وأيد هذا العمل في جزء من المنح باحث (116403-RSG-09-082-01-MGO) من الجمعية الأميركية للسرطان وصناديق جماعية من ويك فورست العلوم الصحية جامعة إلى ع. وأيد DBS بواسطة منحة لجنة التحقيق الوطنية 5T32CA079448 التدريب.

<ol>
	<li>Elbashir, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Duplexes+of+21-nucleotide+RNAs+mediate+RNA+interference+in+cultured+mammalian+cells.">Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells.</a> Nature. 411, 494-498 (2001).</li><li>Sui, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+DNA+vector-based+RNAi+technology+to+suppress+gene+expression+in+mammalian+cells.">A DNA vector-based RNAi technology to suppress gene expression in mammalian cells.</a> Proc. Natl. Acad. Sci. U.S.A. 99, 5515-5520 (2002).</li><li>Brummelkamp, T. R., Bernards, R., Agami, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+system+for+stable+expression+of+short+interfering+RNAs+in+mammalian+cells.">A system for stable expression of short interfering RNAs in mammalian cells.</a> Science. 296, 550-553 (2002).</li><li>Zhang, Q., Stovall, D. B., Inoue, K., Sui, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+oncogenic+role+of+Yin+Yang+1.">The oncogenic role of Yin Yang 1.</a> Crit. Rev. Oncog. 16, 163-197 (2011).</li><li>Wan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yin+Yang+1+plays+an+essential+role+in+breast+cancer+and+negatively+regulates+p27.">Yin Yang 1 plays an essential role in breast cancer and negatively regulates p27.</a> Am. J. Pathol. , (2012).</li><li>Sui, G., Shi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+silencing+by+a+DNA+vector-based+RNAi+technology.">Gene silencing by a DNA vector-based RNAi technology.</a> Methods in Molecular Biology. 309, 205-218 (2005).</li><li>Trower, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+PCR-based+colony+screening+protocol+for+cloned+inserts.">A rapid PCR-based colony screening protocol for cloned inserts.</a> Methods Mol. Biol. 58, 329-333 (1996).</li><li>Lizee, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantitative+reverse+transcriptase-polymerase+chain+reaction+as+a+method+for+determining+lentiviral+vector+titers+and+measuring+transgene+expression.">Real-time quantitative reverse transcriptase-polymerase chain reaction as a method for determining lentiviral vector titers and measuring transgene expression.</a> Hum. Gene Ther. 14, 497-507 (2003).</li><li>Zhang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+significance+of+controlled+conditions+in+lentiviral+vector+titration+and+in+the+use+of+multiplicity+of+infection+(MOI)+for+predicting+gene+transfer+events.">The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events.</a> Genet Vaccines Ther. 2, 6 (2004).</li><li>Geran, R. I., Greenberg, N. H., MacDonald, M. M., Schumacher, A. M., Abbott, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocols+for+screening+chemical+agents+and+natural+products+against+animal+tumors+and+other+biological+systems.">Protocols for screening chemical agents and natural products against animal tumors and other biological systems.</a> Cancer Chemother. Rep. 3, 1-103 (1972).</li><li>Massoud, T. F., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+imaging+in+living+subjects:+seeing+fundamental+biological+processes+in+a+new+light.">Molecular imaging in living subjects: seeing fundamental biological processes in a new light.</a> Genes Dev. 17, 545-580 (2003).</li><li>Inoue, H., Nojima, H., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+transformation+of+Escherichia+coli+with+plasmids.">High efficiency transformation of Escherichia coli with plasmids.</a> Gene. 96, 23-28 (1990).</li><li>Toledo, J. R., Prieto, Y., Oramas, N., Sanchez, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polyethylenimine-based+transfection+method+as+a+simple+and+effective+way+to+produce+recombinant+lentiviral+vectors.">Polyethylenimine-based transfection method as a simple and effective way to produce recombinant lentiviral vectors.</a> Appl. Biochem. Biotechnol. 157, 538-544 (2009).</li><li>Rubinson, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+lentivirus-based+system+to+functionally+silence+genes+in+primary+mammalian+cells,+stem+cells+and+transgenic+mice+by+RNA+interference.">A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference.</a> Nat Genet. 33, 401-406 (2003).</li><li>Sui, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yin+Yang+1+is+a+negative+regulator+of+p53.">Yin Yang 1 is a negative regulator of p53.</a> Cell. 117, 859-872 (2004).</li></ol>

الإعلان عن أي تضارب في المصالح.

هذا البروتوكول وصفا لطريقة لاسقاط الجين باستخدام shRNA ووضع تصور لتأثيره البيولوجية باستخدام التصوير bioluminescent من نمو الخلايا السرطانية في الجسم الحي. الموقع هدفا لshRNA يجب أن لا تحتوي على أي من مواقع تقييد الثلاثة (BamHI، HindIII وEcoRI) المستخدمة لsubcloning. في سيناريو نادر عند...

بالإضافة إلى المعدات المختبرية التي يشيع استخدامها في الحمض النووي الريبي وتحليلات البروتين وزراعة الخلايا، ويطلب من المواد التالية والكواشف لهذا البروتوكول. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td></tr><tr><td> السلامة البيولوجية مجلس الوزراء مناسبة لاحتواء السلامة الأحيائية من المستوى 2 </td></tr><tr><td> PCR thermocycler </td></tr><tr><td> منبذة فائقة </td></tr><tr><td> التخدير التعريفي غرفة مع الزبالين isoflurane </td></tr><tr><td> الرقمية الورنية الفرجار </td></tr><tr><td> IVIS نظام التصوير </td></tr><tr><td> درجة عالية من الكفاءة DH5a كولاي </td></tr><tr><td> الانزيمات EcoRI، BamHI، وتقييد HindIII </td></tr><tr><td> T4 الحمض النووي يغاز </td></tr><tr><td> ثالث البلازميدات التعبئة والتغليف جيل الفيروسة البطيئة VSV-G، pRSV القس وpMDLg / pRRE </td></tr></tbody></table> قائمة المواد

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

تدخل الجيش الملكي النيبالي (رني) يحول دون التعبير الجيني من قبل mRNAs الهدف المهينة على وجه التحديد. منذ اكتشاف المزدوج تقطعت بهم السبل الحمض النووي الريبي التدخل الصغيرة (سيرنا) في إسكات الجينات 1، وأصبح رني أداة بحث قوية في الدراسات وظيفة الجين. بالمقارنة مع حذف الجينية، [رني بوساطة إسكات الجينات تمتلك العديد من المزايا، مثل السهولة التي تتم فيها ومدى ملاءمتها لمعظم خطوط الخلية. وقد أظهرت دراسات متعددة في تطبيقات تكنولوجيا رني في أبحاث السرطان. على وجه الخصوص، جعلت تطوير تكنولوجيا مكافحة ناقلات القائم على الحمض النووي الريبي لانتاج صغير دبوس الشعر (shRNA) مدفوعا U6 أو مروج H1 على المدى الطويل والجينات محرض إسكات 2،3 ممكن. استخدامه في تركيبة مع النواقل الفيروسية وراثيا، مثل الفيروسة البطيئة، تسهل الكفاءة العالية للتسليم shRNA و / أو الاندماج في الحمض النووي الجيني للتعبير shRNA مستقر. نحن تصف detaileد الإجراء باستخدام تكنولوجيا الحمض النووي رني ناقلات القائم على تحديد وظيفة الجينات، بما في ذلك بناء ناقلات lentiviral معربا عن shRNA، وإنتاج الفيروسة البطيئة وإصابة الخلية، والدراسات الفنية باستخدام نموذج الفأر طعم أجنبي. تم الإبلاغ عن الاستراتيجيات المختلفة في توليد بنيات shRNA. وصف بروتوكول هنا توظيف تضخيم PCR وربط 3-شظية يمكن استخدامها لمباشرة وكفاءة توليد shRNA التي تحتوي على ثوابت lentiviral دون أن تترك أي المتاخمة اضافية النوكليوتيدات لتسلسل shRNA الترميز. حيث يمكن خفض الكاسيت shRNA التعبير عن هذه الاستراتيجية التي أنشأتها من قبل إنزيمات التقييد، ويمكن بسهولة انتقلوا إلى ناقلات أخرى مع مختلف علامات الفلورسنت أو المضادات الحيوية. ويمكن استخدام الكواشف ترنسفكأيشن معظم الإنتاج التجاري في الفيروسة البطيئة. ومع ذلك، في هذا التقرير، ونحن نقدم وسيلة اقتصادية باستخدام الكالسيوم فوسفات هطول الأمطار التي يمكن أن تحقق أكثر من 90٪ في كفاءة ترنسفكأيشن293T الخلايا. مقارنة التأسيسي ناقلات التعبير shRNA، نظام shRNA محرض هو مناسبة خاصة لاسقاط الجين ضروري لتكاثر الخلايا. علينا أن نبرهن على إسكات الجينات من يانغ يين 1 (YY1)، وهو الجين الورمي المحتملة في الاصابة بسرطان الثدي 4،5، من تيت، وعلى نظام shRNA محرض وآثارها على تكوين ورم. بحث باستخدام الفيروسة البطيئة يتطلب مراجعة والموافقة على بروتوكول السلامة الاحيائية من قبل لجنة السلامة من مؤسسة الباحث. بحث باستخدام نماذج حيوانية يتطلب مراجعة والموافقة على بروتوكول الحيوان من قبل لجنة رعاية الحيوان واستخدام (ACUC) من مؤسسة الباحث.

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

تدخل الجيش الملكي النيبالي (رني) تمتلك مزايا عديدة أكثر من خروج المغلوب الجينات، واستخدمت على نطاق واسع باعتبارها أداة في دراسات الجينات الوظيفية. جعلت من اختراع تكنولوجيا الحمض النووي رني ناقلات القائم على المدى الطويل، ومحرض ضربة قاضية الجين ممكن، وزادت أيضا في جدوى إسكات الجينات<em&gt; في الجسم الحي</em&gt;.

الحمض النووي الريبي التدخل ناقلات القائم على دراسة وظيفة الجينات في سرطان

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. تدخل الجيش الملكي النيبالي (رني) تمتلك مزايا عديدة أكثر من خروج المغلوب الجينات، واستخدمت على نطاق واسع باعتبارها أداة في دراسات الجينات الوظيفية. جعلت من اختراع تكنولوجيا الحمض النووي رني ناقلات القائم على المدى الطويل، ومحرض ضربة قاضية الجين ممكن، وزادت أيضا...

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.

فيفو الناب في العضلات وظيفة الفحص

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.

و في المختبر النظام الدراسي لاستئصال ورم من الخمول وتبديل للنمو النقيلي

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.

المؤشرات الحيوية في نموذج حيواني لكشف الاقتران العصبية، الدموية، والسلوكية للPTSD

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.

وسرطان المثانة نموذج مثلي للدراسات توصيل الجينات

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.

موسع داخل الرغامى التنبيب لدراسة علم الأمراض وعلم وظائف الأعضاء من الرئة ماوس

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.

ثلاثة الثقافات الأبعاد: أداة لدراسة الهندسة المعمارية عادي عنيبية مقابل التحول السرطاني للخلايا الثدي

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.

ل<em&gt; السابقين فيفو</em&gt; نموذج لدراسة هرمون العمل في الثدي البشري

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.

في فيفو وفيفو السابقين المداخل لدراسة سرطان المبيض النقيلي الاستعمار من الهياكل التبانة بقعة في البريتوني الدهنية

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.

باستخدام Saccadometry مع ديب تحفيز الدماغ لدراسة العادية والمرضية وظيفة الدماغ

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.

يصح جين إسكات: عرض لHeptamer من نوع RNA دليل مكتبة صغيرة للسرطان العلاجية وكلاء المحتملة

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