Ce travail a été financé par des subventions du CIRM # TR-1250, RT1-01108, et CL1-00502.

Remarque: Toutes les procédures d&#39;animaux doit être approuvé par IACUC ou équivalent. Tous les équipements chirurgicaux doivent être stérilisés avant la chirurgie. Des gants stériles, les rideaux de gaze et doit être utilisé. 1. Préparation avant la chirurgie <ol><li> Obtenir 6 semaines vieilles Mus musculus CbySmn.CB17-Prkdc SCID / J chez les souris mâles ou d&#39;autres déformations de la souris immunodéprimée. </li><li> Stériliser tous les instruments chirurgicaux, des gants et gaze. </li><li> Dissocier hPSCs être injectés avec accutase. </li><li> Compter les cellules et remettre en suspension 1.000.000 cellules par 20-30 uL dans matrigel dilué 1:1 dans DMEM/F-12. Gardez les cellules sur la glace jusqu&#39;à la fin. </li><li> Notez que souvent, il est utile de faire une course à travers de l&#39;expérience où Trypan bleu est injecté. Cela aidera à identifier les problèmes potentiels avant que les cellules sont gaspillées. </li><li> Dans le vivarium, anesthésier votre souris selon les procédures acceptées à votre institution. Dans nos expériences, nous avons utilisé un Anesthmachine à EIES avec l&#39;isoflurane. Les souris ont été d&#39;abord mis dans une chambre d&#39;induction avec de l&#39;oxygène 1l/min et de 3-4% l&#39;isoflurane. Une fois anesthésié, un cône de nez avec de l&#39;oxygène 1l/min et 2-3% d&#39;isoflurane a été utilisé. </li><li> Rasage de l&#39;abdomen avec une tondeuse, et nettoyer la paroi antérieure de l&#39;abdomen de la souris, à partir du centre de l&#39;abdomen et de travail dans le sens horaire vers l&#39;extérieur. Première utilisation de povidone-iode solution puis laver avec de l&#39;éthanol 70%. Répétez 3 fois, en changeant à chaque fois des écouvillons. Transfert de l&#39;animal à un coussin chauffant pour garder l&#39;animal chaud à l&#39;intérieur d&#39;une culture tissulaire ou le capot de dissection. </li><li> Faire une incision de 1 cm longitudinale à travers la peau et le péritoine d&#39;stériles ciseaux chirurgicaux juste en dessous du niveau de l&#39;articulation de la hanche. </li><li> Tout en maintenant le péritoine avec des pinces, atteindre vers le bas vers la hanche droite avec un autre forcept stérile et retirer le tissu blanc gras avec des testicules attachés. </li><li> Placez les testicules sur de la gaze stérile. </li><li> Remplissez une tuberculine ou à Hamilton (1cc) seringue wie les hPSCs à injecter. Notez que c&#39;est une bonne idée d&#39;inclure une ligne de commande HPSC que vous savez pluripotentes comme les WA09 (également connu sous le nom H9). De cette façon, vous avez un contrôle positif qui vous pouvez utiliser pour déterminer si l&#39;injection ou la chirurgie était viciée. </li><li> Injecter lentement les hPSCs (20-30 pi) dans le centre de la capsule testicule écart des gros vaisseaux sanguins, l&#39;arrêt si la capsule testiculaire commence à gonfler. </li><li> Retirez l&#39;aiguille lentement pour éviter le reflux des cellules. </li><li> En utilisant des pinces, transférer les testicules et le dos du tissu adipeux à sa position initiale dans l&#39;abdomen. </li><li> Fermez le péritoine avec 2 ou 3 points de suture résorbables et fermer la peau avec autoclips. </li><li> La souris doit être gardé au chaud jusqu&#39;à ce qu&#39;il récupère et étant donné une certaine forme d&#39;analgésique (voir ce qui est accepté dans votre vivarium) après la chirurgie deux fois par jour pendant 1-2 jours. Notez que ni l&#39;anesthésie ni l&#39;analgésie avec interférer avec le développement des tumeurs. </li><li> Surveiller eanimale e pour la croissance tumorale pendant 6-12 semaines. Très rarement, les tumeurs peuvent se développer à 5 mm de diamètre dans les 6 semaines après l&#39;injection, il est donc important de surveiller les animaux. Si cela se produit, les animaux doivent être euthanasiés et les tumeurs traitées et analysées comme d&#39;habitude. </li><li> Une fois que la tumeur est palpable et atteint environ 5 mm de taille, anesthésier la souris et le sacrifier selon les procédures acceptées protocole animaux. </li><li> Retirer de la tumeur et le document en conséquence - la taille photographier, de mesurer, peser. </li><li> Couper en petits morceaux la tumeur et de fixer dans une solution de paraformaldéhyde à 4%. Entreposer dans du paraformaldéhyde à 4% avant que des échantillons sont envoyés à un pathologiste pour la coupe, coloration et de l&#39;analyse. </li></ol> 2. Résultats représentatifs: Lorsque ce protocole est fait tel que décrit et la lignée cellulaire injecté est pluripotentes, une palpable, évidente visuellement, la tumeur doit former dans les 12 semaines au plus. Pour établies lignes HPSC comme WA09, nous observons habituellementtumeurs dans les 6 semaines. Pour les lignes iPSC il n&#39;est pas rare de voir des tumeurs au sein de 8-10 semaines. Il est très important d&#39;injecter des souris avec une ligne qui est connue pour être pluripotentes, comme un contrôle positif, afin d&#39;être sûr que la procédure a été effectuée correctement. Les tumeurs regardent habituellement très hétérogène et ont de nombreux kystes joint (Figure 1). L&#39;analyse des échantillons de tumeurs par un pathologiste doit montrer des tissus différenciés de tous les trois feuillets embryonnaires (Figure 2). <img alt="Figure 1" src="/files/ftp_upload/3177/3177fig1.jpg" /> Figure 1. Typique tératome HPSC dérivée dans la capsule du testicule. En suivant le protocole décrit, un million de WA09 cellules ont été injectées dans la capsule du testicule d&#39;une souris immunodéprimées. Six semaines plus tard, un tératome a été observée. A) Tératome tiré à partir de la souris. B) Gros plan image de la tératome. Notez les structures hétérogénéité et le kyste. <img alt="Figure 2" src = &quot;/ files/ftp_upload/3177/3177fig2.jpg&quot; /&gt; Figure 2. Hématoxyline éosine et les sections colorées de la tératome montrent les tissus de chaque couche de germe. Après fixation, le tératome a été sectionnée et colorées à l&#39;hématoxyline et l&#39;éosine. Analyse par un pathologiste a révélé la présence de cellules provenant de chacune des 3 couches germinales.

<ol>
	<li>Bradley, A., Evans, M., Kaufman, M. H., Robertson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+germ-line+chimaeras+from+embryo-derived+teratocarcinoma+cell+lines.">Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines.</a> Nature. 309, 255-255 (1984).</li><li>Evans, M. J., Kaufman, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+in+culture+of+pluripotential+cells+from+mouse+embryos.">Establishment in culture of pluripotential cells from mouse embryos.</a> Nature. 292, 154 (1981).</li><li>Martin, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+pluripotent+cell+line+from+early+mouse+embryos+cultured+in+medium+conditioned+by+teratocarcinoma+stem+cells.">Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.</a> Proc. Natl. Acad. Sci. U. S. A. 78, 7634-7634 (1981).</li><li>Downing, G. J., Battey, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technical+assessment+of+the+first+20+years+of+research+using+mouse+embryonic+stem+cell+lines.">Technical assessment of the first 20 years of research using mouse embryonic stem cell lines.</a> Stem Cells. 22, 1168-1168 (2004).</li><li>Thomson, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+derived+from+human+blastocysts.">Embryonic stem cell lines derived from human blastocysts.</a> Science. 282, 1145-1145 (1998).</li><li>Reubinoff, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+lines+from+human+blastocysts:+somatic+differentiation+in+vitro.">Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro.</a> Nat Biotechnol. 18, (2000).</li><li>Muller, F. J., Goldmann, J., Loser, P., Loring, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+call+to+standardize+teratoma+assays+used+to+define+human+pluripotent+cell+lines.">A call to standardize teratoma assays used to define human pluripotent cell lines.</a> Cell Stem Cell. 6, 412-412 (2010).</li><li>Brivanlou, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells.+Setting+standards+for+human+embryonic+stem+cells.">Stem cells. Setting standards for human embryonic stem cells.</a> Science. 300, 913-913 (2003).</li><li>Lensch, M. W., Schlaeger, T. M., Zon, L. I., Daley, G. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Teratoma+formation+assays+with+human+embryonic+stem+cells:+a+rationale+for+one+type+of+human-animal+chimera.">Teratoma formation assays with human embryonic stem cells: a rationale for one type of human-animal chimera.</a> Cell Stem Cell. 1, 253-25 (2007).</li><li>Gertow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organized+development+from+human+embryonic+stem+cells+after+injection+into+immunodeficient+mice.">Organized development from human embryonic stem cells after injection into immunodeficient mice.</a> Stem. Cells. Dev. 13, 421-421 (2004).</li><li>Gertow, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+human+embryonic+stem+cell-derived+teratomas+for+the+assessment+of+pluripotency.">Isolation of human embryonic stem cell-derived teratomas for the assessment of pluripotency.</a> Curr Protoc Stem Cell Biol. Chapter 1, Unit1B 4-Unit1B 4 (2007).</li></ol>

Pas de conflits d&#39;intérêt déclarés.

La méthode présentée ici offre une grande fiabilité, des moyens simples de générer des tératomes de hPSCs dans la capsule du testicule. Il ya plusieurs paramètres critiques de cette technique. En particulier, il est important d&#39;injecter des lignées cellulaires HPSC qui sont connus pour être pluripotentes comme un contrôle. D&#39;autres paramètres importants comprennent l&#39;intervalle de temps entre l&#39;injection et l&#39;observation des tumeurs. Pour les lignées cellulaires comme WA09, tératomes devrait être observée dans 6-8 semaines. Pour les lignes iPSC nouvelles, nous constatons que souvent 10 semaines sont nécessaires. Une autre préoccupation est le nombre de cellules injectées. Nous injectons un million de cellules, mais le dosage peut se faire facilement avec moins de cellules. En outre, le milieu d&#39;injection est important. Nous constatons que nous obtenons les meilleurs résultats lorsque les cellules sont injectées dans un mélange 1:1 de matrigel et DMEM/F-12, par opposition à PBS ou DMEM/F-12.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td></tr><tr><td> Accutase </td><td> Invitrogen </td><td> A1110501 </td></tr><tr><td> DMEM/F-12 </td><td> Invitrogen </td><td> 113300-032 </td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354277 </td></tr></tbody></table>

teratoma generation in the testis capsule

Les cellules souches pluripotentes (CSP) ont la caractéristique unique qu&#39;ils peuvent se différencier en cellules de tous les trois feuillets embryonnaires. Cela les rend un outil potentiellement précieux pour le traitement de nombreuses maladies différentes. Avec l&#39;avènement de cellules souches pluripotentes induites (CISP) et de la recherche continue sur les cellules souches embryonnaires (CSEh) il ya un besoin pour des essais qui peuvent démontrer que d&#39;une lignée cellulaire donnée est pluripotentes. La transmission germinale a été l&#39;étalon-or pour démontrer la pluripotence des cellules souches embryonnaires de souris (CESM) lignes 1,2,3. L&#39;utilisation de ce test, les chercheurs peuvent montrer qu&#39;une ligne MESC peut faire tous les types de cellules dans l&#39;embryon, y compris les cellules germinales 4. Avec la nouvelle génération de CSE humaines lignes 5,6, le dosage approprié pour prouver pluripotence de ces cellules était pas claire, car les CES de l&#39;homme ne peut pas être testé pour la transmission germinale. Comme une mère porteuse, le dosage tératome est actuellement utilisé pour démontrer la pluripotrence des cellules souches pluripotentes humaines (hPSCs) 7,8,9. Bien que ce test a récemment fait l&#39;objet de contrôle et de nouvelles technologies sont actuellement à l&#39;étude, le dosage tératome est l&#39;étalon-or actuel 7. Dans cet essai, les cellules en question sont injectés dans une souris immunodéprimées. Si les cellules sont pluripotentes, un tératome finiront par développer et les articles de la tumeur montrent les tissus de tous les 3 couches germinales 10. Dans le dosage tératome, hPSCs peut être injecté dans différentes zones de la souris. Les sites d&#39;injection les plus courants sont la capsule du testicule, de la capsule du rein, le foie, ou dans la jambe soit sous-cutanée ou intramusculaire 11. On décrit ici un protocole robuste pour la génération des tératomes de hPSCs utilisant la capsule testicule comme site de croissance de la tumeur.

Watch this Scientific Journal Video about Teratoma Generation in the Testis Capsule at JoVE.com

Teratoma Generation in the Testis Capsule

Les cellules souches pluripotentes (hPSCs) ont le potentiel pour traiter une myriade de maladies différentes. L&#39;utilité de ces cellules réside dans le fait qu&#39;elles peuvent se différencier en n&#39;importe quel type cellulaire dans le corps. Nous décrivons ici le dosage tératome, qui est utilisé pour démontrer la pluripotence des hPSCs.

Génération Tératome dans la capsule du testicule

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers.  This makes them a ...

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20.6K vues.  Scripps Research Institute. Les cellules souches pluripotentes (hPSCs) ont le potentiel pour traiter une myriade de maladies différentes. L'utilité de ces cellules réside dans le fait qu'elles peuvent se différencier en n'importe quel type cellulaire dans le corps. Nous décrivons ici le dosage tératome, qui est utilisé pour démontrer la pluripotence des hPSCs.

Vidéo: Teratoma Generation in the Testis Capsule

A Strategy to Identify de Novo Mutations in Common Disorders such as Autism and Schizophrenia

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, the de novo mutation rate in humans is relatively high, so new mutations are generated at a high frequency in the population. However, de novo mutations have not been reported in most common diseases. Mutations in genes leading to severe diseases where there is a strong negative selection against the phenotype, such as lethality in embryonic stages or reduced reproductive fitness, will not be transmitted to multiple family members, and therefore will not be detected by linkage gene mapping or association studies. The observation of very high concordance in monozygotic twins and very low concordance in dizygotic twins also strongly supports the hypothesis that a significant fraction of cases may result from new mutations. Such is the case for diseases such as autism and schizophrenia. Second, despite reduced reproductive fitness1 and extremely variable environmental factors, the incidence of some diseases is maintained worldwide at a relatively high and constant rate. This is the case for autism and schizophrenia, with an incidence of approximately 1% worldwide. Mutational load can be thought of as a balance between selection for or against a deleterious mutation and its production by de novo mutation. Lower rates of reproduction constitute a negative selection factor that should reduce the number of mutant alleles in the population, ultimately leading to decreased disease prevalence. These selective pressures tend to be of different intensity in different environments. Nonetheless, these severe mental disorders have been maintained at a constant relatively high prevalence in the worldwide population across a wide range of cultures and countries despite a strong negative selection against them2. This is not what one would predict in diseases with reduced reproductive fitness, unless there was a high new mutation rate. Finally, the effects of paternal age: there is a significantly increased risk of the disease with increasing paternal age, which could result from the age related increase in paternal de novo mutations. This is the case for autism and schizophrenia3. The male-to-female ratio of mutation rate is estimated at about 4&#x2013;6:1, presumably due to a higher number of germ-cell divisions with age in males. Therefore, one would predict that de novo mutations would more frequently come from males, particularly older males4. A high rate of new mutations may in part explain why genetic studies have so far failed to identify many genes predisposing to complexes diseases genes, such as autism and schizophrenia, and why diseases have been identified for a mere 3% of genes in the human genome. Identification for de novo mutations as a cause of a disease requires a targeted molecular approach, which includes studying parents and affected subjects. The process for determining if the genetic basis of a disease may result in part from de novo mutations and the molecular approach to establish this link will be illustrated, using autism and schizophrenia as examples.

Molecular genetic strategy for finding de novo mutations causing common disorders such as autism and schizophrenia.

Une stratégie pour identifier des mutations de novo dans les troubles courants comme l&#39;autisme et la schizophrénie

There are several lines of evidence supporting the role of de novo mutations as a mechanism for common disorders, such as autism and schizophrenia. First, ...

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Médecine

Technical Aspects of the Mouse Aortocaval Fistula

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta and inferior vena cava (IVC) are exposed. The proximal infrarenal aorta and the distal aorta are dissected for clamp placement and needle puncture, respectively. Special attention is paid to avoid dissection between the aorta and the IVC. After clamping the aorta, a 25 G needle is used to puncture both walls of the aorta into the IVC. The surrounding connective tissue is used for hemostatic compression. Successful creation of the AVF will show pulsatile arterial blood flow in the IVC. Further confirmation of successful AVF can be achieved by post-operative Doppler ultrasound.

The goal is to produce an arteriovenous fistula that is simple and reproducible. This method does not use sutures or glue adhesive. Therefore the samples can be used with the least amount of foreign materials for analysis.

Aspects techniques de la fistule aorto-cave de la souris

Technical aspects of creating an arteriovenous fistula in the mouse are discussed. Under general anesthesia, an abdominal incision is made, and the aorta ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

Rénale Capsule xénogreffe sous-cutanée et Pellet implantation pour l&#39;évaluation de la carcinogenèse prostatique et l&#39;hyperplasie prostatique bénigne

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

Generation and 3-Dimensional Quantitation of Arterial Lesions in Mice Using Optical Projection Tomography

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating important information on the pathogenesis of lesion formation and the action of novel therapies. Use of atherosclerosis-prone mice, surgical methods of lesion induction, and dietary modification has dramatically improved understanding of the mechanisms that contribute to disease development and the potential of new treatments.
Classically, analysis of lesions is performed ex vivo using 2-dimensional histological techniques. This article describes application of optical projection tomography (OPT) to 3-dimensional quantitation of arterial lesions. As this technique is non-destructive, it can be used as an adjunct to standard histological and immunohistochemical analyses.
Neointimal lesions were induced by wire-insertion or ligation of the mouse femoral artery whilst atherosclerotic lesions were generated by administration of an atherogenic diet to apoE-deficient mice.
Lesions were examined using OPT imaging of autofluorescent emission followed by complementary histological and immunohistochemical analysis. OPT clearly distinguished lesions from the underlying vascular wall. Lesion size was calculated in 2-dimensional sections using planimetry, enabling calculation of lesion volume and maximal cross-sectional area. Data generated using OPT were consistent with measurements obtained using histology, confirming the accuracy of the technique and its potential as a complement (rather than alternative) to traditional methods of analysis.
This work demonstrates the potential of OPT for imaging atherosclerotic and neointimal lesions. It provides a rapid, much needed ex vivo technique for the routine 3-dimensional quantification of vascular remodelling.

Ex vivo analysis of arterial lesions from animal models of cardiovascular disease classically relies on histological and immunohistochemical techniques. These provide 2-dimensional measurements in 3-dimensional lesions. This manuscript describes the generation of arterial lesions for quantitative analysis in 3-dimensions using optical projection tomography.

Génération et quantification 3-Dimensional des lésions artérielles chez la souris utilisant la tomographie optique de projection

The generation and analysis of vascular lesions in appropriate animal models is a cornerstone of research into cardiovascular disease, generating ...

Collection and Extraction of Saliva DNA for Next Generation Sequencing

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality DNA. However, DNA extraction from saliva can yield high quality DNA with little to no degradation/fragmentation that is suitable for a variety of DNA assays without the expense of a phlebotomist and can even be acquired through the mail. However, at present, no saliva DNA collection/extraction protocols for next generation sequencing have been presented in the literature. This protocol optimizes parameters of saliva collection/storage and DNA extraction to be of sufficient quality and quantity for DNA assays with the highest standards, including microarray genotyping and next generation sequencing.

DNA extraction from saliva can provide a readily available source of high molecular weight DNA, with little to no degradation/fragmentation. This protocol provides optimized parameters for saliva collection/storage and DNA extraction to be of sufficient quality and quantity for downstream DNA assays with high quality requirements.

Collecte et d&#39;extraction de l&#39;ADN de la salive pour le séquençage de prochaine génération

The preferred source of DNA in human genetics research is blood, or cell lines derived from blood, as these sources yield large quantities of high quality ...

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

Une<em&gt; In Vitro</em&gt; Modèle dormance du cancer du sein sensible aux œstrogènes dans la moelle osseuse: un outil pour les études de mécanisme moléculaire et Hypothèse Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is still time consuming. Comparative studies that use recombinant lines with transgenes integrated into safe harbor loci could benefit from approaches that use site-specific targeted recombinases, like Cre or FLPe, which are more rapid and less prone to off-target effects. Such methods have been described, although they do not significantly outperform gene targeting in most aspects. Using Zinc-finger nucleases, we previously created a master cell line in the AAVS1 locus of hPSCs&#160;that contains a GFP-Hygromycin-tk expressing cassette, flanked by heterotypic FRT sequences. Here, we describe the procedures to perform FLPe recombinase-mediated cassette exchange (RMCE) using this line. The master cell line is transfected with a RMCE donor vector, which contains a promoterless Puromycin resistance, and with FLPe recombinase. Application of both a positive (Puromycin) and negative (FIAU) selection program leads to the selection of RMCE without random integrations. RMCE generates fully characterized pluripotent polyclonal transgenic lines in 15 d&#160;with 100% efficiency. Despite the recently described limitations of the AAVS1 locus, the ease of the system paves the way for hPSC transgenesis in isogenic settings, is necessary for comparative studies, and enables semi-high-throughput genetic screens for gain/loss of function analysis that would otherwise be highly time consuming.

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Here we report a rapid and efficient gene editing method based on RMCE in the AAVS1 locus of human Pluripotent Stem Cells (hPSCs) that improves upon previously described systems. Using this technique, isogenic lines can be rapidly and reliably generated for proper comparative studies, facilitating transgenesis-mediated research with hPSCs.

Génération rapide et efficace des Recombinant pluripotentes humaines cellules souches par recombinase médiée Cassette Exchange dans le<em&gt; AAVS1</em&gt; Locus

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Drug Repurposing Hypothesis Generation Using the "RE:fine Drugs" System

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The goal of computational methods for drug repurposing is to enable solutions for safer, cheaper and faster drug discovery. Towards this end, we developed a novel method that integrates genetic and clinical phenotype data from large-scale GWAS and PheWAS studies with detailed drug information on the concept of transitive Drug-Gene-Disease triads. We created "RE:fine Drugs," a freely available, interactive dashboard that automates gene, disease and drug-based searches to identify drug repurposing candidates. This web-based tool supports a user-friendly interface that includes an array of advanced search and export options. Results can be prioritized in a variety of ways, including but not limited to, biomedical literature support, strength and statistical significance of GWAS and/or PheWAS associations, disease indications and molecular drug targets. Here we provide a protocol that illustrates the functionalities available in the "RE:fine Drugs" system and explores the different advanced options through a case study.

Drug Repurposing Hypothesis Generation Using the &quot;RE:fine Drugs&quot; System

Here we describe a protocol using the web-based drug repurposing hypothesis generation tool: "RE:fine Drugs." This protocol can be modified to a user's preferences at the level of the query type (gene, drug or disease) and/or the range of available advanced options.

Système: Drug Repurposing Hypothesis Generation Utilisation de la &quot;Drogues fines RE&quot;

The promise of drug repurposing is that existing drugs may be used for new disease indications in order to curb the high costs and time for approval. The ...

The Generation of Closed Femoral Fractures in Mice: A Model to Study Bone Healing

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies that promote efficient bone healing are non-existent and in high demand. Effective and reproducible animal models of fractures healing are needed to understand the complex biological processes associated with bone regeneration. Many animal models of fracture healing have been generated over the years; however, murine fracture models have recently emerged as powerful tools to study bone healing. A variety of open and closed models have been developed, but the closed femoral fracture model stands out as a simple method for generating rapid and reproducible results in a physiologically relevant manner. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice and facilitate a post-fracture stabilization of the femur by inserting an intramedullary steel rod. Although devices such as a nail or a screw offer greater axial and rotational stability, the use of an intramedullary rod provides a sufficient stabilization for consistent healing outcomes without producing new defects in the bone tissue or damaging nearby soft tissue. Radiographic imaging is used to monitor the progression of callus formation, bony union, and subsequent remodeling of the bony callus. Bone healing outcomes are typically associated with the strength of the healed bone and measured with torsional testing. Still, understanding the early cellular and molecular events associated with fracture repair is critical in the study of bone tissue regeneration. The closed femoral fracture model in mice with intramedullary fixation serves as an attractive platform to study bone fracture healing and evaluate therapeutic strategies to accelerate healing.

The murine closed femoral fracture model is a powerful platform to study fracture healing and novel therapeutic strategies to accelerate bone regeneration. The goal of this surgical protocol is to generate unilateral closed femoral fractures in mice using an intramedullary steel rod to stabilize the femur.

La génération des Fractures fémorales fermées chez la souris : un modèle pour étudier la guérison osseuse

Bone fractures impose a tremendous socio-economic burden on patients, in addition to significantly affecting their quality of life. Therapeutic strategies ...

Generation of Porcine Testicular Organoids with Testis Specific Architecture using Microwell Culture

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs in vivo. Formation of organoids has opened up different avenues of basic and translational research. In recent years, testicular organoids have garnered interest in the field of male reproductive biology. Testicular organoids allow for the study of cell-cell interactions, tissue development, and the germ cell niche microenvironment and facilitate high throughput drug and toxicity screening. A method is needed to reliably and reproducibly generate testicular organoids with testis specific tissue architecture. The microwell culture system contains a dense array of pyramid-shaped microwells. Testicular cells derived from pre-pubertal testes are centrifuged into these microwells and cultured to generate testicular organoids with testis-specific tissue architecture and cell associations. Thousands of homogeneous organoids can be generated via this process. The protocol reported here will be of broad interest to researchers studying male reproduction.

Here, we present a protocol for the reproducible generation of porcine testicular organoids with testis specific tissue architecture using the commercially available microwell culture system.

Génération d'organoïdes testiculaires porcins avec l'architecture spécifique de Testis utilisant la culture de Microwell

Organoids are three dimensional structures composed of multiple cell types that are capable of recapitulating tissue architecture and functions of organs ...