עבודה זו מומנה על ידי מענקים CIRM # TR-1250, RT1-01108, ו CL1-00502.

הערה: כל הנהלים בעלי חיים חייב להיות מאושר על ידי IACUC או שווה ערך. כל הציוד הכירורגי יש לעקר לפני הניתוח. כפפות סטריליות, וילונות גזה יש להשתמש. 1. הכנה לפני הניתוח <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להשיג 6 בן שבוע מ.א.ס. מאסכאלאס CbySmn.CB17-Prkdc SCID / י עכברים ממין זכר או זן immunocompromised השני של העכבר. </li><li style=";text-align:right;direction:rtl"> לעקר את כל מכשירי ניתוח, כפפות, ו גזה. </li><li style=";text-align:right;direction:rtl"> לנתק hPSCs להיות מוזרק עם accutase. </li><li style=";text-align:right;direction:rtl"> ספירת תאים ו resuspend 1,000,000 תאים לכל μL 20-30 ב matrigel בדילול 01:01 ב DMEM/F-12. שמור על תאים על הקרח עד גמר. </li><li style=";text-align:right;direction:rtl"> שים לב כי לעיתים קרובות כדאי לעשות ריצה דרך של ניסוי שבו Trypan כחול מוזרק. זה יעזור לזהות בעיות פוטנציאליות לפני כל התאים מבוזבז. </li><li style=";text-align:right;direction:rtl"> ב ביבר, להרדים את העכבר בהתאם לנהלים המקובלים במוסד שלך. בניסויים שלנו, השתמשנו anesthesia מכונה עם isoflurane. העכברים הוכנסו בתחילה עם חמצן 1L/min ו 3-4% isoflurane בתא אינדוקציה. הרדים פעם, חרוט האף עם חמצן 1L/min ו 2-3% isoflurane היה בשימוש. </li><li style=";text-align:right;direction:rtl"> לגלח את הבטן עם קוצץ, ולנקות את הקיר הקדמי של הבטן העכבר, החל ממרכז הבטן ועבודה עם כיוון השעון כלפי חוץ. הפתרון הראשון Povidone-יוד השימוש מכן לשטוף עם אתנול 70%. לחזור 3 פעמים, שינוי דגימות בכל פעם. להעביר את בעל החיים משטח חימום כדי לשמור על בעל החיים חם בתוך התרבות רקמות או מכסה המנוע לנתח. </li><li style=";text-align:right;direction:rtl"> לעשות חתך אורכי 1 ס&quot;מ דרך העור הצפק עם מספריים כירורגיות סטריליות ממש מתחת לרמה של הירך משותפת. </li><li style=";text-align:right;direction:rtl"> תוך כדי לחיצה הצפק עם מלקחיים, להגיע לעבר הירך הימנית עם forcept אחר סטרילית ולשלוף את רקמת שומן לבן יחד עם האשכים המצורפים. </li><li style=";text-align:right;direction:rtl"> מניחים את האשכים על גזה סטרילי. </li><li style=";text-align:right;direction:rtl"> מלא טוברקולין או המילטון (1cc) מזרק Wiה hPSCs כדי להיות מוזרק. שים לב, זה רעיון טוב לכלול פס בקרה hPSC שאתה יודע שהוא pluripotent כגון WA09 (הידוע גם בשם H9). בדרך זו יש לך ביקורת חיובית שאפשר להשתמש בו כדי לקבוע אם הזריקה שלך או ניתוח היה פגום. </li><li style=";text-align:right;direction:rtl"> לאט לאט להזריק את hPSCs (20-30 μL) למרכז הקפסולה testis הרחק מכל כלי הדם הגדולים, לעצור אם הקפסולה האשכים מתחיל להתנפח. </li><li style=";text-align:right;direction:rtl"> להוציא את המחט לאט לאט כדי למנוע רפלוקס של התאים. </li><li style=";text-align:right;direction:rtl"> באמצעות מלקחיים, להעביר את האשכים והגב רקמת שומן למקומו המקורי בבטן. </li><li style=";text-align:right;direction:rtl"> לסגור את הצפק עם 2 או 3 תפרים reabsorbable ולסגור את העור עם autoclips. </li><li style=";text-align:right;direction:rtl"> העכבר יש לשמור חם עד שהוא מתאושש וקיבל צורה כלשהי של כאבים (ראה מה שמקובל ביבר שלך) לאחר הניתוח פעמיים ביום במשך 1-2 ימים. שים לב, לא הרדמה או משכך כאבים עם להפריע להתפתחות הגידול. </li><li style=";text-align:right;direction:rtl"> צג הדואר חיה על הגידול שבועות 6-12. לעתים נדירות ביותר, גידולים עשוי לגדול ל 5 מ&quot;מ בגודל קודמות עד 6 שבועות לאחר ההזרקה, ולכן חשוב לעקוב אחר בעלי החיים. אם זה יקרה, בעלי החיים צריך להיות מורדמים וגידולים את עיבוד וניתוח כרגיל. </li><li style=";text-align:right;direction:rtl"> לאחר הגידול הוא מוחשי ומגיעה בגודל 5 מ&quot;מ בערך, להרדים את העכבר ולהקריב אותו על פי הנהלים המקובלים פרוטוקול של בעלי חיים. </li><li style=";text-align:right;direction:rtl"> הסרה של הגידול המסמך בהתאם - גודל, מדד צילום, לשקול. </li><li style=";text-align:right;direction:rtl"> גזור הגידול לחתיכות קטנות ולתקן בפתרון paraformaldehyde 4%. חנות paraformaldehyde 4% עד דגימות נשלחות פתולוג עבור מכתים, חתך וניתוח. </li></ol> 2. נציג תוצאות: כאשר בפרוטוקול זה נעשה כפי שתואר לבין הקו התא שהוחדר הוא pluripotent, מוחשי, ברור מבחינה חזותית, הגידול צריך להקים בתוך 12 שבועות לכל היותר. עבור קווי hPSC שהוקמו כמו WA09, אנו בדרך כלל רואיםגידולים בתוך 6 שבועות. עבור קווי iPSC אין זה יוצא דופן לראות גידולים בתוך 8-10 שבועות. חשוב מאוד להכניס עכברים עם קו זה ידוע pluripotent, כביקורת חיובית, על מנת להיות בטוח כי ההליך בוצע בצורה נכונה. גידולים בדרך כלל נראים הטרוגנית מאוד ויש להם ציסטות מצורפים רבים (איור 1). ניתוח של דגימות הגידול על ידי פתולוג צריך להראות רקמות הבדיל מכל שלוש שכבות הנבט (איור 2). <img alt="איור 1" src="/files/ftp_upload/3177/3177fig1.jpg" /> באיור 1. אופיינית תפלצת רוחנית נגזר hPSC בכמוסה testis. בעקבות הפרוטוקול המתואר, WA09 מיליון תאים הוזרקו הקפסולה testis של העכבר נפגעת מערכת החיסון. שישה שבועות לאחר מכן נצפתה תפלצת רוחנית. א) משך תפלצת רוחנית של העכבר. ב) תקריב תמונה של תפלצת רוחנית. הערה המבנים ואת ההטרוגניות ציסטה. <img alt="איור 2" src = &quot;/ files/ftp_upload/3177/3177fig2.jpg&quot; /&gt; איור 2. Hematoxylin וחתכים Eosin מוכתמים מן תפלצת רוחנית להראות רקמות של כל שכבת נבט. לאחר קיבוע, תפלצת רוחנית נותח ומוכתמים Hematoxylin ו Eosin. ניתוח על ידי פתולוג חשף את נוכחותם של תאים כל אחד 3 שכבות הנבט.

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אין ניגוד עניינים הצהיר.

השיטה המוצגת כאן מספקת אמינה מאוד, באמצעים פשוטים של יצירת teratomas מ hPSCs הקפסולה testis. ישנם מספר פרמטרים קריטיים בטכניקה זו. בפרט, חשוב להזריק שורות תאים hPSC שידוע כי הם להיות pluripotent כביקורת. פרמטרים חשובים נוספים כוללים את מרווח הזמן בין הזרקה והתבוננות הגידול. על שורות תאים כמו WA09, teratomas צריך לשמור על 6-8 שבועות. על קווים חדשים iPSC אנו מוצאים כי לעתים קרובות 10 שבועות נדרשים. חשש נוסף הוא מספר התאים המוזרקים. אנחנו מזריקים מיליון תאים, אך assay יכול להיעשות בקלות עם פחות תאים. בנוסף, בינוני זריקה חשובה. אנו מוצאים כי אנחנו מקבלים את התוצאות הטובות ביותר כאשר התאים מוזרקים בתערובת 01:01 של matrigel ו DMEM/F-12, לעומת PBS או DMEM/F-12.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> חברה </td><td> מספר קטלוגי </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

בתאי גזע pluripotent (PSCs) יש מאפיין ייחודי כי הם יכולים להתמיין לתאים מכל שלוש שכבות הנבט. זה גורם להם להיות כלי רב ערך פוטנציאלית לטיפול במחלות שונות. עם כניסתו של המושרה בתאי גזע pluripotent (iPSCs) ומחקר המשך בתאי גזע עובריים אנושיים (hESCs) יש צורך מבחני זה יכול להוכיח כי קו מסוים הוא תא pluripotent. שידור germline כבר את תקן הזהב עבור הוכחת pluripotence של תא גזע עובריים בעכבר (המסקלין) שורות 1,2,3. שימוש assay זה, החוקרים יכולים להראות כי הקו המסקלין יכול לעשות את כל סוגי התאים בעובר כולל בתאי הנבט 4. עם הדור של האדם ESC קווים 5,6, assay המתאים להוכיח pluripotence של תאים אלה לא היה ברור מאז ESCs אדם לא יכול להיבחן לשידור germline. כתחליף, assay תפלצת רוחנית משמשת כיום להפגין pluripotency בתאי גזע האדם גזע pluripotent (hPSCs) 7,8,9. למרות assay זה הגיע לאחרונה תחת בדיקה וטכנולוגיות חדשות נבדקות באופן פעיל, assay תפלצת רוחנית הוא תקן הזהב הנוכחי 7. ב assay זה, התאים המדוברים מוזרקים לעכבר נפגעת מערכת החיסון. אם התאים pluripotent, תפלצת רוחנית בסופו של דבר לפתח קטעי הגידול יראה רקמות מכל 3 שכבות הנבט 10. ב assay תפלצת רוחנית, hPSCs יהיה ניתן להזריק לתוך אזורים שונים של העכבר. האתרים הנפוצים ביותר כוללים הזרקת testis הקפסולה, הקפסולה כליות, כבד, או את הרגל או מתחת לעור או לשריר 11. כאן אנו מתארים את פרוטוקול חזקים עבור הדור של teratomas מ hPSCs באמצעות הקפסולה testis כאתר של הגידול.

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

Teratoma Generation in the Testis Capsule

אדם בתאי גזע pluripotent (hPSCs) יש פוטנציאל לטפל מספר עצום של מחלות שונות. השירות של תאים אלה הוא בכך שהם יכולים להתמיין לכל סוג תא בגוף. כאן אנו מתארים את assay תפלצת רוחנית, אשר משמש כדי להדגים את pluripotence של hPSCs.

תפלצת רוחנית דור הקפסולה testis

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

teratoma-generation-in-the-testis-capsule

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20.6K Views.  Scripps Research Institute. אדם בתאי גזע pluripotent (hPSCs) יש פוטנציאל לטפל מספר עצום של מחלות שונות. השירות של תאים אלה הוא בכך שהם יכולים להתמיין לכל סוג תא בגוף. כאן אנו מתארים את assay תפלצת רוחנית, אשר משמש כדי להדגים את pluripotence של hPSCs.

Video: Teratoma Generation in the Testis Capsule

Making Sense of Listening: The IMAP Test Battery

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the abilities to extract and use the information encoded in the auditory signal. We refer to these as listening skills (or auditory processing AP). Deficits in these skills are associated with delayed language and literacy development, though the nature of the relevant deficits and their causal connection with these delays is hotly debated.
When a child is referred to a health professional with normal hearing and unexplained difficulties in listening, or associated delays in language or literacy development, they should ideally be assessed with a combination of psychoacoustic (AP) tests, suitable for children and for use in a clinic, together with cognitive tests to measure attention, working memory, IQ, and language skills. Such a detailed examination needs to be relatively short and within the technical capability of any suitably qualified professional. Current tests for the presence of AP deficits tend to be poorly constructed and inadequately validated within the normal population. They have little or no reference to the presenting symptoms of the child, and typically include a linguistic component. Poor performance may thus reflect problems with language rather than with AP. To assist in the assessment of children with listening difficulties, pediatric audiologists need a single, standardized child-appropriate test battery based on the use of language-free stimuli.
We present the IMAP test battery which was developed at the MRC Institute of Hearing Research to supplement tests currently used to investigate cases of suspected AP deficits. IMAP assesses a range of relevant auditory and cognitive skills and takes about one hour to complete. It has been standardized in 1500 normally-hearing children from across the UK, aged 6-11 years. Since its development, it has been successfully used in a number of large scale studies both in the UK and the USA. IMAP provides measures for separating out sensory from cognitive contributions to hearing. It further limits confounds due to procedural effects by presenting tests in a child-friendly game-format. Stimulus-generation, management of test protocols and control of test presentation is mediated by the IHR-STAR software platform. This provides a standardized methodology for a range of applications and ensures replicable procedures across testers. IHR-STAR provides a flexible, user-programmable environment that currently has additional applications for hearing screening, mapping cochlear implant electrodes, and academic research or teaching.

A test battery (IMAP) for performing an in-depth assessment of auditory and cognitive abilities contributing to listening skills is described. It is quick to administer, child-friendly and free from linguistic confounds. Stimulus generation and protocol management are controlled via a software platform (IHR-STAR) to ensure replicable procedures.

יצירת תחושה של הקשבה: סוללה מבחן IMAP

The ability to hear is only the first step towards making sense of the range of information contained in an auditory signal. Of equal importance are the ...

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.

אסטרטגיה לזהות מוטציות דה נובו בהפרעות נפוצות כגון אוטיזם וסכיזופרניה

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, ...

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.

היבטים טכניים של מורסת Aortocaval מאוס

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.

כליות קפסולת Xenografting ותת עור מכבישי השרשה להערכת הערמונית Carcinogenesis והיפרפלזיה שפירה של הערמונית

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.

דור וQuantitation 3 ממדים של עורקי פצעים בעכברים באמצעות טומוגרפיה הקרנה אופטית

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.

איסוף והפקת DNA רוק לסידור הדור הבא

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.

<em&gt; במבחנה</em&gt; תרדמת דגם של סרטן השד רגיש לאסטרוגן במוח העצם: כלי ללימודי מנגנון מולקולרי והשערת דור

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.

הדור מהיר ויעיל של תאי גזע פלוריפוטנטיים רקומביננטי אנושי ידי recombinase בתיווך המרת קלטת ב<em&gt; AAVS1</em&gt; לוקוס

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.

דור שערה ותרופות repurposing שימוש &quot;RE: סמים בסדר&quot; מערכת

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.

הדור של שברי הירך סגור בעכברים: מודל ללמוד ריפוי עצם

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