Name: the production of pluripotent stem cells from mouse amniotic fluid cells using a transposon system
Uploaded: 2017-02-28T13:00:00
Description: Watch this Scientific Journal Video about The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System at JoVE.com

This work was supported by CARIPARO Foundation Grant number 13/04 and Fondazione Istituto di Ricerca Pediatrica Citt&#224; della Speranza Grant number 10/02. Martina Piccoli, Chiara Franzin and Michela Pozzobon are funded by Fondazione Istituto di Ricerca Pediatrica Citt&#224; della Speranza. Enrica Bertin is funded by CARIPARO Foundation Grant number 13/04. Paolo De Coppi is funded by Great Ormond Street Hospital Children's Charity.

כל הנהלים היו בהתאם לחוק האיטלקי. דגימות Murine AF נבצרו מעכברים בהריון 13.5 ימים לאחר coitum (DPC) מ C57BL / 6-Tg (UBC-GFP) 30Scha / J עכברים בשם GFP. 1. ייצור transposon הערה: וקטורי ביטוי transposon נוצרו באמצעות נהלי שיבוט סטנדרט...

השיטה שנבחרה כדי להשיג את האינדוקציה של pluripotency רלוונטית לבטיחות קלינית תא ביחס השתלה לטווח ארוכה. כיום, קיימות מספר שיטות מתאימות תכנות מחדש. בין השיטות הלא-אינטגרטיבי יותר, ויראלי סנדאי (SEV) הווקטור הוא וירוס RNA אשר יכול לייצר כמויות גדולות של חלבון ללא להשתלב בגרעין ...

אבחון טרום לידתי הוא כלי קליני חשוב להעריך מחלות גנטיות (סטיות כרומוזומליות כלומר, monogenetic או polygenetic / מחל מולטיפקטוריאלית) ו מומים מולדים (כלומר מולד בקע סרעפתי, נגעי ריאות פיברוזיס, exomphalos, gastroschisis). מי שפיר (AF) תאים הם פשוט להשיג מנהלים מתוכננים באופן שגרתי במהלך השליש השני של הריון (כלומר מי שפיר ו amnioreduction) או ניתוחים קיסריים 1, 2. הזמינות של תאי AF מחולים טרום לידתי או בילוד מספק את האפשרות של שימוש מקור זה עבור רפואה רגנרטיבית, וכמה החוקרים בדקו את האפשרות לטיפול נזקים ברקמות שונות או מחלות באמצעות האוכלוסייה בתאי גזע שבודדו 3 AF, 4, 5, 67, 8, 9, 10, 11, 12. האפשרות של קבלה בקלות תאי AF מחולים חולים, בחלון הזמן בו המחלה היא לעתים קרובות נייחת, פותחת את הדרך אל הרעיון של שימוש מקור התא הזה למטרות תכנות מחדש. ואכן, גזע מושר (iPS) תאים שמקורם בתאי AF יכולים להיות מובחנים בתאים של ריבית עבור בדיקות סמים במבחנה או גישות הנדסת רקמות, כדי להכין טיפול למטופל ספציפי נאות לפני הלידה. מחקרים רבים כבר הוכיחו את יכולתם של תאי AF כדי לתכנת מחדש בדיל לתוך מגוון רחב של סוגי תאי 13, 14, 15, 16, 17 &lt;/ sup&gt;, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27. מאז גילויו על ידי טקהאשי יאמאנאקה 28 תאים סומטיים לתכנות מחדש דרך הביטוי הכפוי של ארבעה גורמי שעתוק (Oct4, Sox2, cMyc ו Klf4), הושג התקדמות בתחום של תכנות מחדש. בהתחשב בשיטות שונות, אנו יכולים להבחין בין גישות ויראלי ולא ויראלי. הראשון מצפה השימוש של וקטורים ויראליים (רטרווירוסים ו lentiviruses), אשר יש יעילות גבוהה, אך בדרך כלל השתקה שלמה של transgene retroviral, הוא עם התוצאה של שורת תאים לתכנות מחדש באופן חלקי את הסיכוןinsertional mutagenesis 29, 30, 31. השיטה הלא-ויראלי משתמשת באסטרטגיות שונות: פלסמידים כלומר, וקטורים, mRNA, חלבון, transposons. על הגזרה של תאי iPS ללא רצפים מהונדסים שואפת לעקוף את השפעות מזיקי ביטוי transgene דולף mutagenesis insertional. בין כל האסטרטגיות הלא-ויראלי הנ&quot;ל, את PiggyBac (PB) מערכת transposon / transposase דורשת חוזרת מסוף ההפוך רק חובק transgene ו ביטוי חולף של אנזים transposase לזרז אירועי כניסה או כריתה 32. היתרון בשימוש transposons על פני שיטות אחרות לייצור תאי שב&quot;ס הוא האפשרות לקבל תאי iPS ללא וקטור עם גישת וקטור שאינו ויראלי שמראה אותה היעילות של וקטורי retroviral. זה אפשרי על ידי כריתת עקבות-פחות של קידוד transposon המשולב עבור reprogramming גורמים הבאים ביטוי חולף חדש של transposase בתאי שב&quot;ס 33. בהתחשב בכך PB הוא יעיל בסוגי תאים שונים 34, 35, 36, 37, הוא מתאים יותר עבור גישה קלינית ביחס וקטורים ויראליים, ומאפשר את ייצור תאי iPS ללא קסנו בניגוד פרוטוקולי ייצור ויראלי הנוכחיים משתמשות xenobiotic תנאים, מערכת זו משמשת להשיג תאי שב&quot;ס מן בעכברים AF. כאן אנו מציעים פרוטוקול מפורט לאחר עבודה שכבר פורסם להראות לייצור שיבוטי שב&quot;ס פלוריפוטנטיים מתאי AF עכבר (תאי iPS-AF) 38.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>100 mm Bacterial-grade Petri Dishes&#160;</td>
			<td>BD Falcon</td>
			<td>351029</td>
			<td>For in vitro differentiation</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol&#160;</td>
			<td>Sigma</td>
			<td>M6250</td>
			<td>For mouse AF, iPS-AF cells and differentiation medium</td>
		</tr>
		<tr>
			<td>Alexa568-conjugated goat anti-mouse IgM&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21043</td>
			<td>Secondary antibody (immunofluorescence)</td>
		</tr>
		<tr>
			<td>Alexa594-conjugated chicken anti-goat IgG&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21468</td>
			<td>Secondary antibody (immunofluorescence)</td>
		</tr>
		<tr>
			<td>Alexa594-conjugated chicken anti-rabbit IgG&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A21442</td>
			<td>Secondary antibody (immunofluorescence)</td>
		</tr>
		<tr>
			<td>Alexa594-conjugated goat anti-mouse IgG&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>A11005</td>
			<td>Secondary antibody (immunofluorescence)</td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase kit&#160;</td>
			<td>Sigma</td>
			<td>85L1</td>
			<td>Alkaline Phosphatase&#160; staining</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Sigma</td>
			<td>A0166</td>
			<td>For bacterial selection</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin&#160;</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td>BSA, for blocking solution. Diluted in PBS 1X</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>C2432</td>
			<td>For RNA extraction</td>
		</tr>
		<tr>
			<td rowspan="2">DH5&#945; cells</td>
			<td rowspan="2">Thermo Fisher Scientific</td>
			<td rowspan="2">18265-017</td>
			<td rowspan="2">Bacteria for cloning procedure</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td>Thermo Fisher Scientific</td>
			<td>41965039</td>
			<td>For MEF, mouse AF, iPS-AF cells and differentiation medium</td>
		</tr>
		<tr>
			<td>Doxycycline&#160;</td>
			<td>Sigma</td>
			<td>D9891</td>
			<td>For exogenous factors expression</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes (1.5 mL)&#160;</td>
			<td>Sarstedt&#160;</td>
			<td>72.706</td>
			<td>For PB production&#160;</td>
		</tr>
		<tr>
			<td>ES FBS&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>10439024</td>
			<td>For mouse AF, iPS-AF cells and differentiation medium</td>
		</tr>
		<tr>
			<td>FBS&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>10270106</td>
			<td>For MEF medium</td>
		</tr>
		<tr>
			<td>Fine point forceps</td>
			<td>F.S.T</td>
			<td>Dumont #5&#160;</td>
			<td>AF isolation</td>
		</tr>
		<tr>
			<td>Gelatin</td>
			<td>J.T.Baker</td>
			<td>131</td>
			<td>Used 0.1%, diluted in PBS 1X</td>
		</tr>
		<tr>
			<td>Glycine</td>
			<td>Bio-Rad</td>
			<td>161-0718</td>
			<td>For blocking solution. Diluted in PBS 1X</td>
		</tr>
		<tr>
			<td>Haematoxylin QS</td>
			<td>Vector Laboratories</td>
			<td>H3404</td>
			<td>Nuclei detection</td>
		</tr>
		<tr>
			<td>HE&#160;</td>
			<td>Bio-Optica</td>
			<td>04-061010</td>
			<td>Histological analysis of teratoma</td>
		</tr>
		<tr>
			<td>Hoechst&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3570</td>
			<td>Nuclei detection</td>
		</tr>
		<tr>
			<td>Horse Serum&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>16050-122</td>
			<td>For blocking solution</td>
		</tr>
		<tr>
			<td>HRP-conjugated goat anti-mouse IgG</td>
			<td>SantaCruz</td>
			<td>sc2005</td>
			<td>Secondary antibody (immunoperoxidase)</td>
		</tr>
		<tr>
			<td>ImmPACT NovaRED&#160;</td>
			<td>Vector Laboratories</td>
			<td>SK4805</td>
			<td>Peroxidase substrate</td>
		</tr>
		<tr>
			<td>Insulin syringe with needle (25G)</td>
			<td>Terumo</td>
			<td>SS+01H25161</td>
			<td>Amniocentesis procedure</td>
		</tr>
		<tr>
			<td>Klf4&#160;</td>
			<td>SantaCruz</td>
			<td>sc-20691</td>
			<td>Rabbit polyclonal IgG</td>
		</tr>
		<tr>
			<td>L-glutamine&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030</td>
			<td>For mouse AF, iPS-AF cells and differentiation medium</td>
		</tr>
		<tr>
			<td>LB broth (Lennox)</td>
			<td>Sigma</td>
			<td>L3022</td>
			<td>For bacterial growth</td>
		</tr>
		<tr>
			<td>LIF&#160;</td>
			<td>Sigma</td>
			<td>L5158</td>
			<td>For mouse AF and iPS-AF cells medium</td>
		</tr>
		<tr>
			<td>Matrigel&#160;</td>
			<td>BD</td>
			<td>354234</td>
			<td>For in vitro differentiation. Diluted 1:10 in DMEM</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma</td>
			<td>32213</td>
			<td>Peroxidase blocking</td>
		</tr>
		<tr>
			<td>MULTIWELL 24 well plate</td>
			<td>BD Falcon</td>
			<td>353047</td>
			<td>For in vitro differentiation</td>
		</tr>
		<tr>
			<td>MULTIWELL 6 well plate</td>
			<td>BD Falcon</td>
			<td>353046</td>
			<td>For MEF, mouse AF and iPS-AF cells culture</td>
		</tr>
		<tr>
			<td>Nanog&#160;</td>
			<td>ReproCELL</td>
			<td>RCAB0002P-F</td>
			<td>Rabbit polyclonal IgG</td>
		</tr>
		<tr>
			<td>Non-essential amino acids&#160;</td>
			<td>Sigma</td>
			<td>M7145</td>
			<td>For mouse AF, iPS-AF cells and differentiation medium</td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Vector Laboratories</td>
			<td>S2000</td>
			<td>For blocking solution. Diluted in PBS 1X</td>
		</tr>
		<tr>
			<td>NP-40</td>
			<td>Sigma</td>
			<td>12087-87-0</td>
			<td>For cell permeabilization. Diluted in PBS 1X</td>
		</tr>
		<tr>
			<td rowspan="2">Oct4</td>
			<td rowspan="2">SantaCruz</td>
			<td rowspan="2">sc-5279</td>
			<td rowspan="2">Mouse monoclonal IgG2b</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Oligo (dT)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>18418012</td>
			<td>For RT-PCR</td>
		</tr>
		<tr>
			<td>Paraformaldehyde (solution)</td>
			<td>Sigma</td>
			<td>441244</td>
			<td>PFA, fixative, diluted in PBS</td>
		</tr>
		<tr>
			<td>PBS 10X</td>
			<td>Thermo Fisher Scientific</td>
			<td>14200-067</td>
			<td>D-PBS, free of Ca2+/Mg2+. Diluted with sterile water to obtain PBS 1X</td>
		</tr>
		<tr>
			<td>Penicillin - Streptomycin&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>15070063</td>
			<td>For MEF, mouse AF, iPS-AF cells and differentiation medium</td>
		</tr>
		<tr>
			<td>Petri Dish (150mm)</td>
			<td>BD Falcon</td>
			<td>353025</td>
			<td>For MEF culture, tissue culture</td>
		</tr>
		<tr>
			<td>PiggyBac transposase expression plasmid&#160;</td>
			<td>Provided by professor Andras Nagy laboratory</td>
			<td>-</td>
			<td>mPBase</td>
		</tr>
		<tr>
			<td>PiggyBac-tetO2-IRES-OKMS transposon plasmid</td>
			<td>Provided by professor Andras Nagy laboratory</td>
			<td>-</td>
			<td>PB-tetO2-IRES-OKMS</td>
		</tr>
		<tr>
			<td>QIAprep Spin Maxiprep Kit</td>
			<td>Qiagen</td>
			<td>12663</td>
			<td>For plasmids purification</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>For plasmids purification</td>
		</tr>
		<tr>
			<td>Reverse tetracycline transactivator transposon plasmid&#160;</td>
			<td>Provided by professor Andras Nagy laboratory</td>
			<td>-</td>
			<td>rtTA</td>
		</tr>
		<tr>
			<td>RNeasy Mini Kit&#160;</td>
			<td>Qiagen</td>
			<td>74134</td>
			<td>For RNA extraction</td>
		</tr>
		<tr>
			<td>Sox2&#160;</td>
			<td>SantaCruz</td>
			<td>sc-17320</td>
			<td>Goat polyclonal IgG</td>
		</tr>
		<tr>
			<td>SSEA1&#160;</td>
			<td>Abcam</td>
			<td>ab16285</td>
			<td>Mouse monoclonal IgM</td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>18064-014</td>
			<td>For RT-PCR</td>
		</tr>
		<tr>
			<td>T&#160;</td>
			<td>Abcam</td>
			<td>ab20680</td>
			<td>Rabbit polyclonal IgG</td>
		</tr>
		<tr>
			<td>Taq DNA Polymerase</td>
			<td>Thermo Fisher Scientific</td>
			<td>10342020</td>
			<td>PCR</td>
		</tr>
		<tr>
			<td>Trypsin&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>25300-054</td>
			<td>Cell culture passaging</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Bio-Rad</td>
			<td>161-047</td>
			<td>For cell permeabilization, diluted in PBS 1X</td>
		</tr>
		<tr>
			<td rowspan="2">TRIzol Reagent</td>
			<td rowspan="2">Thermo Fisher Scientific</td>
			<td rowspan="2">15596-026</td>
			<td rowspan="2">For RNA extraction</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td rowspan="2">Tubb3&#160;&#160;</td>
			<td rowspan="2">Promega&#160;</td>
			<td rowspan="2">G712A</td>
			<td rowspan="2">Mouse monoclonal IgG1</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>TWEEN-20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td>For cell permeabilization, diluted in PBS 1X</td>
		</tr>
		<tr>
			<td>&#945;fp&#160;&#160;&#160;</td>
			<td>R&#38;D Systems</td>
			<td>MAB1368</td>
			<td>Mouse Monoclonal IgG1</td>
		</tr>
		<tr>
			<td rowspan="2">&#945;SMA&#160;</td>
			<td rowspan="2">Abcam</td>
			<td rowspan="2">ab7817</td>
			<td rowspan="2">Mouse Monoclonal IgG2a</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td>Transfection Reagent (FuGENE HD)</td>
			<td>Promega&#160;</td>
			<td>E2311</td>
			<td>For AF cells transfection</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td>SM2645</td>
			<td>To perform amniocentesis&#160;</td>
		</tr>
		<tr>
			<td>200 ul tips</td>
			<td>Sarstedt&#160;</td>
			<td>70.760012</td>
			<td>To pick bacteria colonies</td>
		</tr>
		<tr>
			<td>Scissor</td>
			<td>F.S.T</td>
			<td>14094-11 stainless 25U</td>
			<td>To perform amniocentesis&#160;</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma</td>
			<td>2860</td>
			<td>To clean the abdominal wall of the pregnant dam</td>
		</tr>
		<tr>
			<td>Tissue culture petri dish (150 mm)&#160;</td>
			<td>BD Falcon</td>
			<td>353025</td>
			<td>For MEF expansion</td>
		</tr>
		<tr>
			<td>Mitomycin C</td>
			<td>Sigma</td>
			<td>M4287-2MG</td>
			<td>For MEF inactivation</td>
		</tr>
		<tr>
			<td>MULTIWELL 96 well plate</td>
			<td>BD Falcon</td>
			<td>353071</td>
			<td>For iPS-AF culture</td>
		</tr>
	</tbody>
</table>

the production of pluripotent stem cells from mouse amniotic fluid cells using a transposon system

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using different methods. Here, we produced iPS cells from mouse amniotic fluid cells, using a non-viral-based transposon system. All obtained iPS cell lines exhibited characteristics of pluripotent cells, including the ability to differentiate toward derivatives of all three germ layers in vitro and in vivo. This strategy opens up the possibility of using cells from diseased fetuses to develop new therapies for birth defects.

כדי להעריך את היכולת של תכנות מחדש, בתאי עכבר AF נאספו מעוברים של עכברים GFP. התאים היו transfected עם הפלסמיד transposon עגול PB-tetO2-IRES-OKMS, המבטא את הגורמים יאמאנאקה (Oct4, Sox2, cMyc ו Klf4) צמוד חלבון פלואורסצנטי mCherry באופן דוקסיציקלין-מושרה, הפוכה transactivator טטרציקלין (PB- CAG-rt...

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The Production of Pluripotent Stem Cells from Mouse Amniotic Fluid Cells Using a Transposon System

In this study, we generate induced pluripotent stem cells from mouse amniotic fluid cells, using a non-viral-based transposon system.

את ייצור תאי גזע פלוריפוטנטיים מתאי מי שפירים עכבר באמצעות מערכת transposon

Induced pluripotent stem (iPS) cells are generated from mouse and human somatic cells by forced expression of defined transcription factors using ...

The overall goal of this procedure is to isolate and reprogram murine amniotic fluid cells using a transposon system. The main advantage of this technique is that it is easily performed. In a therapeutic setting, cells from human disease fetuses can be differentiated to cells of interest for drug testing or tissue engineering, in order to prepare another way the patient specific therapy before childbirth.This method can help answer key questions of programming. The transposon system is efficient in different cell types. It's more suitable for clinical approach with respect to viral vectors and allow the production of oxino-free IPS cells.To begin the isolation, clean the abdominal wall of the dam using 70 percent ethanol. Using scissors, perform a midline laparotomy, incising the length of the abdominal wall to gain access to the abdominal cavity. Use forceps to expose the uterus and then excise it using scissors.Transfer the uterus into a 100 millimeter dish filled with sterile 1xPBS and keep on ice. Place the dish under a stereomicroscope at 10X magnification and hold the uterus with forceps whilst removing the uterine wall with scissors, taking care to avoid damaging the fetal membranes. Collect the fetuses into a 100 millimeter dish filled with sterile 1XPBS and place on ice.Grasp the placenta of one fetus with the fine point forceps and move it to a clean 100 millimeter dish. Here, carefully remove the yolk sack. Disrupt the amnion using the forceps and using an insulin syringe, collect 30 microliters of amniotic fluid.Transfer the fluid to a 15 milliliter conical vial. Repeat this process for each fetus, collecting the fluid into the same vial each time. Next, wash each fetus with sterile 1XPBS supplemented with one percent fetal bovine serum and collect this into the 15 milliliter conical tube along with the amniotic fluid.Centrifuge the tube at 145 times G for five minutes and then under a laminar flow hood, remove the supernatant. Re-suspend the pelleted cells in two milliliters of amniotic fluid culture medium. Take a zero point one percent gelatin-coated six well plate containing myomycin-C treated mouse embryonic fibroblasts or MEF and then seed two milliliters of cells onto the plate.Cultivate the cells at 37 degrees Celsius and five percent carbon dioxide, changing the medium every other day. Continue culture for seven days until the cells are ready for transfection. To begin transfection, in a one point five millimiter microcentrifuge tube, mix one microgram of transposon plasmid with zero point five micrograms of transposase expression plasmid and zero point five micrograms of reverse tetracycline transactivator plasmid.Dilute to 100 microliters using d-man. Add eight microliters of transfection reagent into the microcentrifuge tube containing the DNA. Incubate this mix for 15 minutes at room temperature.Taking the six well plate with mouse AF cells, dropwise add 100 microliters of the transfection agent DNA mixture per well, distributing by gentle swirling with a final volume of two milliliters per well. Leave cells for 24 hours at 37 degrees Celsius in five percent carbon dioxide. The following day, remove the culture medium and then feed each well of cells with two milliliters of fresh iPS AF medium supplemented with one point five micrograms per milliliter of doxycycline to induce the expression of Yamanaka's factors.Feed the cells daily with fresh dox-containing medium without passage from this point forward. Observe the cells under the florescence microscope within 24 hours of the first doxycylcine treatment to look for any mCherry expression and daily from this point on for colony formation. Once colony formation is observed, pick single colonies into 50 microliters of cell culture medium.Next, transfer them sequentially into separate wells of a 96 well tissue culture plate with mytomycin c treated MEF. The following day, add 50 microliters of trypsin to each well and incubate for five minutes at 37 degrees Celsius to dissociate the colonies into single cells. Following incubation, pipette the liquid up and down to further disaggregate the colonies.Transfer 50 microliters of the trypsin cell mix into a new well containing inactivated MEF and then return the cells to the incubator. When cells reach 60 to 70 percent confluence, split them into a new well of a 24 well plate and then continue incubation at room temperature. Once these cells reach 60 to 70 percent confluence, flip the cells one to two into two wells, one containing doxycycline and one without to verify that colonies will also grow in the absence of doxycycline.Continue to maintain the IPS AF colonies and doxycycline independent medium on inactivated MEF at 37 degrees Celsius in five percent carbon dioxide. Change the medium daily until cells are ready for staining, imaging, and RNA extraction. Here, the stable doxycycline independent induced pleuripotent mouse amniotic GFP positive cells express the pleuripotency markers required to confirm successful reprogramming.RTPCR analysis using R1 ES cells as a control showed expression of the induced pleuripotency markers in the presence or absence of doxycycline. The expression of the housekeeping gene, beta two microglobulin, was also observed in all three cell types. Teratomas isolated from mice injected with the induced pleuropotent cells were immunostained.Detection of alpha fetal protein, alpha smooth muscle actin, and beta three tubulin in the masses confirm cell differentiation into all three germ layers, ectoderm, mesoderm, and endoderm. Further, RTPCR analysis of in vitro embryoid bodies and in vivo teratoma cells confirm the expression of germ layer specific marker genes for the mesoderm, endoderm, and ectoderm. After watching this video, you should have a good understanding of how to isolate and reprogram murine amniotic fluid cells.This is a simple, safe procedure that can be applied also to human amniotic fluid cells from a sample obtained from routine diagnoses or cesarean sections.

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