עבודה זו נתמכה על ידי מענק מניו Innovator תכנית פרס של מנהל ה- NIH (ED). AA נתמך בחלקו על ידי פרסים לתואר ראשון מהמכון הלאומי לסרטן והקרן הלאומי למדע.

1. ביצוע הווקטורים ויראליים רצפי הקידוד עבור הגן של עניין, כמו גם בקרות החיוביות ושליליות, מוכנסים לתוך pBABEpuro על ידי שיטות מסורתיות שיבוט (PCR הגברה, עיכול הגבלה וקשירה). ישנם ארבעה אתרי הגבלה על הווקטור שבו ניתן להכניס את ה- DNA: BamHI, SnaBI, EcoRI וסאלי. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן את ה- DNA פלסמיד כיתה transfection מהתאים מוסמכים באמצעות ערכת midi פלסמיד QIAGEN. </li><li style=";text-align:right;direction:rtl"> למדוד ריכוז ה- DNA באמצעות ספקטרופוטומטר NanoDrop2000. </li></ol> 2. רטרו-וירוס הפקה קו תא אריזת Plat-E ישמש להפקת רטרו-וירוס ecotropic שיספוק את cDNA של עניין לתאי NIH 3T3. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הקמת תרבויות Plat-E מ תאים קפואים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> במהירות להפשיר תאי Plat-E באמבט מים 37 מעלות צלזיוס ולהעביר צינור חרוטי 15 מיליליטר. לאט לאט להוסיף 9 מיליליטר של מדיום Plat-E (Duהנשר בינוני של lbecco שונה (DMEM) + 10% שור עוברי סרום (FBS) + פניצילין וסטרפטומיצין). </li><li style=";text-align:right;direction:rtl"> צנטריפוגות הצינור ב 180 x גרם במשך 5 דקות וזורקים supernatant. </li><li style=";text-align:right;direction:rtl"> להשעות מחדש את התאים עם 10 מיליליטר של מדיום Plat-E והעברה לצלחת תרבות 10 סנטימטרים. </li><li style=";text-align:right;direction:rtl"> דגירה התאים ב-37 מעלות צלזיוס, 5% CO 2 באינקובטור עד שהם% ומחוברות 80-90 (כ 2 ימים). </li></ol></li><li style=";text-align:right;direction:rtl"> פיצול תא <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשאוב את המדיום ולשטוף תאי פעם עם PBS. </li><li style=";text-align:right;direction:rtl"> הוסף 2 מיליליטר של 0.05% טריפסין-EDTA דגירה דקות 1 ב RT. </li><li style=";text-align:right;direction:rtl"> לנתק את התאים על ידי הקשה על אצבע, להוסיף 10 מיליליטר של מדיום Plat-E, ולהעביר את ההשעיה תא צינור 15 מיליליטר. </li><li style=";text-align:right;direction:rtl"> צנטריפוגות הצינור ב 180 x גרם במשך 5 דקות וזורקים supernatant. </li><li style=";text-align:right;direction:rtl"> Resuspend התאים עם 10 מיליליטר של מדיום Plat-E וזרעם במנות חדשות ב1: 4-1: 6 דילולים. </li></ol></li><li style=";text-align:right;direction:rtl"> Plat-E זריעה וTransfection <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> זרע 2 x 10 6תאים לכל צלחת תרבות 10 סנטימטרים באמצעות מדיום Plat-E ללא כל אנטיביוטיקה. </li><li style=";text-align:right;direction:rtl"> דגירה התאים O / N ב37 ° C, חממה 5% CO 2. </li><li style=";text-align:right;direction:rtl"> למחרת, להעביר 300 μl של Opti-ממ לתוך צינור 1.5 מיליליטר microfuge. </li><li style=";text-align:right;direction:rtl"> להוסיף 27 μl של polyethylenimine (PEI), מגיב transfection חסכוני 12, אל הצינור מוכן עם Opti-MEM. מערבבים בעדינות על ידי הקשה על אצבע ודגירה של 5 דקות ב RT. </li><li style=";text-align:right;direction:rtl"> הוסף 9 מיקרוגרם של ה- DNA פלסמיד כיתה transfection לתוך צינור Opti-ממ / PEI, מערבב בעדינות על ידי vortexing, ודגירה במשך 15 דקות ב RT. </li><li style=";text-align:right;direction:rtl"> מוסיף את ה- DNA / PEI ירידה מבחינת מורכבת לתוך צלחת Plat-E, ודגירת O / N ב 37 מעלות צלזיוס, 5% CO 2. </li><li style=";text-align:right;direction:rtl"> למחרת, לשאוב את המדיום המכיל את מגיב transfection ולהוסיף 10 מיליליטר של מדיום Plat-E הטרי (ללא אנטיביוטיקה). </li><li style=";text-align:right;direction:rtl"> להחזיר את התאים לחממה. </li></ol></li></ol> 3. תאי NIH 3T3 וזיהום <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הקמת NIHתרבויות 3T3 וזריעה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> במהירות להפשיר תאי NIH 3T3 באמבט מים 37 מעלות צלזיוס ולהעביר לצינור 15 מיליליטר. לאט לאט להוסיף 9 מיליליטר של מדיום NIH 3T3 (DMEM + 10% FBS). </li><li style=";text-align:right;direction:rtl"> צנטריפוגות הצינור ב 180 x גרם במשך 5 דקות וזורקים supernatant. </li><li style=";text-align:right;direction:rtl"> Resuspend התאים עם 10 מיליליטר של מדיום NIH 3T3 והעברה לצלחת תרבות 10 סנטימטרים. </li><li style=";text-align:right;direction:rtl"> דגירה התאים ב37 ° C, חממה 5% CO 2. זה מאוד חשוב כי תאי NIH 3T3 תמיד נשמרים underconfluent (50-60%), כתדר של שינוי ספונטני (ולכן רמות בסיסיות של היווצרות מוקדים) מגביר פעם בתרבות מגיעה confluency. </li><li style=";text-align:right;direction:rtl"> זרע 3 x 10 5 תאים לכל צלחת 10 סנטימטרים ודגירת O / N לזיהום למחרת. </li></ol></li><li style=";text-align:right;direction:rtl"> זיהום <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> קציר supernatant retroviral מצלחת Plat-E באמצעות מזרק חד פעמי מיליליטר 10, סינונו דרך מסנן קרום ניילון גודל 0.45 מיקרומטר נקבובית, והעברתו ל-15 מיליליטרצינור. </li><li style=";text-align:right;direction:rtl"> הוסף 10 מיליליטר של מדיום Plat-E הטרי לתאים (ללא אנטיביוטיקה), ולהחזיר אותם לחממה. </li><li style=";text-align:right;direction:rtl"> לשאוב את המדיום מצלחת תא NIH 3T3 להיות נגועות, ולהוסיף 5 מיליליטר של מדיום NIH 3T3 רגיל ו -5 מיליליטר של supernatant המסונן המכיל וירוס. </li><li style=";text-align:right;direction:rtl"> להוסיף polybrene לצלחת בריכוז של 6 מיקרוגרם / מיליליטר. </li><li style=";text-align:right;direction:rtl"> דגירה התאים O / N ב 37 מעלות צלזיוס, 5% CO 2. </li><li style=";text-align:right;direction:rtl"> למחרת, חזור על השלבים 3.2.1 - 3.2.5 לסיבוב שני של זיהום. </li><li style=";text-align:right;direction:rtl"> החלף את המדיום המכיל וירוס עם מדיום NIH 3T3 רגיל. </li><li style=";text-align:right;direction:rtl"> אפשר תאי NIH 3T3 לגדול במשך 2-3 שבועות, החלפה הבינוני במידת צורך. בדוק ביטוי של החלבון של עניין על ידי אלקטרופורזה הקונבנציונלית החלבון וimmunoblot על lysates תא שלם מצלחות העתק. </li></ol></li></ol> 4. מכתים וכימות <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> קריסטל ויולט מכתים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לשאוב את מדיום NIH 3T3 ולמקם את הכליםעל קרח. </li><li style=";text-align:right;direction:rtl"> לשטוף הכלים פעמיים עם PBS קר כקרח. </li><li style=";text-align:right;direction:rtl"> תקן את התאים עם מתנול קר כקרח במשך 10 דקות. </li><li style=";text-align:right;direction:rtl"> הסר את המנות מהקרח, לשאוב מתנול, ולהוסיף 3 מיליליטר של 0.5% פתרון סגול גביש עשה ב 25% מתנול (RT). </li><li style=";text-align:right;direction:rtl"> דגירה המנות במשך 5 דקות בRT. </li><li style=";text-align:right;direction:rtl"> לשאוב את הפתרון סגול גביש ולשטוף היטב את המנה עם מילי- Q H 2 O עד שלא צבע יורד בשטיפה. </li><li style=";text-align:right;direction:rtl"> לאפשר את הכלים לייבוש O / N בbenchtop (שנחשף). </li></ol></li><li style=";text-align:right;direction:rtl"> כימות מוקדים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ברגע שהמנות יבשות, להשתמש בסרגל למדוד את האוספים צבעוניים הכהה של תאים על הצלחת. לספור רק את אלה שהם יותר מ 5 מ&quot;מ בקוטר כ&quot; מוקדים &quot;. רשום את מספר המוקדים ולחשב ממוצעים והמשמעות בהשוואה לקבוצת הביקורת. </li></ol></li></ol>

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יש לי המחברים אין לחשוף.

FFA מספק שיטה קלה ומהירה כדי להעריך שינוי ממאיר במבחנה. זה ניתן להקרנה של מספר גדול יחסית של גני מועמדים, והדרישות הטכניות הצנועים שלה לעשות את זה יעיל וחסכוני. יתר על כן, ניתן coexpressed שתיים או יותר גנים (המכונה לעתים assay &quot;שיתוף פעולה&quot;) כדי להעריך את הפוטנציאל הסר...

ניתן להבחין בתאי גידול מעמיתיהם הרגילים על ידי מגוון רחב של שינויים, מדפוסי ביטוי גנים לepigenomics לשינויים מורפולוגיים ושגשוג. בקרב האחרון, הפחית את התלות בסרום, אובדן קשר עיכוב (צפיפות), הרכישה של התפשטות מעגן-עצמאי וסופו של דבר, את היכולת ליצור גידולים כאשר הוזרקו לבעלי חיים הם אינדיקטורים שימושיים, הניתנים למדידה של שינוי הממאיר 3. כמה במבחנה ובמבחני vivo פותחו לשינוי סלולארי. במבחנה מבחני מכוונים לזיהוי ומדידת שינויים במורפולוגיה תרבות (assay היווצרות מוקד), דינמיקת התרבות (שיעור צמיחה, צפיפות רוויה) וגורם גדילה (צמיחה בסרום מופחת) או מעגן (צמיחה באגר רכה) דרישות. תקן הזהב לקביעת האופי הממאיר של סוג תא נשאר היווצרות גידול (xenografts) בחיות מעבדה. עם זאת, לאהוא יעלה ואורך של מחקרי in vivo לא תמיד להפוך אותם מוצדק כצעד ראשון אימות או הקרנה של אונקוגנים מועמד. למרות שאף assay במבחנה מספק הערכה ברורה של הפוטנציאל שבעור של גן, הם מספקים תובנה פוטנציאל שבעור שעשויה לצמצם את העתיד במחקרי vivo. אחת מהמערכות הנפוצות ביותר להערכת פוטנציאל סרטני במבחנה הוא Assay גיבוש הפוקוס 2. גישה זו מסתמכת על השימוש בפיברובלסטים עכבר NIH 3T3, קו תאים לא שינה שמראה עיכוב קשר חזק. ביטוי יתר של תוצאות אונקוגן באובדן צמיחת צפיפות תלויה; תאים הופכים יכולים לגדול אז במספר שכבות, ויצרו &quot;מוקדים&quot;, דמיינו בקלות נגד monolayer הרקע של תאים-שינה הלא. גיבוש הפוקוס Assay, אז, מודד את היכולת של אונקוגן מועמד לגרום לשינוי ממאיר, כפי שמעיד על אובדן inhib קשרition כפנוטיפ למדידה. FFA נעשה שימוש כדי להעריך שינוי בביטוי יתר של החלבון קינאז (למשל, 4 Src, BRAF 5), גורמי שעתוק (למשל, N-myc 6) קולטנים, G-מצמידים חלבון (לדוגמא, 7 P2RY8) וGTPases (למשל , ראס 1), בין יתר. הקלות היחסית של assay זה הופכת אותו לבחירה טובה שתספק תשובה מהירה וחזותי ברורה אם ביטוי יתר של הגן די בכך כדי להפוך את תאי פיברובלסטים עכבר NIH 3T3 במבחנה. FFA המתואר בפרוטוקול זה משתמש בקו Plat-E תא אריזה 8, המספק חלבוני אריזה נגיפיים, וpBABEpuro retroviral הווקטור 9 (פלסמיד Addgene 1764) כדי לייצר רטרו-וירוס. לאחר transfection עם מבנה pBABEpuro המכיל את הגן של עניין, שורת תאי Plat-E תפיק רטרו-וירוס ecotropic שיכול לשמש כדי להדביק תאי NIH 3T3. Tהשיטה הנגיפית של מסירת הגן היא יעילה יותר מאשר שיטות transfection הכימי מסורתיות והיא מציעה דרך לבטא את הגן 10-קיימא. התאגד ברגע שלתוך הגנום של תאי NIH 3T3, ביטוי יתר של הגן של עניין הוא מונע על ידי האמרגן חוזר מסוף ארוך הנגיפי (LTR) 11. ביטוי קבוע זה יכול לשמש כדי לקבוע אם הגן של עניין יש לו פעילות שבעור, כפי שהיא נמדדת על ידי יצירת מוקדים, על תאי NIH 3T3.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">pBABE-puro vector</td>
			<td style="width:127px;">Addgene</td>
			<td style="width:123px;">Plasmid 1764</td>
			<td style="width:171px;">cloning vector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Platinum-E Retroviral Packaging Cell Line, Ecotropic</td>
			<td style="width:127px;">Cell Biolabs, Inc.</td>
			<td style="width:123px;">RV-101</td>
			<td>cell line for viral production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">NIH 3T3 Cell Line murine</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:123px;">93061524</td>
			<td>cell line for focus formation assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">10 mL BD Luer-Lok tip syringe</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:123px;">309604</td>
			<td>viral production reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">0.45 &#956;m Puradisc Syringe Filter</td>
			<td style="width:127px;">Whatman</td>
			<td style="width:123px;">6750-2504</td>
			<td>viral production reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Polyethylenimine (PEI)</td>
			<td style="width:127px;">Polysciences, Inc.</td>
			<td style="width:123px;">23966-2</td>
			<td>cell transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Polybrene Infection / Transfection Reagent</td>
			<td style="width:127px;">EMD Millipore</td>
			<td style="width:123px;">TR-1003-G</td>
			<td>cell transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Crystal Violet</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:123px;">C581-25</td>
			<td>cell stain reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Plasmid Plus Midi Kit</td>
			<td style="width:127px;">QIAGEN</td>
			<td style="width:123px;">12945</td>
			<td>plasmid purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">BD Falcon Tissue Culture Dishes</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:123px;">353003</td>
			<td>cell culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">11995-065</td>
			<td>cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">0.05% Trypsin-EDTA</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">25300-054</td>
			<td>cell culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Opti-MEM I Reduced Serum Medium</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">31985-062</td>
			<td>cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">16000-044</td>
			<td>cell culture media</td>
		</tr>
	</tbody>
</table>

focus formation: a cell-based assay to determine the oncogenic potential of a gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

MXD3 הוא גורם שעתוק הרוכסן לאוצין סליל לולאה, סליל בסיסי (bHLHZ) שהוא חבר של MYC / מקס / רשת MAD. זה חבר טיפוסי של משפחת MAD 13-15, וזה כבר דיווח להיות מעורב בהיווצרות הסרטן 16,17. בהשוואה לpBABEpuro (בקרה שלילית) וMYC (ביקורת חיובית), היו מנות NIH 3T3 בי MXD3 היה ביטוי יתר באופן משמעותי ?...

Watch this Scientific Journal Video about Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene at JoVE.com

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Assay גיבוש הפוקוס מספק שיטה פשוטה להעריך את הפוטנציאל להפוך לאונקוגן מועמד.

להתמקד גיבוש: Assay מבוסס תאים כדי לקבוע את הפוטנציאל שבעור של ג&#39;ין

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

focus-formation-cell-based-assay-to-determine-oncogenic-potential

Research

JoVE Journal

Medicine

27.7K Views.  University of California, Davis.  Assay גיבוש הפוקוס מספק שיטה פשוטה להעריך את הפוטנציאל להפוך לאונקוגן מועמד. 

Video: Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

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 ...

A Novel Surgical Approach for Intratracheal Administration of Bioactive Agents in a Fetal Mouse Model

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and acquired diseases. Apart from congenital or inherited abnormalities with the requirement for long-term expression of the delivered gene, several non-inherited perinatal conditions, where short-term gene expression or pharmacological intervention is sufficient to achieve therapeutic effects, are considered as potential future indications for this kind of approach. Candidate diseases for the application of short-term prenatal therapy could be the transient neonatal deficiency of surfactant protein B causing neonatal respiratory distress syndrome1,2 or hyperoxic injuries of the neonatal lung3. Candidate diseases for permanent therapeutic correction are Cystic Fibrosis (CF)4, genetic variants of surfactant deficiencies5 and &alpha;1-antitrypsin deficiency6.
Generally, an important advantage of prenatal gene therapy is the ability to start therapeutic intervention early in development, at or even prior to clinical manifestations in the patient, thus preventing irreparable damage to the individual. In addition, fetal organs have an increased cell proliferation rate as compared to adult organs, which could allow a more efficient gene or stem cell transfer into the fetus. Furthermore, in utero gene delivery is performed when the individual's immune system is not completely mature. Therefore, transplantation of heterologous cells or supplementation of a non-functional or absent protein with a correct version should not cause immune sensitization to the cell, vector or transgene product, which has recently been proven to be the case with both cellular and genetic therapies7.
In the present study, we investigated the potential to directly target the fetal trachea in a mouse model. This procedure is in use in larger animal models such as rabbits and sheep8, and even in a clinical setting9, but has to date not been performed before in a mouse model. When studying the potential of fetal gene therapy for genetic diseases such as CF, the mouse model is very useful as a first proof-of-concept because of the wide availability of different transgenic mouse strains, the well documented embryogenesis and fetal development, less stringent ethical regulations, short gestation and the large litter size.
Different access routes have been described to target the fetal rodent lung, including intra-amniotic injection10-12, (ultrasound-guided) intrapulmonary injection13,14 and intravenous administration into the yolk sac vessels15,16 or umbilical vein17. Our novel surgical procedure enables researchers to inject the agent of choice directly into the fetal mouse trachea which allows for a more efficient delivery to the airways than existing techniques18.

We developed a novel surgical approach for intratracheal administration of bioactive agents into the mouse fetus. The delivery route is more efficient in targeting the fetal mouse lungs than the commonly used intra-amniotic injection. This procedure has to date not been described in a mouse model.

גישה ניתוחית חדשנית למנהל Intratracheal של סוכני יו במודל של עכברים עוברי

Prenatal pulmonary delivery of cells, genes or pharmacologic agents could provide the basis for new therapeutic strategies for a variety of genetic and ...

High-throughput Flow Cytometry Cell-based Assay to Detect Antibodies to N-Methyl-D-aspartate Receptor or Dopamine-2 Receptor in Human Serum

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases in children and adults. These antibodies have been used to guide diagnosis and treatment. Cell-based assays have improved the detection of antibodies in patient serum. They are based on the surface expression of brain antigens on eukaryotic cells, which are then incubated with diluted patient sera followed by fluorochrome-conjugated secondary antibodies. After washing, secondary antibody binding is then analyzed by flow cytometry. Our group has developed a high-throughput flow cytometry live cell-based assay to reliably detect antibodies against specific neurotransmitter receptors. This flow cytometry method is straight forward, quantitative, efficient, and the use of a high-throughput sampler system allows for large patient cohorts to be easily assayed in a short space of time. Additionally, this cell-based assay can be easily adapted to detect antibodies to many different antigenic targets, both from the central nervous system and periphery. Discovering additional novel antibody biomarkers will enable prompt and accurate diagnosis and improve treatment of immune-mediated disorders.

Over the recent years, live cell-based assays have been used successfully to detect antibodies against surface and conformational antigens. Here, we describe a method using high-throughput flow cytometry enabling the analysis of large cohorts of patients. Detection of novel antibodies will improve diagnosis and treatment of immune-mediated disorders.

Assay המבוסס תא זרימת cytometry תפוקה גבוהה לזיהוי נוגדנים לN-מתיל-D-aspartate קולטן דופמין או-2 קולטן בסרום אדם

Over the recent years, antibodies against surface and conformational proteins involved in neurotransmission have been detected in autoimmune CNS diseases ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

השתנה<em&gt; במבחנה</em&gt; פלישת Assay כדי לקבוע את התפקיד הפוטנציאלי של הורמונים, ציטוקינים ו / או גורמי גדילה בפלישת תאי הסרטן המתווך

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

רומן<em&gt; In vivo</em&gt; טכניקה וין העברה<em&gt; במבחנה</emמבחני תא בהתבסס&gt; לחקר אובדן עצם בפרעות השלד והשרירים

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Assay (3D) תלת ממדי גידול ספרואיד הפלישה

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

ניצול של assay כינונה רך אגר מושבה לזיהוי מעכבי tumorigenicity בתאי סרטן השד

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

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 ...

Injection of Syngeneic Murine Melanoma Cells to Determine Their Metastatic Potential in the Lungs

Approximately 90% of human cancer deaths are linked to metastasis. Despite the prevalence and relative harm of metastasis, therapeutics for treatment or prevention are lacking. We report a method for the establishment of pulmonary metastases in mice, useful for the study of this phenomenon. Tail vein injection of B57BL/6J mice with B16-BL6 is among the most used models for melanoma metastases. Some of the circulating tumor cells establish themselves in the lungs of the mouse, creating "experimental" metastatic foci. With this model it is possible to measure the relative effects of therapeutic agents on the development of cancer metastasis. The difference in enumerated lung foci between treated and untreated mice indicates the efficacy of metastases neutralization. However, prior to the investigation of a therapeutic agent, it is necessary to determine an optimal number of injected B16-BL6 cells for the quantitative analysis of metastatic foci. Injection of too many cells may result in an overabundance of metastatic foci, impairing proper quantification and overwhelming the effects of anti-cancer therapies, while injection of too few cells will hinder the comparison between treated and controls.

Metastasis plays a profound role in the virulence of cancer, accounting for an estimated 90% of deaths. We report a protocol for a metastatic melanoma model in mice that is useful for determining the efficacy of therapeutic agents against this clinical phenomenon.

הזרקה של תאים syngeneic Murine מלנומה מנת לקבוע את פוטנציאל גרורתי שלהם בתוך הריאות

Approximately 90% of human cancer deaths are linked to metastasis.  Despite the prevalence and relative harm of metastasis, therapeutics for treatment or ...

A Choroid Plexus Epithelial Cell-based Model of the Human Blood-Cerebrospinal Fluid Barrier to Study Bacterial Infection from the Basolateral Side

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The BCSFB functions in separating the cerebrospinal fluid (CSF) from the blood and restricting the molecular exchange to a minimum extent. An in vitro model of the BCSFB is based on cells derived from a human choroid plexus papilloma (HIBCPP). HIBCPP cells display typical barrier functions including formation of tight junctions (TJs), development of a transepithelial electrical resistance (TEER), as well as minor permeabilities for macromolecules. There are several pathogens that can enter the central nervous system (CNS) via the BCSFB and subsequently cause severe disease like meningitis. One of these pathogens is Neisseria meningitidis (N. meningitidis), a human-specific bacterium. Employing the HIBCPP cells in an inverted cell culture filter insert system enables to study interactions of pathogens with cells of the BCSFB from the basolateral cell side, which is relevant in vivo. In this article, we describe seeding and culturing of HIBCPP cells on cell culture inserts. Further, infection of the cells with N. meningitidis along with analysis of invaded and adhered bacteria via double immunofluorescence is demonstrated. As the cells of the CP are also involved in other diseases, including neurodegenerative disorders like Alzheimer`s disease and Multiple Sclerosis, as well as during the brain metastasis of tumor cells, the model system can also be applied in other fields of research. It provides the potential to decipher molecular mechanisms and to identify novel therapeutic targets.

The epithelial cells of the choroid plexus (CP) form the blood-cerebrospinal fluid barrier (BCSFB). An in vitro model of the BCSFB employs human choroid plexus papilloma (HIBCPP) cells. This article describes culturing and basolateral infection of HIBCPP cells using a cell culture filter insert system.

מודל אפיתל דמה מקלעת מבוססת תאים של הגדר ודם-השדרתי נוזל לחקר זיהום חיידקים מצד basolateral

The epithelial cells of the choroid plexus (CP), located in the ventricular system of the brain, form the blood-cerebrospinal fluid barrier (BCSFB). The ...