We thank Dr. John Ohlfest and Dr. Stacey Decker13 for the generous gift of plasmids and for the training provided to master this method. We thank Marta Dzaman for help with standardizing the Hematoxylin-Eosin staining procedure of paraffin-embedded sections. We also thank Molly Dahlgren for perusing this manuscript and providing helpful suggestions. This work is supported by NIH/NINDS grants to MGC and PRL, Leah&#8217;s Happy Hearts Foundation grant to MCG and PRL. Alex&#8217;s Lemonade Foundation young Investigator Award and the St. Baldrick&#8217;s Foundation Fellowship to CK.

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The authors have nothing to disclose.

במאמר זה, אנו פירוט שיטה תכליתית ושחזור ליצירת מודלים חדשים של GBM באמצעות שילוב transposase- SB בתיווך של פלסמיד דנ&quot;א שעור לתאים המקיפים את אזור subventricular של עכברים בילוד. אנו מציגים פרוטוקול להפקת פלסמידים transposon עם גנים חדשים של עניין, להמחיש כיצד לפקח על ההתקדמות של הגידו?...

multiforme גליובלסטומה (GBM) הוא (60%) הגידול הנפוץ ביותר העיקרי המוח במבוגרים, עם חציון הישרדות של 15-21 חודשים, כאשר טופל בניתוח, הקרנות, וכימותרפיה 1. טיפולים חדשניים לGBM הם הכרחיים. טיפולים ניסיוניים דורשים בדיקה במודלים של בעלי חיים שכראוי לשחזר את המאפיינים הבולטים של המחלה האנושית. אסטרטגיות כדי לגרום GBM במכרסמים כוללים mutagenesis הכימי עם סוכני אלקילציה, בתאי מין או שינויים גנטיים סומטיים, או השתלה באמצעות תא גליומה קווים 2. הנפוץ ביותר המודלים להעסיק את ההשתלה של שורות תאי גליומה, או orthotopically, למוח או תת עורי באמצעות תאי syngeneic בבעלי חיים עם גנוטיפ הזהה; או תאי xenogeneic, קווים הנפוצים ביותר אדם GBM תא, שהושתלו בעכברים חיסוניים חלש 3. Xenografts מציע יתרונות רבים למחקר של גידולים תוך-גולגולתי: נוחות של שחזור, standaשיעורי rdized צמיחה, זמן של לוקליזציה מוות וגידול. עם זאת, יש לי מודלים אלה מגבלות בשל הגישה המלאכותית, פולשני כירורגי המשמשת להשתלות ויכולת מוגבלת לשחזר במדויק היסטולוגית כולל מאפיין של GBM האנושי (IV הכיתה WHO): נמק פסאודו-pallisading, atypias הגרעיני, פלישה מפוזרת, ריבוי מיקרו-וסקולרית והיווצרות של כלי דם glomeruloid חריגות 4-7. אינדוקציה של GBM על ידי שינוי הגנום של תאים סומטיים עם DNA שעור, או עם וקטורים ויראליים 8-12, או עם שילוב בתיווך transposon 13, מתרבה באופן הדוק יותר אטיולוגיה של מחלות בבני אדם ומשחזרת תכונות histo-פתולוגי של GBM האנושי. מבחינה הסטורית, גידולים של מערכת העצבים המרכזית שמאורגנים על בסיס התא הנתפס של מקור, שבו נצפה, יהיה גורם מנבא להישרדות 14. GBMs מסווג ליסודי ותיכוני. טוד ראיות המתעורריםאיי מצביע על אופי הטרוגני מאוד של גליובלסטומה העיקרית 15. GBM המשני (15%), כתוצאה משינוי הממאיר של astrocytomas הנמוך כיתה (WHO אני כיתה) וastrocytomas anaplasic (WHO כיתה II), המשויכים תחילת מוקדמת של המחלה, הפרוגנוזה טובה יותר ודפוס &quot;proneural&quot; של ביטוי גנים , ואילו GBM הראשוני (85%) מראה תחילת סוף, הפרוגנוזה וגליה (קלסית) עלובות, עצבית או דפוסי ביטוי mesenchymal. אם דפוסי ביטוי גנים אלה לתאם עם התא בפועל המוצא של הגידול הוא עדיין נחקר באופן פעיל. צבירת נתונים מראה כי השילוב של מוטציות גנטיות הקשורות לGBMs מנבא להישרדות. לדוגמא, אובדן heterozygocity (LOH) של כרומוזומים 1p / 19q, מוטציות IDH1, amplifications PDGFRα, המשויכים GBMs המשני, דפוס ביטוי proneural ופרוגנוזה טובה יותר, ואילו EGFR ביטוי היתר, Notch והפעלת מסלול קיפוד סוניק, NFאובדן ומוטציות של p53 1 וPTEN מתואמים עם עצבי, קלאסי, או GBM הראשוני mesenchymal ופרוגנוזה גרועה 16,17. כניסתו של פרויקטים לקביעת רצף בקנה מידה גדולים וההצטברות של דגימות מטופל רבות זמינות לבדיקה מביאה שפע של מידע החדש ביחס למוטציות גנטיות ומסלולים מעורבים בפתוגנזה GBM והתקדמות והאפשרות של רפואה מותאמת אישית, שבו טיפולים יכולים להיות מותאמים באופן ספציפי ל מומים הגנטיים של המטופל. סופו של דבר, כדי להעריך את הערך המנבא של מוטציות אלה ומסלולים באופן שיטתי, ולבחון טיפולים אפשריים בכל מקרה ומקרה, דורש מודלים של בעלי חיים של GBM עם שינויים גנטיים שנקבע מראש. אינטגרציה בתיווך transposon של הדנ&quot;א הגנומי מציעה גישה אפשרית. המערכת היפיפייה הנרדמת transposon, חבר Tc1 ספן הכיתה / של transposons, הייתה &quot;מתעורר&quot; (נבנה) בproce רב שלביםss של mutagenesis הספציפי אתר מגן transposase salmonoid, שהפך לפני רדום יותר מ -10 מ&#39;שנים 18,19. בעיקרו של דבר, transposons DNA מוקף רצפים ספציפיים (חוזר הפוך / חזרות ישירות: IR / DR) ניתן לשלב לתוך הגנום באופן &quot;גזור והדבק&quot; באמצעות הפעילות של transposase היפיפה הנרדם. Transposase מכיר את הקצוות של אתרי IR, בולה transposon ומשלב אותו באופן אקראי לאתר DNA אחר בין הבסיסים T ו, בסיסים שישוכפלו בכל קצה של transposon בשכול (איור 1 א). Transposase היפיפה הנרדם מורכב משלושה תחומים, תחום transposon מחייב, רצף לוקליזציה גרעיני ותחום קטליטי. ארבע מולקולות transposase נדרשות להביא את שני קצוות של transposon יחד ולאפשר לשכול, לעומת זאת, אם מולקולות רבות מדי של transposase נמצאות, הם יכולים dimerize וtetramerize לעכבתגובת טרנספוזיציה 20. תגובת טרנספוזיציה יעילה דורשת יחס אופטימלי של transposase לtransposons. DNA קידוד transposase יכול להיות מועבר באותו פלסמיד עם transposon (בcis) או על פלסמיד שונה (בטראנס). כדי להבטיח את היחס האופטימלי בין transposase וtransposons, ניתן לבחור אמרגן עם פעילות מתאימה לביטוי של transposase (למודל &quot;ציס&quot;) או היחס של פלסמידים בפתרון ההזרקה יכולה להיות מותאם (ל מודל &quot;טראנס&quot;). מערכת transposon היפיפייה הנרדמת יכולה לשמש בהצלחה לגנומיקה הפונקציונלית, mutagenesis insertional, transgenesis וריפוי גנטי סומטיים 21. להיות מבנה סינטטי מחדש הנדסת מולקולה פונקציונלית מגרסת salmonoid רדומה, transposase היפיפה הנרדם לא להיקשר transposons האחר בבני אדם או יונקים אחרים 20. מאז גילויו, יש הנדסה מולקולרית העשרתed את היעילות של מערכת transposon SB טרנספוזיציה דרך שינויים ברצפי IR ותוספת של dinucleotides TATA איגוף transposon, וכתוצאה מכך transposons pt2. transposons אלה מותאמים מחייב של SB לאתר IR והגדיל את היעילות של כריתה. Transposase SB גם עבר שיפור משמעותי; transposase המשמש בניסויים שהוצגו במסמך זה SB100X, transposase היפראקטיבי שנוצר על ידי אסטרטגית דשדוש DNA ואחריו מסך גנטי בקנה מידה גדולה בתאי יונקים 22. בדוח זה אנו מציגים שיטה מהירה, תכליתית ושחזור כדי לגרום GBM המהותי בעכברים עם שילוב הלא-נגיפי, בתיווך transposon של פלסמיד דנ&quot;א לתוך תאים של האזור תת-חדרית של עכברי יילודים 23. אנו מציגים דרך פשוטה ליצור transposons עם גני רומן נוח לשילוב הגנומי באמצעות transposase היפיפה הנרדם ומדגים כיצד לפקח על ספיגת פלסמיד והתקדמות מחלה באמצעות פליטת אור. אנחנו גם לאפיין תכונות היסטולוגית והחיסונית histochemical של GBM לשכפל עם המודל הזה. בנוסף, אנו מציגים שיטה מהירה ליצירת שורות תאים ראשוניות GBM מגידולים אלה. המודל היפיפה הנרדם, שבו גידולים הנגרמים מהתאים מקוריים לבעלי החיים, מאפשר הערכה התפקודית של התפקיד של גני GBM מועמד באינדוקציה וההתפתחות של גידולים. מערכת זו גם מתאימה גם לבדיקות של טיפולי GBM רומן, כוללים טיפולים חיסוניים בעכברים חיסוניים מוסמכת, ללא הצורך בניתוחים פולשניים דלקתיים, אשר עשוי לשנות את המיקרו-הסביבה המקומית.

<table border="0" cellpadding="0" cellspacing="0" width="952">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Name of Material/ Equipment</td>
			<td style="width:199px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:267px;">Comments/Description</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:367px;">Stoelting&#39;s Lab Standard with Rat and Mouse Adaptors</td>
			<td style="width:199px;">Stoelting</td>
			<td style="width:119px;">51670</td>
			<td style="width:267px;">Other companies produce similar frames, any of them with small mouse adaptors are suitable&#160;</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:367px;">Quintessential Stereotaxic Injector (QSI)</td>
			<td style="width:199px;">Stoelting</td>
			<td style="width:119px;">53311</td>
			<td style="width:267px;">Injections can be made without it, ,but&#160;&#160;&#160; the automatic injector allows for increased reproducibility and convenience</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:367px;">10 &#956;L 700 series hand fitted MICROLITER syringe</td>
			<td style="width:199px;">Hamilton</td>
			<td style="width:119px;">Model 701</td>
			<td style="width:267px;">This syringe model will deliver from 0.5 to 500 ul of solution reliably. Other syrnges (fro example 5 ul, Model 75) may also be used</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:367px;">30 gauge gauge hypodermic needle 
			(1.25&#34;, 15o bevel)&#160;</td>
			<td style="width:199px;">Hamilton</td>
			<td style="width:119px;">S/O# 197462</td>
			<td style="width:267px;">This needle is ideal to pierce the skin and the skull of a neonatal mouse without the need of other invasive procedures. If it gets dull (doesn&#39;t easily enter the skin), it needs to be replaced.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">in vivo-jetPEI</td>
			<td style="width:199px;">Polyplus Transfection</td>
			<td style="width:119px;">201-10G</td>
			<td style="width:267px;">Aliquot in small volumes and keep at -20oC&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">D-Luciferin, Potassium Salt</td>
			<td style="width:199px;">Goldbio.com</td>
			<td style="width:119px;">LuckK-1g</td>
			<td style="width:267px;">Other sources of firefly lucifierase are just as adequate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Hematoxylin Solution, Harris Modified</td>
			<td style="width:199px;">Sigma Aldrich</td>
			<td style="width:119px;">HHS128-4L</td>
			<td style="width:267px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:367px;">Tissue-Tek O.C.T. Compound</td>
			<td style="width:199px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">62550-01</td>
			<td style="width:267px;">for embedding brains for cryosectioning and immunohistovhemistry</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Advanced DMEM/F-12, no glutamine</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:119px;">12634-010</td>
			<td style="width:267px;">for the culture of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">B-27&#168; Supplement (50X), serum free&#160;</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:119px;">17504-044</td>
			<td style="width:267px;">serum free supplement for the culture of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">N2 Supplement (100x)</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:119px;">17502-048</td>
			<td style="width:267px;">serum free supplement for the culture of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Normocyn</td>
			<td style="width:199px;">InvivoGen</td>
			<td style="width:119px;">ant-nr-1</td>
			<td style="width:267px;">anti-mycoplasma agent for the culture of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Recombinant Human EGF</td>
			<td style="width:199px;">PEPROTECH</td>
			<td style="width:119px;">AF-100-15</td>
			<td style="width:267px;">growth factor supplement for the culture of&#160; neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Recombinant Human FGF-basic</td>
			<td style="width:199px;">PEPROTECH</td>
			<td>100-18B</td>
			<td style="width:267px;">growth factor supplement for the culture of&#160; neurospheres</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">HyClone HyQTase&#160; Cell Detachment Reagent&#160;</td>
			<td style="width:199px;">Thermo Scientific</td>
			<td style="width:119px;">SV3003001</td>
			<td style="width:267px;">for the dissociation of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Kimble-Chase Kontes Pellet Pestle</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td>K749510-0590</td>
			<td style="width:267px;">for the dissociation of freshly dissected tumors</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Falcon Cell Strainers mesh size 70um</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td>08-771-2</td>
			<td style="width:267px;">for generating single cell suspensions of dissociated neurospheres</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Xylenes Histological grade</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:119px;">C8H10</td>
			<td style="width:267px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Protocol Harris Hematoxylin Mercury free ( acidified)</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:119px;">245-678</td>
			<td style="width:267px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Protocol Eosyn Y Solution ( intensified)</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:119px;">314-631</td>
			<td style="width:267px;"></td>
		</tr>
	</tbody>
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transposon mediated integration of plasmid dna into the subventricular zone of neonatal mice to generate novel models of glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

לאפיין תכונות histo-פתולוגי של glioblastomas המושרה SB, עכברים בילוד C57 / BL6 הוזרקו בP1 עם לוציפראז קידוד פלסמיד (pt2 / SB100x-לוק) בשילוב עם פלסמידים קידוד transposons עם DNA שעור, כלומר, NRAS (PT / CAGGS -NRASV12) וSV40 LGT (PT / CMVSV40-LGT) (איור 3c) או קידוד פלסמיד p53 קצר סיכת ראש עם PDGFβ וכתב ה- GFP (pT2shp53 /...

Watch this Scientific Journal Video about Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma at JoVE.com

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

Transposon מתווך האינטגרציה של פלסמיד דנ&quot;א לתוך אזור Subventricular של עכברים בילוד ליצירת מודלים חדשניים של גליובלסטומה

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary ...

transposon-mediated-integration-plasmid-dna-into-subventricular-zone

Research

JoVE Journal

Medicine

12.8K Views.  University of Michigan School of Medicine. Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

Video: Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

Biochemical Measurement of Neonatal Hypoxia

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to cause/exacerbate well documented neonatal disorders including neurological impairment 1-3. Decreased adenosine triphosphate production occurs due to a lack of oxidative phosphorylation. To compensate for this energy deprived state molecules containing high energy phosphate bonds are degraded 2. This leads to increased levels of adenosine which is subsequently degraded to inosine, hypoxanthine, xanthine, and finally to uric acid. The final two steps in this degradation process are performed by xanthine oxidoreductase. This enzyme exists in the form of xanthine dehydrogenase under normoxic conditions but is converted to xanthine oxidase (XO) under hypoxia-reperfusion circumstances 4, 5. Unlike xanthine dehydrogenase, XO generates hydrogen peroxide as a byproduct of purine degradation 4, 6. This hydrogen peroxide in combination with other reactive oxygen species (ROS) produced during hypoxia, oxidizes uric acid to form allantoin and reacts with lipid membranes to generate malondialdehyde (MDA) 7-9. Most mammals, humans exempted, possess the enzyme uricase, which converts uric acid to allantoin. In humans, however, allantoin can only be formed by ROS-mediated oxidation of uric acid. Because of this, allantoin is considered to be a marker of oxidative stress in humans, but not in the mammals that have uricase.
We describe methods employing high pressure liquid chromatography (HPLC) and gas chromatography mass spectrometry (GCMS) to measure biochemical markers of neonatal hypoxia ischemia. Human blood is used for most tests. Animal blood may also be used while recognizing the potential for uricase-generated allantoin. Purine metabolites were linked to hypoxia as early as 1963 and the reliability of hypoxanthine, xanthine, and uric acid as biochemical indicators of neonatal hypoxia was validated by several investigators 10-13. The HPLC method used for the quantification of purine compounds is fast, reliable, and reproducible. The GC/MS method used for the quantification of allantoin, a relatively new marker of oxidative stress, was adapted from Gruber et al 7. This method avoids certain artifacts and requires low volumes of sample. Methods used for synthesis of MMDA were described elsewhere 14, 15. GC/MS based quantification of MDA was adapted from Paroni et al. and Cighetti et al. 16, 17. Xanthine oxidase activity was measured by HPLC by quantifying the conversion of pterin to isoxanthopterin 18. This approach proved to be sufficiently sensitive and reproducible.

A method is described to measure biochemical markers of neonatal hypoxia-ischemia. The approach utilizes high pressure liquid chromatography (HPLC) and Gas Chromatography Mass Spectrometry (GC/MS).

מדידה ביוכימית של היפוקסיה ילודים

Neonatal hypoxia ischemia is characterized by inadequate blood perfusion of a tissue or a systemic lack of oxygen. This condition is thought to ...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

שימוש במודל של בעלי חיים אלח דם להערכת טיפולים צמחים רומן

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

The Measurement and Treatment of Suppression in Amblyopia

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current estimates put the prevalence of amblyopia at approximately 1-3%1-3, the majority of cases being monocular2. Amblyopia is most frequently caused by ocular misalignment (strabismus), blur induced by unequal refractive error (anisometropia), and in some cases by form deprivation.
Although amblyopia is initially caused by abnormal visual input in infancy, once established, the visual deficit often remains when normal visual input has been restored using surgery and/or refractive correction. This is because amblyopia is the result of abnormal visual cortex development rather than a problem with the amblyopic eye itself4,5 . Amblyopia is characterized by both monocular and binocular deficits6,7 which include impaired visual acuity and poor or absent stereopsis respectively. The visual dysfunction in amblyopia is often associated with a strong suppression of the inputs from the amblyopic eye under binocular viewing conditions8. Recent work has indicated that suppression may play a central role in both the monocular and binocular deficits associated with amblyopia9,10 .
Current clinical tests for suppression tend to verify the presence or absence of suppression rather than giving a quantitative measurement of the degree of suppression. Here we describe a technique for measuring amblyopic suppression with a compact, portable device11,12 . The device consists of a laptop computer connected to a pair of virtual reality goggles. The novelty of the technique lies in the way we present visual stimuli to measure suppression. Stimuli are shown to the amblyopic eye at high contrast while the contrast of the stimuli shown to the non-amblyopic eye are varied. Patients perform a simple signal/noise task that allows for a precise measurement of the strength of excitatory binocular interactions. The contrast offset at which neither eye has a performance advantage is a measure of the "balance point" and is a direct measure of suppression. This technique has been validated psychophysically both in control13,14 and patient6,9,11 populations.
In addition to measuring suppression this technique also forms the basis of a novel form of treatment to decrease suppression over time and improve binocular and often monocular function in adult patients with amblyopia12,15,16 . This new treatment approach can be deployed either on the goggle system described above or on a specially modified iPod touch device15.

Amblyopia is a developmental disorder of the visual cortex that is often accompanied by strong suppression of one eye. We present a new technique for measuring and treating interocular suppression in patients with amblyopia that can be deployed using virtual reality goggles or a portable iPod Touch device.

המדידה והטיפול בדיכוי בעין עצלה

Amblyopia, a developmental disorder of the visual cortex, is one of the leading causes of visual dysfunction in the working age population. Current ...

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1).

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

זיהוי<em&gt; יפהפה נרדם</em&gt; הוספות Transposon בגידולים מוצקים באמצעות PCR לינקר בתיווך

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows for positive pressure bulk flow to deliver greater quantities of therapeutics to an intracranial target than traditional drug delivery methods. The clinical utility of real time MRI guided CED (rCED) lies in the ability to accurately target, monitor therapy, and identify complications. With training, rCED is efficient and complications may be minimized. The agarose gel model of the brain provides an accessible tool for CED testing, research, and training. Simulated brain rCED allows practice of the mock surgery while also providing visual feedback of the infusion. Analysis of infusion allows for calculation of the distribution fraction (Vd/Vi) allowing the trainee to verify the similarity of the model as compared to human brain tissue. This article describes our agarose gel brain phantom and outlines important metrics during a CED infusion and analysis protocols while addressing common pitfalls faced during CED infusion for the treatment of neurological disease.

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. In order to prepare health care professionals for adoption of CED, accessible training models are needed. We describe the use of agarose gel as such a model of the human brain for testing, research, and training.

משלוח משופר הסעה מודרכת תמונה לתוך מודלים Agarose ג&#39;ל של המוח

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

הזרקת orthotopic של תאי סרטן השד לחלב שומן Pad של עכברים לחקר התפתחות גידול.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

השתלת כליה עכבר: מודלים של דחיית השתל

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

דגם חזרזיר של ילודים חוסר חמצן-איסכמי אנצפלופתיה

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

מודלי כירורגי של Gastroesophageal Reflux עם עכברים

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

אוטם שריר לב עכברים ילודים, מודל התחדשות לב

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...