עבודה זו נתמכה בחלקה על ידי מענקי מחקר המלומדת (116403-RSG-09-082-01-MgO) של האגודה האמריקנית למלחמה בסרטן וקופות המיפוי של אוניברסיטת Wake Forest למדעי הבריאות ל GS. DBS נתמך על ידי NCI אימון מענק 5T32CA079448.

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אין ניגוד עניינים הצהיר.

פרוטוקול זה מתאר שיטה להפיל הגן באמצעות shRNA ולדמיין ההשפעה הביולוגית שלה באמצעות הדמיה bioluminescent גידול תאים סרטניים in vivo. אתר היעד של shRNA לא יכילו כל אחד משלושת אתרי הגבלה (BamHI, HindIII ו EcoRI) משמש עבור subcloning. בתרחיש נדיר כאשר כל אחד מהאתרים הללו קיים, אנזים הגבלה נוספת שנ...

בנוסף ציוד מעבדה נפוצה המשמשת RNA וניתוח חלבון ו תרבית תאים, חומרים הבאים ריאגנטים נדרשים עבור פרוטוקול זה. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td></tr><tr><td> בטיחות ביולוגית מתאים הבלימה ברמת בטיחות ביולוגית 2 הממשלה </td></tr><tr><td> PCR thermocycler </td></tr><tr><td> Ultracentrifuge </td></tr><tr><td> הרדמה, אינדוקציה קאמרית עם אוכלי נבלות isoflurane </td></tr><tr><td> Digital Vernier קליפר </td></tr><tr><td> IVIS הדמיה המערכת </td></tr><tr><td> המוסמכות ביותר DH5a החיידק אשריכיה קולי </td></tr><tr><td> EcoRI, BamHI, &amp; HindIII הגבלה אנזימים </td></tr><tr><td> האנזים T4 DNA </td></tr><tr><td> הדור השלישי lentivirus אריזה פלסמידים VSV-G, pRSV-Rev ו pMDLg / pRRE </td></tr></tbody></table> רשימת חומרים

dna vector-based rna interference to study gene function in cancer

התערבות RNA (RNAi) מעכב ביטוי גנים על ידי mRNAs יעד משפילים במיוחד. מאז גילויו של פעמיים תקועים התערבות RNA קטן (siRNA) בגן ההשתקה 1, RNAi הפך לכלי רב עוצמה המחקר במחקרים תפקוד הגן. לעומת מחיקה גנטית, RNAi בתיווך להשתקת גנים בעל יתרונות רבים, כגון הקלות שבה היא מתבצעת והתאמתו שורות תאים ביותר. מחקרים רבים הדגימו את היישומים של טכנולוגיית RNAi בחקר הסרטן. בפרט, פיתוח טכנולוגיית ה-DNA וקטור מבוסס לייצר סיכת ראש RNA קטן (shRNA) מונע על ידי U6 או האמרגן H1 הפכה לטווח ארוך הגן מושרה להשתיק 2,3 אפשרי. השימוש בו בשילוב עם וקטורים ויראליים מהונדסים גנטית, כמו lentivirus, מקלה על יעילות גבוהה של משלוח shRNA ו / או אינטגרציה לתוך הדנ&quot;א הגנומי לביטוי shRNA יציב. אנו מתארים detaileהליך D באמצעות וקטור מבוססי טכנולוגיית ה-DNA RNAi לקבוע תפקוד הגן, כולל בנייה של וקטורים lentiviral המבטאים shRNA, ייצור lentivirus וזיהום התא, ומחקרים תפקודית באמצעות מודל xenograft העכבר. אסטרטגיות שונות דווחו ביצירת מבנים shRNA. פרוטוקול המתואר כאן העסקת הגברה PCR ו קשירת 3-שבר יכול לשמש ישירות וביעילות ליצור shRNA המכילים מבנים lentiviral מבלי לעזוב כל הצמוד נוקלאוטיד נוסף ברצף shRNA קידוד. מאז shRNA ביטוי קלטות שנוצרו על ידי אסטרטגיה זו ניתן לגזור על ידי אנזימי הגבלה, הם יכולים להעביר בקלות וקטורים אחרים עם סמנים ניאון או אנטיביוטיקה שונים. ריאגנטים transfection המסחריים יכולים לשמש בייצור lentivirus. עם זאת, בדו&quot;ח זה, אנו מספקים שיטה כלכלית באמצעות סידן זרחתי משקעים כי ניתן להשיג על יעילות transfection של 90%293T תאים. לעומת מכוננת וקטורים shRNA הביטוי, מערכת shRNA מושרה מתאים במיוחד כדי לדפוק את הגן חיוני התפשטות תאים. אנחנו מדגימים להשתקת גנים של ין יאנג 1 (YY1), oncogene פוטנציאל בסרטן השד 4,5, על ידי ט במערכת מושרה shRNA והשפעתו על היווצרות הגידול. מחקר באמצעות lentivirus מחייב סקירה ואישור פרוטוקול בטיחות ביולוגית על ידי ועדת בטיחות ביולוגית של המוסד של החוקר. מחקרים באמצעות מודלים של בעלי חיים מחייבת בדיקה ואישור של פרוטוקול בעלי חיים על ידי טיפול בבעלי חיים ועדת שימוש (ACUC) במוסד של החוקר.

Watch this Scientific Journal Video about DNA Vector-based RNA Interference to Study Gene Function in Cancer at JoVE.com

DNA Vector-based RNA Interference to Study Gene Function in Cancer

התערבות RNA (RNAi) הוא בעל יתרונות רבים על פני בנוקאאוט הגן נעשה שימוש נרחב ככלי במחקרים גנים תפקודיים. המצאת ה-DNA וקטור מבוססי RNAi הטכנולוגיה הפכה לטווח ארוך הגן מושרה מציאה אפשרי, גם הגדילה את הכדאיות של השתקת הגן<em&gt; In vivo</em&gt;.

וקטור מבוסס DNA התערבות RNA ללמוד לתפקד גנים סרטן

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA ...

dna-vector-based-rna-interference-to-study-gene-function-in-cancer

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20.3K Views.  Wake Forest University School of Medicine. התערבות RNA (RNAi) הוא בעל יתרונות רבים על פני בנוקאאוט הגן נעשה שימוש נרחב ככלי במחקרים גנים תפקודיים. המצאת ה-DNA וקטור מבוססי RNAi הטכנולוגיה הפכה לטווח ארוך הגן מושרה מציאה אפשרי, גם הגדילה את הכדאיות של השתקת הגן In vivo.

Video: DNA Vector-based RNA Interference to Study Gene Function in Cancer

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
contractions. The pelvic limb of an anesthetized dog is immobilized in a stereotactic frame to align the tibia at a right angle to the femur. Adhesive wrap affixes the 
paw to a pedal mounted on the shaft of a servomotor to measure torque. Percutaneous nerve stimulation activates pelvic limb muscles of the paw to either push (extend) or 
pull (flex) against the pedal to generate isometric torque. Percutaneous tibial nerve stimulation activates tibiotarsal extensor muscles. Repeated eccentric (lengthening) 
contractions are induced in the tibiotarsal flexor muscles by percutaneous peroneal nerve stimulation. The eccentric protocol consists of an initial isometric contraction 
followed by a forced stretch imposed by the servomotor. The rotation effectively lengthens the muscle while it contracts, e.g., an eccentric contraction. During 
stimulation flexor muscles are subjected to an 800 msec isometric and 200 msec eccentric contraction. This procedure is repeated every 5 sec. To avoid fatigue, 4 min rest 
follows every 10 contractions with a total of 30 contractions performed.

In Vivo Canine Muscle Function Assay

We describe a minimally-invasive and painless method to measure canine hindlimb muscle strength and muscle response to repeated eccentric contractions.

In vivo Assay שרירים כלב פונקציה

We describe a minimally-invasive and reproducible method to measure canine pelvic limb muscle strength and muscle response to repeated eccentric 
...

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

במבחנה מערכת לחקר תרדמת גידול ואת החלף ל גידול גרורתי

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

Biomarkers in an Animal Model for Revealing Neural, Hematologic, and Behavioral Correlates of PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for objective diagnosis but also for the evaluation of therapeutic efficacy and resilience to trauma. Ongoing research is directed at identifying molecular biomarkers for PTSD, including traumatic stress induced proteins, transcriptomes, genomic variances and genetic modulators, using biologic samples from subjects' blood, saliva, urine, and postmortem brain tissues. However, the correlation of these biomarker molecules in peripheral or postmortem samples to altered brain functions associated with psychiatric symptoms in PTSD remains unresolved. Here, we present an animal model of PTSD in which both peripheral blood and central brain biomarkers, as well as behavioral phenotype, can be collected and measured, thus providing the needed correlation of the central biomarkers of PTSD, which are mechanistic and pathognomonic but cannot be collected from people, with the peripheral biomarkers and behavioral phenotypes, which can.
Our animal model of PTSD employs restraint and tail shocks repeated for three continuous days - the inescapable tail-shock model (ITS) in rats. This ITS model mimics the pathophysiology of PTSD 17, 7, 4, 10. We and others have verified that the ITS model induces behavioral and neurobiological alterations similar to those found in PTSD subjects 17, 7, 10, 9. Specifically, these stressed rats exhibit (1) a delayed and exaggerated startle response appearing several days after stressor cessation, which given the compressed time scale of the rat's life compared to a humans, corresponds to the one to three months delay of symptoms in PTSD patients (DSM-IV-TR PTSD Criterian D/E 13), (2) enhanced plasma corticosterone (CORT) for several days, indicating compromise of the hypothalamopituitary axis (HPA), and (3) retarded body weight gain after stressor cessation, indicating dysfunction of metabolic regulation.
The experimental paradigms employed for this model are: (1) a learned helplessness paradigm in the rat assayed by measurement of acoustic startle response (ASR) and a charting of body mass; (2) microdissection of the rat brain into regions and nuclei; (3) enzyme-linked immunosorbent assay (ELISA) for blood levels of CORT; (4) a gene expression microarray plus related bioinformatics tools 18. This microarray, dubbed rMNChip, focuses on mitochondrial and mitochondria-related nuclear genes in the rat so as to specifically address the neuronal bioenergetics hypothesized to be involved in PTSD.

We describe a rat model of post traumatic stress disorder (PTSD) that reveals the persistent alterations in neuroendocrine function and the delayed long-term, exaggerated fear response, characteristic of PTSD patients. The animal model and methods described here are useful for correlating biomarkers in brain nuclei, which are mechanistic but cannot be measured in patients, with biomarkers in peripheral white blood cells, which can.

סמנים ביולוגיים במודל חיה לחשיפה קושרת עצבית, המטולוגית, והתנהגות של PTSD

Identification of biomarkers representing the evolution of the pathophysiology of Post Traumatic Stress Disorder (PTSD) is vitally important, not only for ...

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 ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

פולשנית Intratracheal אינטובציה ללמוד פתולוגיה והפיזיולוגיה של עכבר ריאות

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

שלוש תרבויות ממדים: כלי ללמוד רגיל אדריכלות acinar לעומת שינוי ממאיר של תאי שד

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

<em&gt; Ex vivo</em&gt; דגם לחקר הורמוני פעולה בשד האדם

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

In vivo לשעבר vivo גישות לחקר סרטן השחלות גרורתי התיישבות של מבני ספוט החלב בהצפק שומני

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Using Saccadometry with Deep Brain Stimulation to Study Normal and Pathological Brain Function

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including Parkinson's and Huntington's can disrupt it. People with Parkinson's disease, for example, tend to have increased saccadic latencies. Consequently, the quantitative measurement of saccadic eye movements has received considerable attention as a potential biomarker for neurodegenerative conditions. A lot more can be learned about the brain in both health and disease by observing what happens to eye movements when the function of specific brain areas is perturbed. Deep brain stimulation is a surgical intervention used for the management of a range of neurological conditions including Parkinson's disease, in which stimulating electrodes are placed in specific brain areas including several sites in the basal ganglia. Eye movement measurements can then be made with the stimulator systems both off and on and the results compared. With suitable experimental design, this approach can be used to study the pathophysiology of the disease being treated, the mechanism by which DBS exerts it beneficial effects, and even aspects of normal neurophysiology.

This paper describes the use of quantitative measurement of eye movements in conjunction with stimulation of focal areas of the deep brain in order to study physiology, pathophysiology, and the mechanisms of deep brain stimulation.

שימוש Saccadometry עם דיפ גירוי המוח לחקר תפקוד מוח נורמלי ופתולוגיים

The oculomotor system involves a large number of brain areas including parts of the basal ganglia, and various neurodegenerative diseases including ...

TRUE Gene Silencing: Screening of a Heptamer-type Small Guide RNA Library for Potential Cancer Therapeutic Agents

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3&#8242; processing endoribonuclease, tRNase ZL, to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.

Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers.

ג&#39;ין השתקה נכונה: הקרנת ספריית RNA מדריך Heptamer מסוג Small בתרופות סרטן פוטנציאליות

TRUE gene silencing (termed after tRNase ZL-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes ...