עבודה זו מומנה על ידי בפרס קרן המדע הלאומית הקריירה (CBET-0846395) ואת המחלקה לרפואה השוואתי, אוניברסיטת טוליין.

1. בעלי חיים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> נקבה C57BL/6J עכברים (Mus שריר) מאומתים ללא ectoparasites מדבקת, endoparasites helminth, ונוגדנים ל 17 וירוסים Murine על ידי הספק לפני המשלוח. עכברים הם שוכנו במתקן AAALAC-מוכר את החיה של אוניברסיטת טוליין בהתאם מדריך לטיפול ושימוש בחיות מעבדה. 1 </li><li style=";text-align:right;direction:rtl"> באופן ספציפי, עכברים הם הקבוצה שוכנו (4-5 עכברים / לכלוב) צמרות פוליקרבונט מאוורר בנפרד cageswith microisolation לסנן על מצע עץ. הם סיפקו את המודעה מכרסם pelleted כרצונך מזון ומים מהברז. </li><li style=";text-align:right;direction:rtl"> עכברים מותר 7 ימים להסתגל לפני תחילת המחקר. </li></ol> 2. תאים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> קרצינומה של תא תא Murine מעבר לקו MB49 משמש לייצור גידולים בשלפוחית ​​השתן. </li><li style=";text-align:right;direction:rtl"> הם תאים בתרבית בינוני שונה Dulbecco של הנשר בתוספת סרום 10% שור עוברית, 100 יחידות / מ&quot;ל ​​פניצילין, ו 100 יחידות / מ&quot;ל ​​סטרפטומיצין. </li><li style=";text-align:right;direction:rtl"> תרבויות מתוחזקים על 37 ° C ו 5% CO 2 עם התקשורת משתנה כל יומיים שלושה. </li></ol> 3. גידול בעלי חיים דגם orthotopic החדרה של המודל שלפוחית ​​השתן הגידול המתואר כאן הוא שינוי של ההליך שתואר על ידי גינטר ואח&#39; 2. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הרדמה מושרה עם 2% isoflurane (MWI / VetOne, מרידיאן, מזהה, חתול # 501017) חמצן ומתוחזק על 0.75-1.25%. (חיות מרובות ניתן לטפל בו זמנית, אשר יחייב עלייה ברמות isoflurane.) כדי למנוע התייבשות הקרנית, העיניים משומנים במשחה דמעות מלאכותיות עיניים. חום הגוף נשמר עם כרית מחוממת תחת לעטוף נייר. </li><li style=";text-align:right;direction:rtl"> הפרווה של הגב התחתון נחתך כדי לקדם את הקשר עם לוחית מתכת הארקה, ואזור מגולח מצופה בג&#39;ל electroconduction. </li><li style=";text-align:right;direction:rtl"> עכבר כל כך להציב בנפרד על צלחת הארקה מתכת recumbancy הגב, ואת שלפוחית ​​השתן היא באמצעות צינתור transurethrally משומנים 24-מד, 3 / 4 אינץ&#39;, Over-the-מחט קטטר IV עם stylet הסיר. </li><li style=";text-align:right;direction:rtl"> בשלפוחית ​​השתן מנוקז של שתן, חוט פלטינה מוחדר דרך קטטר לתוך שלפוחית ​​השתן משאיר חוט חשוף בכל צד של הקטטר כ - 2 מ&quot;מ על קצה lumenal של הקטטר. חוט עשוי פלטינה בגלל ההתנגדות של החומר חמצון. </li><li style=";text-align:right;direction:rtl"> הנוכחי 2.5 W מוחל אז שלפוחית ​​השתן לרגע כ 0.5 ידי נגיעה את החלק החיצוני של חוט עם יחידת electrocautery מוגדר קרישה. </li><li style=";text-align:right;direction:rtl"> חוט לאחר מכן מועבר למיקום השני שלפוחית ​​השתן ועוד תשלום 2.5 W מיושם. </li><li style=";text-align:right;direction:rtl"> החוט מוסר, ו 100 μl של תאים MB49 בריכוז של 1x10 6 תאים / מ&quot;ל היא החדירו דרך קטטר לתוך שלפוחית ​​השתן באמצעות מזרק 1ml. </li><li style=";text-align:right;direction:rtl"> מזרקים הם שמאל המחוברים צנתרים כדי למנוע בזרימת של המתלים התא העכברים צינתור נשמרות תחת הרדמה במשך 90 דקות כדי לאפשר לתאים לצרף את האתרים לשרוף. </li><li style=";text-align:right;direction:rtl"> 100-200 μl של תמיסת מלח סטרילית נורמלי הוא בזריקה IP עכבר אחד לפני התאוששות. </li><li style=";text-align:right;direction:rtl"> בתום תקופת הדגירה צנתרים מוסרים את בעלי החיים מותר להתאושש על משטח מחומם עד רפלקסים ליישר לחזור, ובנקודה העכברים מוחזרים כלובים לביתם. </li></ol> 4. אולטראסאונד <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> גידולים מותר לגדול במשך 3-5 ימים לפני העכברים לעבור בדיקת אולטרסאונד של שלפוחית ​​השתן כדי לפקח על הגידול. </li><li style=";text-align:right;direction:rtl"> עבור בדיקות אולטרסאונד ראשוני, בעלי חיים מורדמים כמתואר לעיל. עכברים ממוקמים recumbancy הגב, הבטן והעור הוא הוכן על ידי גזירת הפרוות. </li><li style=";text-align:right;direction:rtl"> קטטר 24 IV מד מוכנס לתוך transurethrally בשלפוחית ​​השתן. בשלב זה, שלפוחית ​​השתן ניתן לנקז שתן שנאספו לניתוח נוסף. </li><li style=";text-align:right;direction:rtl"> 100 μl של תמיסת מלח סטרילית נורמלי הוא החדירו לתוך שלפוחית ​​השתן באמצעות קטטר כדי לשפר את הנראות שלפוחית ​​השתן באולטראסאונד. </li><li style=";text-align:right;direction:rtl"> הולכה ג&#39;ל אולטראסאונד מוחל על הבטן, אולטרסאונד מבוצע על מנת להעריך את קיר שלפוחית ​​השתן לומן. </li></ol> 5. נציג תוצאות: רקמת הגידול יתחילו להופיע 3-5 ימים לאחר innoculation. מניסיוננו, קבוצה של 12 עכברים, 50% או יותר שגרתי יש מסת רקמת לזיהוי בתוך השלפוחית ​​(0.5-1.0 μl, באמצעות V = π / 6 ° L ° W · H) על ידי חמישה ימים. בעת שימוש MB 49 תאים, גידולים ימשיכו לגדול המתת חסד יידרש על ידי 24 יום אם ללכת העכברים שלא טופלו. המתת חסד מומלץ כאשר העכברים להיות רדום או cachectic, להציג פרווה פרועות, מכנס, שאינם מגיבים התנהגות, או כאשר הם מאבדים 20% ממשקל הגוף הראשוני שלהן. &lt;/ P&gt; הגידולים לגדול במהירות יהיה מרכזי נמקי לאחר שהם מגיעים המוני של 200 מ&quot;ג או יותר, 3 לכאורה כי התאים בגידול בפיתוח מתרבים מהר יותר מאשר מה שניתן לתיקון על ידי אספקת הדם angiogenic. אפשר גם לעקוב אחר התקדמות הגידול בעקיפין דרך רמת hematurea, אשר קשורה כנראה אנגיוגנזה מהירה בתוך גידולים מתפתחות. צוין באופן עקבי כי שתן של נושאות גידולים עכברים שלא טופלו בחומר הכלול דם ביום 14. ריכוז כדוריות הדם האדומות הופיעו להגדיל לאורך זמן אצל עכברים אלו, הכרכים של שתן שנאספו פחתה. גידולים עודף של 50 מ&quot;ג יכול להיות גם מישש בקלות יחסית, בדרך כלל על ידי innoculation 14 ימים הודעה.

<ol>
	<li>Institute of Laboratory Animal Resources. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide for the Care and Use of Laboratory Animals. , (1996).</li><li>Gunther, J. H., Jurczok, A., Wulf, T., Brandau, S., Deinert, I., Jocham, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+syngeneic+orthotopic+murine+bladder+cancer+(MB49).">Optimizing syngeneic orthotopic murine bladder cancer (MB49).</a> Cancer Res. 59, 2834-2837 (1999).</li><li>Zhang, X., Atala, A., Godbey, W. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression-targeted+gene+therapy+for+the+treatment+of+transitional+cell+carcinoma.">Expression-targeted gene therapy for the treatment of transitional cell carcinoma.</a> Cancer Gene Ther. 15, 543-552 (2008).</li></ol>

ניסויים בבעלי חיים בוצעו בהתאם להנחיות והתקנות שנקבעו על ידי טיפול המוסדי ועדת שימוש אוניברסיטת טוליין.

שיטה זו פולשנית של הקמת גידולים בשלפוחית ​​השתן בעכברים orthotopic הנשי יכול להיות מיושם גם על סוגי תאים אחרים מאשר MB49, למרות תוצאות שונות צריך להיות צפוי. כאשר פחות אגרסיבי תאים סרטניים נמצאים בשימוש, הגדלת חלשות במקצת (באמצעות במרווחים של 0.5 W להקים את אופטימלית חדש) תני...

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<td>C57Bl/6J mice</td>
<td>&nbsp;</td>
<td>Jackson Laboratories</td>
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an orthotopic model of murine bladder cancer

בהליך זה פשוט, גידולים בשלפוחית ​​השתן הן הוקמה בשנת C57 נקבות עכברים באמצעות צנתור, צריבה המקומי, הידבקות התא הבאים. לאחר שלפוחיות השתן שלהם הם צינתור transurethrally והריק, בעלי חיים צינתור שוב כדי לאפשר החדרת חוט פלטינה לתוך שלפוחיות מבלי לפגוע השופכה או בשלפוחית ​​השתן. צנתרים עשויים טפלון לשמש כמבודד על חוט, אשר תנהל זרם חשמלי לתוך השלפוחית ​​כדי ליצור פגיעה לשרוף. יחידת electrocautery משמש כדי לספק 2.5W עד סוף חשוף של הכבל, שתשרוף את שכבות תאי ומתן אתרי מצורף קרצינומה של תאים, אשר נשלחו על ההשעיה לשלפוחית ​​השתן דרך צנתור מאוחר יותר. תאים נשארים בשלפוחית ​​במשך 90 דקות, שלאחריו צנתרים יוסרו שלפוחיות המותר כדי לנקז באופן טבעי. הפיתוח של הגידול מנוטר באמצעות אולטרסאונד. תשומת לב ספציפית משולמת טכניקת צנתור בסרטון הנלווה.

Watch this Scientific Journal Video about An Orthotopic Model of Murine Bladder Cancer at JoVE.com

An Orthotopic Model of Murine Bladder Cancer

גידולים בשלפוחית ​​השתן הן הוקמה בשנת נקבות העכברים בצורה פולשנית דרך צנתור, צריבה המקומי, בעקבות הידבקות של תאים קרצינומה לאתרים לשרוף.

דגם orthotopic של סרטן שלפוחית ​​השתן Murine

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

an-orthotopic-model-of-murine-bladder-cancer

Research

JoVE Journal

Medicine

15.8K Views.  Tulane University. גידולים בשלפוחית ​​השתן הן הוקמה בשנת נקבות העכברים בצורה פולשנית דרך צנתור, צריבה המקומי, בעקבות הידבקות של תאים קרצינומה לאתרים לשרוף.

Video: An Orthotopic Model of Murine Bladder Cancer

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-פוטון הדמיה של הפלישה תאים סרטניים במודל עכבר orthotopic של קרצינומה של תאים קשקשיים אוראלי

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

שילוב הקרנות של מודל העכבר Orthotopic המוח גידול

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

Development of Obliterative Bronchiolitis in a Murine Model of Orthotopic Lung Transplantation

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and standardized not only for the study of allo-rejection but also between syngeneic strains for examining mechanisms of ischemia-reperfusion injury after lung transplantation. Although the application of the rat and other large animal model 2 contributed significantly to the elucidation of these studies, the scope of those investigations is limited by the scarcity of knockout and transgenic rats. Due to no effective therapies for obliterative bronchiolitis, the leading cause of death in lung transplant patients, there has been an intensive search for pre-clinical models that replicate obliterative bronchiolitis. The tracheal allograft model is the most widely used and may reproduce some of the histopathologic features of obliterative bronchiolitis 3. However, the lack of an intact vasculature with no connection to the recipient's conducting airways, and incomplete pathologic features of obliterative bronchiolitis limit the utility of this model 4. Unlike transplantation of other solid organs, vascularized mouse lung transplants have only recently been reported by Okazaki and colleagues for the first time in 2007 5. Applying the basic principles of the rat lung transplant, our lab initiated the obliterative bronchiolitis model using minor histoincompatible antigen murine orthotopic single-left lung transplants which allows the further study of obliterative bronchiolitis immunopathogenesis6.

Obliterative bronchiolitis is the key impediment to the long-term survival of lung transplant recipients and the lack of a robust preclinical model precludes examining obliterative bronchiolitis immunopathogenesis. Unlike other solid organ transplants, vascularized mouse lung transplantation has only recently been developed. Here we show our independently developed obliterative bronchiolitis model after murine orthotopic single-lung transplantation.

פיתוח Bronchiolitis סותם במודל Murine של השתלת ריאות Orthotopic

Orthotopic lung transplantation in rats was first reported by Asimacopoulos and colleagues in 1971 1. Currently, this method is well accepted and ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

שלפוחית ​​השתן Orthotopic גידול דגם ואת הערכת טיפול סרנה השלפוחית

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

מודל עכבר orthotopic של קרצינומה של בלוטת התריס אנפלסטית

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

סרטן שלפוחית ​​השתן לדגם orthotopic ג&#39;ין משלוח לימודים

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

באמצעות PCR כמוני בזמן אמת לקביעת engraftment תא התורם במודל תחרותי Murine מח עצם השתלה

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

דגם orthotopic bioluminescent של התקדמות סרטן הלבלב

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

מודל Murine orthotopic של אדם סרטן ערמונית גרורה

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

פולשנית הקמת Murine Orthotopic שלפוחית ​​השתן xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

דגם orthotopic של סרטן שלפוחית ​​השתן Murine

דגם orthotopic של סרטן שלפוחית השתן Murine