This work was supported by grants from the Novartis Foundation for Medical-Biological Research and from the Research Pool of the University of Fribourg (to MW). We thank Li Zhao, Zhan Xu, and Solange Kharoubi Hess for technical support, as well as Radu Aricescu and Yvonne Jones (Oxford University, UK) for providing the pHLsec plasmid, and Thomas Sch&#252;rpf (Harvard Medical School) for helpful discussions.

1. הפקה של פלסמיד הביטוי pHLsec-Gzm <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן רנ&quot;א הכל מתאי NK אנושיים (תאים ראשוניים שהוכנו כב -26 או שורת תאי NK NK-92 המבטא את כל חמש Gzms האנושי) בשיטת בידוד RNA מתאימה ולהפוך לתמלל באמצעות ערכה לסנתז cDNA ראשונה גדיל, הבאים manufacturer` המלצות של. להגביר Gzm cDNA באמצעות PCR ושיבוט לpHLsec כפי שתואר ב -23 (מתנת סוג של ראדו Aricescu והאיבון ג&#39;ונס, אוניברסיטת אוקספורד, בריטניה), תוך שימוש באתרי AgeI וKpnI (איור 1). הערה: השתמש בפריימרים הבאים שצוינו בטבלה 1. </li><li style=";text-align:right;direction:rtl"> אשר מוסיף נכון על ידי רצף. הרחב את פלסמידים ביטוי בתאי DH5α ולטהר באמצעות ערכת בידוד פלסמיד ללא רעלן פנימי ולעקוב אחר ההוראות של היצרן. </li><li style=";text-align:right;direction:rtl"> Resuspend פלסמידים המטוהרים ב, מים סטריליים ללא רעלן פנימי בריכוז של 2 מ&quot;ג / מ&quot;ל ​​ולאחסן ב -20 ° C untiשימוש l. </li></ol> 2. ביטוי של Gzms בתאי HEK293T <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנה של חומרים כימיים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להכין מדיום תרבות סטנדרטי. לנשר בינוני השתנה Dulbecco (DMEM) גלוקוז המכיל גבוה (25 מ&quot;מ), Glutamax (4 מ&quot;מ), פירובט נתרן (1 מ&quot;מ) להוסיף 10% מסרום סטנדרטי עגל עוברי (FCS), פניצילין (100 יחידות / מיליליטר), סטרפטומיצין ( 100 מיקרוגרם / מיליליטר). </li><li style=";text-align:right;direction:rtl"> הכן בינוני transfection. למדיום תרבות ללא פניצילין, סטרפטומיצין להוסיף 25 מיקרוגרם / מיליליטר chloroquine (להוסיף טרי ביום transfection מ1,000x מניות בPBS, מאוחסנות ב -20 ° C). </li><li style=";text-align:right;direction:rtl"> הכן בינוני תרבות סרום ללא: עד בינוני סרום ללא לHEK293 תאים להוסיף גלוטמין (4 מ&quot;מ), פניצילין (100 יחידות / מיליליטר), סטרפטומיצין (100 מיקרוגרם / מיליליטר), ו2.5mg / ב amphotericin L </li><li style=";text-align:right;direction:rtl"> הכן את הפתרונות שמתוארים בטבלה 2 ומסנן עם מסנן 0.45 מיקרומטר לפני שימוש: </li></ol></li><li style=";text-align:right;direction:rtl"> הרחבת תאי HEK293T <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לגדל תאי HEK293T ב 20 מיליליטר כתבינוני יור באמצעות 150 x 25 מנות בתרבית רקמת מ&quot;מ. </li><li style=";text-align:right;direction:rtl"> פיצול תאים ב 80% confluency (מפוצל ביחס של 1: 4, בדרך כלל בכל יום 3 rd). תאים באופן רופף לצרף, הם יכולים להיות מכניים מנותקים ללא trypsinization ידי קפדנות pipetting למעלה ולמטה. </li><li style=";text-align:right;direction:rtl"> תאי צלחת בלילה שלפני transfection לתת confluency 60-70% ביום transfection (צפיפות זריעה ~ 2 x 10 5 תאים / מיליליטר). גודל הכנה רגיל מורכב של 20 עד 25 מנות תרבות. </li></ol></li><li style=";text-align:right;direction:rtl"> transfection סידן פוספט <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> 1 שעה לפני transfection, לשנות את התרבות בינונית הסטנדרטי למדיום transfection 20 מיליליטר. לקראת סוף שלב דגירה שעה 1 זה, ​​לערבב 400 מיקרוגרם של ה- DNA פלסמיד עם 10.95 DDH מיליליטר 2 0 ו1.55 מיליליטר של 2M CaCl 2 בצינורות 50 מיליליטר (צינור 1/5 מנות). </li><li style=";text-align:right;direction:rtl"> 1 עד 2 דקות לפני transfection, להוסיף 12.5 מיליליטר 2x HBS ל12.5 מיליליטר של פתרון ה- DNA-סידן, לערבב על ידי היפוך של הצינורות ודגירה במשך 30 שניות בRT. </li><li style=";text-align:right;direction:rtl"> להוסיף התערובת מוכנה בדואר 2.3.2 ישירות לתאים (5 מ&quot;ל לכל צלחת), ירידה מבחינת לתוך המדיום. מפזרים באופן שווה על פני כל השטח, הופכים את המדיום לצבע כתום מעט. </li><li style=";text-align:right;direction:rtl"> דגירה תרבות המנות ל7-11 שעות בתרבית רקמת חממה (37 מעלות צלזיוס, 5% CO 2 באוויר humidified). לאחר הדגירה, משקע בסדר גלוי. הסר את מדיום transfection, לשטוף בזהירות עם PBS מראש חימם ולהוסיף בינוני תרבות סרום ללא 20 מיליליטר מוכן ב2.1.3. דגירה של 72 שעות בתרבית רקמת חממה. </li><li style=";text-align:right;direction:rtl"> לנתח את יעילות transfection וביטוי על ידי הפעלת מדגם (~ 20 μl) של supernatant התא על SDS-PAGE וצביעה עם כחול מבריק Coomassie לדמיין להקת granzyme. </li></ol></li><li style=";text-align:right;direction:rtl"> טיהור של Gzms מsupernatant התרבות על ידי ניקל-IMAC <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> לאחר הדגירה, למזוג את supernatants תרבית תאים למ&quot;ל 250 צינורות וברורים על ידי צנטריפוגה. לבצע ספין ראשון (400 XG, 10 דקות, 4 °ג) שינקה את המדיום מהתאים מנותקים. מעבירים את supernatant ב 250 מיליליטר צינורות וספין טריים ב 4000 XG, 30 דקות ב 4 ° C כדי להסיר כל פסולת תא שנותרה. </li><li style=";text-align:right;direction:rtl"> הוסף 5 מיליליטר של 5 M NaCl, 6.25 מיליליטר של 2 מ &#39;טריס-בסיס (pH 8) ו 1 מיליליטר של 0.25 מ&#39; 4 Niso ל250 מיליליטר של supernatant פינה. סנן את supernatant באמצעות יחידת 500 מיליליטר מסנן אבק (0.45 מ&quot;מ). </li><li style=";text-align:right;direction:rtl"> לאזן טור ניקל-iMac עימו מחייב חיץ (כרכי טור 10, CV) באמצעות מערכת FPLC מתאימה. </li><li style=";text-align:right;direction:rtl"> החל supernatant פינה לעמודת equilibrated בקצב זרימה של 0.5 מיליליטר / דקה על 4 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> לאחר supernatant יושם, לשטוף טור עם החיץ-מחייב עד לנקודת התחלת ספיגת UV (בדרך כלל 10 קורות חיים). </li><li style=";text-align:right;direction:rtl"> Gzms Elute עם 20 מ&quot;ל שיפוע ליניארית (0 עד 250 מ&quot;מ imidazole) בקצב זרימה של 0.5 מיליליטר / דקה בעת איסוף 2 מ&quot;ל שברים. לנתח את השברים elution על ידי SDS-עמוד וצביעת Coomassie. </li></ol></li><li style=";text-align:right;direction:rtl"> Enterokinase (EK)טיפול וניקוי סופי על ידי כרומטוגרפיה החלפת קטיון <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> בריכת Gzm מכיל שברים בצינור dialyzing עם ≤10 MWCO. אחסן מדגם קטן ב -20 ° C כשליטה מראש EK. </li><li style=";text-align:right;direction:rtl"> להוסיף EK ביחידת 0.02 / 50 מיליליטר של supernatant הראשוני ישירות לשבריר נקווה בצינור הדיאליזה ודיאליזת O / N (לפחות 16 שעות) ב RT בEK-חיץ (4 L, שינוי חיץ פעם אחת). </li><li style=";text-align:right;direction:rtl"> למחרת, לנתח EK טופל Gzms בהשוואה לEK מראש השליטה על ידי SDS-עמוד וצביעת Coomassie. </li><li style=";text-align:right;direction:rtl"> כאשר עיבוד N-מסוף הוא מלא, לשנות חיץ דיאליזה לS חיץ (4 ליטר) ודיאליזה במשך 4 שעות נוספות בRT. dialysate מסנן (0.45 מיקרומטר). </li><li style=";text-align:right;direction:rtl"> לאזן S-טור עם א &#39;חיץ S טען את המדגם על העמודה עם בקצב זרימה של 0.5 מיליליטר / דקה על 4 מעלות צלזיוס. </li><li style=";text-align:right;direction:rtl"> לאחר טעינת מדגם, לשטוף את העמודה עם חיץ S עד לנקודת התחלת ספיגת UV (כ -10 קורות חיים). Elute Gzms עם שיפוע 30 מיליליטר ליניארי (150 ל -1,000 מ&quot;מ NaCl). דואר GzmAלאוטות ב ~ 650 מ&quot;מ NaCl, GzmB ב ~ 700 מ&quot;מ NaCl וGzmM ב ~ 750 מ&quot;מ NaCl. </li><li style=";text-align:right;direction:rtl"> לנתח שברי elution ידי SDS-עמוד ומבחני colorimetric (ראה להלן). שברים בריכה המכילים Gzms ולהתרכז (על פי 30, לריכוז של כ -100 מיקרומטר) במסנני ספין (15 מיליליטר, 10 MWCO). Aliquot ההכנות מרוכזות Gzm ולאחסן ב -80 ° C. </li></ol></li></ol> פעילות 3. הבדיקה Gzm <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכנה של חומרים כימיים <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הכן חיץ assay colorimetric: 50 מ&quot;מ טריס-בסיס, pH 7. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> למדידה GzmA, להוסיף Na-CBZ-L ליזין thiobenzyl אסתר (S-Bzl) (BLT) למאגר assay לריכוז סופי של 0.2 מ&quot;מ ו5,5&#39;-dithio-bis (חומצה 2-nitrobenzoic) (DTNB) לריכוז של 0.22 מ&quot;מ. לGzmB, כולל 0.2 מ&quot;מ בוק-עלא-עלא-Asp-S-Bzl (AAD) ו0.22 מ&quot;מ DTNB. לGzmM, כולל 0.2 מ&quot;מ SUC-עלא-עלא-Pro Leu-P-nitroanilide (PNA) (AAPL). פפטידים סינטטיים מתווספים מפתרונות מניות מתאימים (בDMSO, Storאד ב -20 ° C) למאגר טרי לפני assay. </li></ol></li><li style=";text-align:right;direction:rtl"> הכן חיץ assay מחשוף: 100 מ&quot;מ NaCl, 50 מ&quot;מ טריס-בסיס, pH 7.5 </li></ol></li><li style=";text-align:right;direction:rtl"> מבחני colorimetric <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> להפיץ דגימות קטנות Gzm (&lt;μl 5) משברי elution או מניות מרוכזות בצלחות 96 היטב שטוח תחתון. כולל בקרה חיובית (הכנות Gzm נבדקו או גולמי lysates תא NK) ובקרה שלילית (חיץ בלבד). </li><li style=";text-align:right;direction:rtl"> להוסיף של חיץ assay 100 μl היטב כל אחד (בעזרת פיפטה רב-ערוצים). דגירה של 10 דקות על 37 מעלות צלזיוס. למדוד OD ב 405 ננומטר בקורא microplate. </li></ol></li><li style=";text-align:right;direction:rtl"> assay המחשוף <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> עבור מבחני מחשוף באמצעות מצעים מטוהרים, שיתוף דגירה מצע חלבון (יליד רקומביננטי או, 100-400 ננומטר; כמה דוגמאות של מצעי Gzm יעילים מוצגות בסעיף התוצאה ובהערה בסוף הסעיף זה) עם דילולים סידוריים של Gzm (החל מ -400 ננומטר) במאגר assay לזמנים שונים.ניתוח על ידי SDS-עמוד וCoomassie או צביעת כסף; או על ידי מערבי סופג באמצעות נוגדנים ספציפיים. </li><li style=";text-align:right;direction:rtl"> עבור מבחני מחשוף באמצעות lysates תא, להשעות 10 6 תאי הלה (או כל קו תאים סרטני זמין) ב 1 מיליליטר של חיץ assay וlyse על ידי הקפאה באתנול / אמבט קרח יבש והפשרה על 37 מעלות צלזיוס פעמים שלוש. הסר פסולת תא על ידי צנטריפוגה (15,000 XG במשך 10 דקות ב 4 ° C). </li><li style=";text-align:right;direction:rtl"> שיתוף דגירה lysate פינה עם Gzms בריכוזים ובזמנים שונים כאמור לעיל. מחשוף הוא זוהה על ידי immunoblotting באמצעות נוגדנים מתאימים. הערה: כמה דוגמאות של מצעים בתום לב שלנוגדנים מסחריים זמינות: הצעה (BH3 אינטראקצית אגוניסט מות תחום) וcaspase 3 ביקע ידי GzmB 27,28; ribonucleoproteins מרובה הטרוגנית הגרעיני (hnRNPA1, A2 ו- U) ביקע ידי GzmA 29; phosphoprotein ציטומגלווירוס 71 על ידי 30 GzmM. </li></ol></li></ol>

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The authors have nothing to disclose.

התפקיד הקלאסי ונחקר בהרחבה של GzmA וGzmB הוא האינדוקציה של אפופטוזיס בתאי יונקים לאחר לידת cytosolic על ידי חלבון יוצרים הנקבובית 1 PFN. לאחרונה, הספקטרום ציטוטוקסיות של Gzms הורחב באופן משמעותי מתאי יונקים לחיידקים 9,10, כמו גם לטפילים מסוימים 11. יתר על כן, הפונק?...

Gzms היא משפחה של פרוטאזות סרין ההומולוגית מאוד מקומי בlysosomes מיוחד של CTL ותאי NK 1. הגרגרים הרעילים לתאים של תאי רוצח אלה מכילים גם חלבוני שיבוש-קרום PFN וGNLY שפורסמו בו זמנית עם Gzms על הכרת תא מטרה המיועד לחיסול 2,3. ישנם חמש Gzms בבני אדם (GzmA, B, H, K ו- M), ו -10 Gzms בעכברים (GzmA - G, K, M ו- N). GzmA וGzmB הם נפוצים ביותר ונחקר בהרחבה בבני אדם ועכברים 1. עם זאת, מחקרים שנעשה לאחרונה יותר החלו לחקור את מסלולי תא-מוות, כמו גם את ההשפעות ביולוגיות נוספות בתיווך Gzms האחר, שנקרא היתום בבריאות ובחוליים 4. הפונקציה הידועה ביותר של Gzms, בפרט של GzmA וGzmB, היא האינדוקציה של מות תאים מתוכנת בתאי יונקים, כאשר נמסרו לתאי המטרה על ידי PFN 5,6. עם זאת, יותר מחקרים שנעשה לאחרונה הראו גם השפעות תאית של Gzms עם השפעה עמוקה על רגולציה ודלקת חיסוניות, באופן בלתי תלויים על ידי משלוח cytosolic PFN 7,8. הספקטרום של תאים שנהרגו ביעילות לאחר כניסת cytosolic של Gzms גם התרחב לאחרונה מתאי יונקים לחיידקי 9,10 ואפילו טפילים מסוימים 11. הגילויים האחרונים אלה נפתחו שדה חדש לגמרי לחוקרי Gzm. לכן,, מערכת יונקים ביטוי תשואה גבוהה יעילה וחסכונית באופן משמעותי להקל את הדרך למחקרים עתידיים אלה. Gzms האנושי, עכבר והחולדה ילידים כבר מטוהר בהצלחה מחלק גרגיר של קווי תאי CTL וNK 12-14. עם זאת, בידיים שלנו התשואה של טכניקות טיהור כזה היא בטווח של תרבות פחות מ -0.1 מ&quot;ג / L תא (תצפית לא פורסם ו -12). יתר על כן, החלטת chromatographic של Gzm יחיד ללא זיהום על ידי טיהורGzms ו / או חלבונים שנמצאים גם בגרגרים r מאתגר (נתונים שלא פורסם ו12,14). Gzms רקומביננטי הופק בחיידקי 15, שמרי 16, תאי חרקים 17 ובאפילו בתאי יונקים כגון 293 HEK 18,19. רק מערכות ביטוי היונקים לשאת הפוטנציאל לייצר אנזימי רקומביננטי עם שינויי posttranslational זהים לחלבון הרעיל לתאים המקומי. שינויי Posttranslational היו מעורבים עם הספיגה הספציפית על ידי אנדוציטוזה והלוקליזציה תאית של פרוטאז בתוך תאי יעד 20-22. לכן, על ידי השימוש בpHLsec 23 (מתנת סוג של ראדו Aricescu והאיבון ג&#39;ונס, אוניברסיטת אוקספורד, בריטניה) כעמוד שדרת פלסמיד ביטוי Gzm, הקמנו מערכת פשוטה, זמן ועלות-יעילה לייצור חלבון תשואה גבוהה ב תאי HEK293T. pHLsec משלב משפר CMV עם אמרגן Β אקטין עוף; יחד, מרכיבים אלה demonstraטד פעילות האמרגן החזק ביותר בשורות תאים שונות 24. בנוסף, מכיל פלסמיד אינטרון ארנב Β-גלובין, אותות קוזאק והפרשה מותאמים, ליס-6xHis-תג ופולי-אות. ניתן לשכפל מוסיף נוחות בין אות ההפרשה ויס-6xHis התג (איור 1) להבטיח ביטוי אופטימלי ויעילות הפרשה לחלבונים חסרי תחומים N-מסוף מתאימים. לביטוי של Gzms, החלפנו את רצף אות הפרשת אנדוגני עם אות ההפרשה הניתנת על ידי הווקטור אחרי enterokinase אתר (EK) (DDDDK), כך שטיפול EK הופעל Gzms המופרש (Gzms הפעיל להתחיל עם N-המסוף רצף חומצות אמינו IIGG 25). בנוסף לטובת לשיטה זו, תאי HEK293T לצמוח במהירות בינוני במחיר נמוך, כגון בינוני של הנשר (DMEM) השתנה Dulbecco, ומתאימים גם לשיטת transfection סידן-פוספט עלות-יעילה.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>TRIzol Reagent</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td>Total RNA isolation kit</td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase</td>
			<td>Invitrogen</td>
			<td>18064-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>NEB</td>
			<td>M0530L</td>
			<td></td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td>12362</td>
			<td></td>
		</tr>
		<tr>
			<td>EX-CELL 293 Serum-Free Medium for HEK 293 Cells</td>
			<td>Sigma</td>
			<td>14571C</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM, high glucose, GlutaMAX Supplement, pyruvate</td>
			<td>Gibco</td>
			<td>31966-021</td>
			<td></td>
		</tr>
		<tr>
			<td>SnakeSkin Dialysis Tubing, 10K MWCO</td>
			<td>Thermo Scientific</td>
			<td>68100</td>
			<td></td>
		</tr>
		<tr>
			<td>Enterokinase from bovine intestine</td>
			<td>Sigma</td>
			<td>E4906</td>
			<td>recombinant, &#8805;20&#160;units/mg protein</td>
		</tr>
		<tr>
			<td>HisTrap Excel 5ml-column</td>
			<td>GE Healthcare</td>
			<td>17-3712-06&#160;</td>
			<td>Nickel IMAC</td>
		</tr>
		<tr>
			<td>HiTrap SP HP 5 ml-column</td>
			<td>GE Healthcare</td>
			<td>17-1152-01&#160;</td>
			<td>S column</td>
		</tr>
		<tr>
			<td>N-&#945;-Cbz-L-lysine thiobenzylester (BLT)</td>
			<td>Sigma</td>
			<td>C3647</td>
			<td>GzmA substrate</td>
		</tr>
		<tr>
			<td>Boc-Ala-Ala-Asp-S-Bzl (AAD)</td>
			<td>MP Biomedicals</td>
			<td>2193608 _10mg</td>
			<td>GzmB substrate</td>
		</tr>
		<tr>
			<td>Suc-Ala-Ala-Pro-Leu-p-nitroanilide (AAPL)</td>
			<td>Bachem</td>
			<td></td>
			<td>GzmM substrate</td>
		</tr>
		<tr>
			<td>5,5&#8242;-dithio-bis(2-nitrobenzoic acid) (DTNB)</td>
			<td>Sigma</td>
			<td>D8130</td>
			<td>Ellman`s reagent</td>
		</tr>
	</tbody>
</table>

a high yield and cost-efficient expression system of human granzymes in mammalian cells

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their cytotoxic granule content to eliminate the target cells and the intracellular pathogen. Death of the host cells and intracellular pathogens is triggered by the granule serine proteases, granzymes (Gzms), delivered into the host cell cytosol by the pore forming protein perforin (PFN) and into bacterial pathogens by the prokaryotic membrane disrupting protein granulysin (GNLY). To investigate the molecular mechanisms of target cell death mediated by the Gzms in experimental in-vitro settings, protein expression and purification systems that produce high amounts of active enzymes are necessary. Mammalian secreted protein expression systems imply the potential to produce correctly folded, fully functional protein that bears posttranslational modification, such as glycosylation. Therefore, we used a cost-efficient calcium precipitation method for transient transfection of HEK293T cells with human Gzms cloned into the expression plasmid pHLsec. Gzm purification from the culture supernatant was achieved by immobilized nickel affinity chromatography using the C-terminal polyhistidine tag provided by the vector. The insertion of an enterokinase site at the N-terminus of the protein allowed the generation of active protease that was finally purified by cation exchange chromatography. The system was tested by producing high levels of cytotoxic human Gzm A, B and M and should be capable to produce virtually every enzyme in the human body in high yields.

בסעיף הבא נציג תיעוד של הכנת GzmA שלם כדי להאיר את השיטה. אנחנו גם מטוהרים בהצלחה GzmB וGzmM, הפקת תוצאות דומות בכל קשור ליעילות טיהור ופעילות. עם זאת, מהכנות מאוחר יותר אלה שרק מראים כמה חתיכות נבחרות של נתונים. הכנות GzmB מתאי 293T הבאים הפרוטוקול הנוכחי שמשו במספר מחקרים שפור?...

Watch this Scientific Journal Video about A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells at JoVE.com

A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease.

תשואה גבוהה ומערכת ביטוי עלות-יעילה של Granzymes האדם בתאי יונקים

When cytotoxic T lymphocytes (CTL) or natural killer (NK) cells recognize tumor cells or cells infected with intracellular pathogens, they release their ...

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JoVE Journal

Biology

9.7K Views.  Boston Children’s Hospital and Harvard Medical School. We describe here a cost-efficient granzyme expression system using HEK293T cells that produces high yields of pure, fully glycosylated and enzymatically active protease. 

Video: A High Yield and Cost-efficient Expression System of Human Granzymes in Mammalian Cells

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

שימוש ג&#39;ין Pulser מערכת MXcell electroporation כדי Transfect תאים ראשוניים עם יעילות גבוהה

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

שימוש מונה תא אוטומטי כדי לפשט את הביטוי מחקרים ג&#39;ין: siRNA מציאה הביטוי IL 4-Gene תלויים תאים Namalwa

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Chromatin Immunoprecipitation (ChIP) to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited from their latent state in the cytoplasm to cell surface receptors where they are activated by phosphorylation at a single tyrosine residue1. They then dimerize and translocate to the nucleus to drive the transcription of target genes, affecting growth, differentiation, homeostasis and the immune response. Not surprisingly, given their widespread involvement in normal cell processes, dysregulation of STAT function contributes to human disease, particularly to cancers2 and autoimmune diseases3. It is well established that transcription is regulated by alterations to the chromatin template4,5. These alterations include the activities of ATP-dependent complexes, as well as covalent histone modifications and DNA methylation6. Because STAT activation of gene expression is both rapid and transient, it requires specific mechanisms for modulating the chromatin template at STAT-dependent gene loci. To define these mechanisms, we characterize the histone modifications and the enzymatic activities that generate them at gene loci that respond to STAT signaling. This protocol describes chromatin immunoprecipitation, a method that is valuable for the study of STAT signaling to chromatin in activated gene expression.

Chromatin Immunoprecipitation ChIP to Assay Dynamic Histone Modification in Activated Gene Expression in Human Cells

This protocol describes how chromatin immunoprecipitation (ChIP) is used to study the dynamic alterations to the chromatin template that regulate transcription induced by a signal transduction pathway.

הכרומטין immunoprecipitation (שבב) לשינוי Assay דינמי היסטון בביטוי הגנים המופעלת בתא האדם

In response to a variety of extracellular ligands, the STAT (signal transducer and activator of transcription) transcription factors are rapidly recruited ...

Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying potassium (referred to as GIRK) channels, which are important for regulating the excitability of neurons. There are four different mammalian GIRK channel subunits (GIRK1-GIRK4) - we focus on GIRK2 because it forms a homotetramer. Stimulation of different types of G protein-coupled receptors (GPCRs), such as the muscarinic receptor (M2R), leads to activation of GIRK channels. Alcohol also directly activates GIRK channels. We will show how to mutate one amino acid by specifically changing one or more nucleotides in the cDNA for the GIRK channel. This mutated cDNA sequence will be amplified in bacteria, purified, and the presence of the point mutation will be confirmed by DNA sequencing. The cDNAs for the mutated and wild-type GIRK channels will be transfected into human embryonic kidney HEK293T cells cultured in vitro. Lastly, whole-cell patch-clamp electrophysiology will be used to study the macroscopic potassium currents through the ectopically expressed wild-type or mutated GIRK channels. In this experiment, we will examine the effect of a L257W mutation in GIRK2 channels on M2R-dependent and alcohol-dependent activation.

We will demonstrate how to study the effect of a single point mutation on the function of an ion channel.

Mutagenesis וניתוח פונקציונלי של תעלות יונים הביעו Heterologously בתאי יונקים

We will demonstrate how to study the functional effects of introducing a point mutation in an ion channel. We study G protein-gated inwardly rectifying ...

Absolute Quantum Yield Measurement of Powder Samples

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and research of illumination, AV equipment, organic EL material, films, filters and fluorescent probes for bio-industry.
Quantum yield is calculated as the ratio of the number of photons absorbed, to the number of photons emitted by a material. The higher the quantum yield, the better the efficiency of the fluorescent material.
For the measurements featured in this video, we will use the Hitachi F-7000 fluorescence spectrophotometer equipped with the Quantum Yield measuring accessory and Report Generator program. All the information provided applies to this system. 
Measurement of quantum yield in powder samples is performed following these steps:
<ol>
 <li>Generation of instrument correction factors for the excitation and emission monochromators. This is an important requirement for the correct measurement of quantum yield. It has been performed in advance for the full measurement range of the instrument and will not be shown in this video due to time limitations.</li>
 <li>Measurement of integrating sphere correction factors. The purpose of this step is to take into consideration reflectivity characteristics of the integrating sphere used for the measurements.</li>
 <li>Reference and Sample measurement using direct excitation and indirect excitation.</li>
 <li>Quantum Yield calculation using Direct and Indirect excitation. Direct excitation is when the sample is facing directly the excitation beam, which would be the normal measurement setup. However, because we use an integrating sphere, a portion of the emitted photons resulting from the sample fluorescence are reflected by the integrating sphere and will re-excite the sample, so we need to take into consideration indirect excitation. This is accomplished by measuring the sample placed in the port facing the emission monochromator, calculating indirect quantum yield and correcting the direct quantum yield calculation.</li>
 <li>Corrected quantum yield calculation.</li>
 <li>Chromaticity coordinates calculation using Report Generator program.</li>
</ol>
The Hitachi F-7000 Quantum Yield Measurement System offer advantages for this
application, as follows:
<ul>
 <li>High sensitivity (S/N ratio 800 or better RMS). Signal is the Raman band of water measured under the following conditions: Ex wavelength 350 nm, band pass Ex and Em 5 nm, response 2 sec), noise is measured at the maximum of the Raman peak. High sensitivity allows measurement of samples even with low quantum yield. Using this system we have measured quantum yields as low as 0.1 for a sample of salicylic acid and as high as 0.8 for a sample of magnesium tungstate.</li>
 <li>Highly accurate measurement with a dynamic range of 6 orders of magnitude allows for measurements of both sharp scattering peaks with high intensity, as well as broad fluorescence peaks of low intensity under the same conditions. </li>
 <li>High measuring throughput and reduced light exposure to the sample, due to a high scanning speed of up to 60,000 nm/minute and automatic shutter function. </li>
 <li>Measurement of quantum yield over a wide wavelength range from 240 to 800 nm.</li>
 <li>Accurate quantum yield measurements are the result of collecting instrument spectral response and integrating sphere correction factors before measuring the sample.</li>
 <li>Large selection of calculated parameters provided by dedicated and easy to use software.
</li>
</ul> 
During this video we will measure sodium salicylate in powder form which is known to have a quantum yield value of 0.4 to 0.5.

In this video we will demonstrate measuring and calculating absolute quantum yield and chromaticity coordinates directly in powder samples using the Hitachi F-7000 Quantum Yield Measuring System.

מדידה אבסולוטית קוונטית תשואה של דוגמאות אבקת

Measurement of fluorescence quantum yield has become an important tool in the search for new solutions in the development, evaluation, quality control and ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

גזירת יעיל של מבשרי לב cardiomyocytes האדם מן האדם תאים pluripotent גזע עובריים עם אינדוקציה מולקולה קטנה

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Analysis of Cell Cycle Position in Mammalian Cells

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of cell proliferation underlies the pathology of diseases like cancer. As such there is great need to be able to investigate cell proliferation and quantitate the proportion of cells in each phase of the cell cycle. It is also of vital importance to indistinguishably identify cells that are replicating their DNA within a larger population. Since a cell&prime;s decision to proliferate is made in the G1 phase immediately before initiating DNA synthesis and progressing through the rest of the cell cycle, detection of DNA synthesis at this stage allows for an unambiguous determination of the status of growth regulation in cell culture experiments.
DNA content in cells can be readily quantitated by flow cytometry of cells stained with propidium iodide, a fluorescent DNA intercalating dye. Similarly, active DNA synthesis can be quantitated by culturing cells in the presence of radioactive thymidine, harvesting the cells, and measuring the incorporation of radioactivity into an acid insoluble fraction. We have considerable expertise with cell cycle analysis and recommend a different approach. We Investigate cell proliferation using bromodeoxyuridine/fluorodeoxyuridine (abbreviated simply as BrdU) staining that detects the incorporation of these thymine analogs into recently synthesized DNA. Labeling and staining cells with BrdU, combined with total DNA staining by propidium iodide and analysis by flow cytometry1 offers the most accurate measure of cells in the various stages of the cell cycle. It is our preferred method because it combines the detection of active DNA synthesis, through antibody based staining of BrdU, with total DNA content from propidium iodide. This allows for the clear separation of cells in G1 from early S phase, or late S phase from G2/M. Furthermore, this approach can be utilized to investigate the effects of many different cell stimuli and pharmacologic agents on the regulation of progression through these different cell cycle phases.
In this report we describe methods for labeling and staining cultured cells, as well as their analysis by flow cytometry. We also include experimental examples of how this method can be used to measure the effects of growth inhibiting signals from cytokines such as TGF-&beta;1, and proliferative inhibitors such as the cyclin dependent kinase inhibitor, p27KIP1. We also include an alternate protocol that allows for the analysis of cell cycle position in a sub-population of cells within a larger culture5. In this case, we demonstrate how to detect a cell cycle arrest in cells transfected with the retinoblastoma gene even when greatly outnumbered by untransfected cells in the same culture. These examples illustrate the many ways that DNA staining and flow cytometry can be utilized and adapted to investigate fundamental questions of mammalian cell cycle control.

Determining the cell cycle position of a population of cells, or understanding how signals affect proliferation, can be readily measured by flow cytometry using this protocol. We report a simple experimental approach to staining cells and quantifying their position in the cell cycle.

ניתוח של עמדת מחזור התא בתאי יונקים

The regulation of cell proliferation is central to tissue morphogenesis during the development of multicellular organisms. Furthermore, loss of control of ...

Expression Analysis of Mammalian Linker-histone Subtypes

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure. H1 is essential for mammalian development1 and regulates specific gene expression in vivo2-4. Among the highly conserved histone proteins, the family of H1 linker histones is the most heterogeneous group. There are 11 H1 subtypes in mammals that are differentially regulated during development and in different cell types. These H1 subtypes include 5 somatic H1s (H1a-e), the replacement H10, 4 germ cell specific H1 subtypes, and H1x5. The presence of multiple H1 subtypes that differ in DNA binding affinity and chromatin compaction ability6-9 provides an additional level of modulation of chromatin function. Thus, quantitative expression analysis of individual H1 subtypes, both of mRNA and proteins, is necessary for better understanding of the regulation of higher order chromatin structure and function.
Here we describe a set of assays designed for analyzing the expression levels of individual H1 subtypes (Figure 1). mRNA expression of various H1 variant genes is measured by a set of highly sensitive and quantitative reverse transcription-PCR (qRT-PCR) assays, which are faster, more accurate and require much less samples compared with the alternative approach of Northern blot analysis. Unlike most other cellular mRNA messages, mRNAs for most histone genes, including the majority of H1 genes, lack a long polyA tail, but contain a stem-loop structure at the 3' untranslated region (UTR)10. Therefore, cDNAs are prepared from total RNA by reverse transcription using random primers instead of oligo-dT primers. Realtime PCR assays with primers specific to each H1 subtypes (Table 1) are performed to obtain highly quantitative measurement of mRNA levels of individual H1 subtypes. Expression of housekeeping genes are analyzed as controls for normalization. 
The relative abundance of proteins of each H1 subtype and core histones is obtained through reverse phase high-performance liquid chromatography (RP-HPLC) analysis of total histones extracted from mammalian cells11-13. The HPLC method and elution conditions described here give optimum separations of mouse H1 subtypes. By quantifying the HPLC profile, we calculate the relative proportion of individual H1 subtypes within H1 family, as well as determine the H1 to nucleosome ratio in the cells.

We describe a set of assays to analyze expression levels of H1 linker histones. mRNA of individual H1 genes are quantitatively measured by random primer based reverse transcription followed by real-time PCR, whereas protein quantification of H1 histones is achieved by HPLC analysis.

הביטוי ניתוח Linker-היסטון תת סוגים יונקים

Linker histone H1 binds to the nucleosome core particle and linker DNA, facilitating folding of chromatin into higher order structure.  H1 is essential ...

Visualization of Endoplasmic Reticulum Localized mRNAs in Mammalian Cells

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum (ER). However, the visualization of these mRNAs can be challenging. This is especially true when only a fraction of the mRNA is ER-associated and their distribution to this organelle is obstructed by non-targeted (i.e. "free") transcripts. In order to monitor ER-associated mRNAs, we have developed a method in which cells are treated with a short exposure to a digitonin extraction solution that selectively permeabilizes the plasma membrane, and thus removes the cytoplasmic contents, while simultaneously maintaining the integrity of the ER. When this method is coupled with fluorescent in situ hybridization (FISH), one can clearly visualize ER-bound mRNAs by fluorescent microscopy. Using this protocol the degree of ER-association for either bulk poly(A) transcripts or specific mRNAs can be assessed and even quantified. In the process, one can use this assay to investigate the nature of mRNA-ER interactions.

Here we describe a method to visualize endoplasmic reticulum-associated mRNAs in mammalian tissue culture cells. This technique involves the selective permeabilization of the plasma membrane with digitonin to remove cytoplasmic contents followed by fluorescent in situ hybridization to detect either bulk poly(A) mRNA or specific transcripts.

ויזואליזציה של mRNAs לוקליזציה reticulum endoplasmic בתאי יונקים

In eukaryotes, most of the messenger RNAs (mRNAs) that encode secreted and membrane proteins are localized to the surface of the endoplasmic reticulum ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

הקרנת siRNA לזיהוי יוביקוויטין ורגולטורים מערכת דמויות יוביקוויטין של מסלולים ביולוגיים בתאי יונקים בתרבית

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...