist das Herz auf der linken Seite des Empfängers Bauch gelegt und mit Gaze abgedeckt. </li><li style="text-align: left"> Der Aufenthalt Nähte an den proximalen und distalen Spitzen des Empfängers Bauchaorta mit der Spenderin aufsteigenden Aorta platziert. </li><li style="text-align: left"> Die Anastomose der rechten Seite des Empfängers Bauchaorta und des Spenders aufsteigenden Aorta sind mit anhaltenden Nähten abgeschlossen. </li><li style="text-align: left"> Zwei Haltefäden werden zuerst an den proximalen und distalen Spitze Spitze des Empfängers untere Hohlvene mit der Spenderin Lungenarterie für die Anastomose des Spenders Lungenarterie und des Empfängers untere Hohlvene gemacht. </li><li style="text-align: left"> Die linke Wand der Vena cava inferior und Spender Lungenarterie ist mit anhaltenden Nähte in der Innenseite der unteren Hohlvene geschlossen. </li><li style="text-align: left"> Nach einem Knoten mit dem proximalen Spitze Standnaht, sind die rechte Wand der Vena cava inferior und des Spenders Lungenarterie mit anhaltenden Nähte außerhalb der Vena Höhle geschlossen. </li><li style="text-align: left"> Der Spender ist das Herz auf die rechte Seite des Empfängers Bauch gedreht. Die rechte Wand des Empfängers Bauchaorta und des Spenders aufsteigenden Aorta Anastomose mit weiterhin Nähten verschlossen. </li><li style="text-align: left"> Nach Anastomose vorgenommen wird, wird die distale Ligatur entfernt, um die Blutung aus der Anastomose zu überprüfen. </li><li style="text-align: left"> Wenn es wenig bis gar keine Blutungen aus der Anastomose wird die proximale Ligatur entfernt. Der Spender das Herz mit Blut füllt sofort, die Farbe des Herzens wird rot, und es beginnt wieder zu schlagen. </li><li style="text-align: left"> Der Darm ist auf den Bauch, die mit 6-0 kontinuierlichen Nähten verschlossen wird zurückgegeben. </li><li style="text-align: left"> Der Empfänger der Maus auf einen warmen Raum gebracht. Nach einer Stunde den Empfänger erholt. </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>

Ergebnisse Wir haben erfolgreich mehr als 1000 Fälle von heterotopen Herztransplantation in Mäusen durchgeführt und erreicht über 90% Erfolgsquote in verschiedene Stämme von Mäusen, einschließlich Wildtyp transgenen und Knockout-Mäusen. Diskussion Da Drs. Corry und Russell eingeführt heterotope Herztransplantation an Mäusen in 1973, hat dieses Modell erweist sich als besonders wertvoll für das Studium Immunabwehr Reaktion und die Entwicklung neuartiger Immunsuppressiva Str...

heterotopic heart transplantation in mice

Die Maus heterotope Herztransplantation wurde in großem Umfang, da es von den Doktoren eingeführt wurde. Corry und Russell 1973. Es ist besonders wertvoll für das Studium Ablehnung und Immunantwort jetzt, dass neuere transgenen und Knockout-Mäuse zur Verfügung stehen, und eine große Anzahl von immunologischen Reagenzien entwickelt worden. Die Herztransplantation Modell ist weniger streng als die Hauttransplantat Modelle, obwohl technisch immer anspruchsvoller. Wir haben eine modifizierte Technik entwickelt und mehr als 1000 erfolgreichen Fällen der heterotopen Herztransplantation in Mäusen abgeschlossen. Bei der Anastomose der Aorta ascendens und abdominalen Aorta, sind zwei Haltefäden am proximalen und distalen Spitzen des Empfängers Bauchaorta mit dem Spender s aufsteigenden Aorta gelegt, dann mit 11-0 Naht für die Anastomose auf beiden Seiten der Aorta mit anhaltenden Nähten. Die Haltefäden machen die Anastomose einfacher und 11-0 ist ein idealer Naht Größe zu Blutungen und Thrombosen zu vermeiden. Bei der Anastomose Lungenarterie und untere Hohlvene, sind zwei Haltefäden am proximalen und distalen Spitze Spitze des Empfängers s untere Hohlvene mit dem Spender s Lungenarterie gemacht. Die linke Wand der Vena cava inferior und Spender s Lungenarterie ist mit anhaltenden Nähte in der Innenseite der unteren Hohlvene geschlossen, nachdem, Naht einen Knoten mit dem proximalen Spitze bleiben der rechten Wand der Vena cava inferior und der Spender s Lungenarterie sind mit anhaltenden Nähte außerhalb der Vena Höhle mit 10-0 Nähten verschlossen. Diese Methode ist einfacher durchzuführen, da Anastomose nur auf der einen Seite der unteren Hohlvene und 10-0 Fäden gemacht wird, ist die richtige Größe zu Blutungen und Thrombosen zu vermeiden. In diesem Artikel geben wir Details der Technik, um das Video zu ergänzen.

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

Heterotopic Heart Transplantation in Mice

Die Maus heterotope Herztransplantation Modell wurde von vielen Forschern nachgewiesen worden, eine wichtige Methode zur Untersuchung von Mechanismen der Abstoßung und Immunantwort werden. Allerdings sind die eingesetzten Techniken erforderlich noch eine Herausforderung. Durch die Modifikation Standard-Techniken haben wir Erfolg mit mehr als 1000 Transplantationen.

Heterotope Herztransplantation in Mäuse

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 Aufrufe.  University of California, San Francisco - UCSF. Die Maus heterotope Herztransplantation Modell wurde von vielen Forschern nachgewiesen worden, eine wichtige Methode zur Untersuchung von Mechanismen der Abstoßung und Immunantwort werden. Allerdings sind die eingesetzten Techniken erforderlich noch eine Herausforderung. Durch die Modifikation Standard-Techniken haben wir Erfolg mit mehr als 1000 Transplantationen.

Serie: 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.

Dünndarmtransplantation In Mice

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

Forschung

Biologie

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.

Transplantation von Pankreas-Inseln in die Niere Capsule von diabetischen Mäusen

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 Hauttransplantation

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.

Visualisierung des schlagenden Herzens in 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.

Heterotope und orthotopen Tracheal Transplantation in Mäuse als Modelle benutzt, um die Entwicklung von obliterans Airway Disease Study

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.

Vorbereitung der Embryonen für Elektronenmikroskopie der Drosophila Embryonale Herz Rohr

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.

Die Quantifizierung der γH2AX Foci in Response to ionisierender Strahlung

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 &#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.

Auswertung der räumlichen Verteilung der γH2AX folgenden ionisierender Strahlung

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.

Klonogene Test: adhärenter Zellen

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.

Germ Cell Transplantation und Hodengewebe in Mäusen Xenografting

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