Name: technique to target microinjection to the developing xenopus kidney
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עבודה זו נתמכה על ידי המכונים הלאומיים לבריאות מענק NIDDK (K01DK092320) ומימון אתחול מתוך מחלקת ילדים בבית הספר לרפואה של אוניברסיטת טקסס מקגוורן.

הפרוטוקול הבא אושר על ידי האוניברסיטה למדעי בריאות מרכז טקסס המרכז של יוסטון ועדת צער בעלי חיים לרפואה בחיות מעבדה, אשר ממשמשת לטיפול המוסדי ועדת שימוש (# פרוטוקול: HSC-AWC-13-135). זיהוי 1. בחירה של בלסטומרים עבור זריקות כליות במיקוד <ol style=";text-align:ri...

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מיקוד כִּליַת רֵאשִׁית לפתח עוברי Xenopus מסתמכת על זיהוי הזרקת blastomere הנכון. הזרקה של blastomere V2 של עוברים 8 תאים מטרות כִּליַת רֵאשִׁית שמאל 18. זה משאיר את כִּליַת רֵאשִׁית התקינה הנגדית כמו שליטה פנימית. אם morpholino מציאה או ביטוי יתר RNA משמש המשנה את ההתפתחות בכלי?...

הכליה העוברית Xenopus, את כִּליַת רֵאשִׁית, הוא מודל טוב ללימוד פיתוח כליות ומחלות. עובר לפתח חיצוני, הם גדולים, יכולים להיות מיוצרים בכמויות גדולות, והם מניפולציות בקלות באמצעות microinjection או הליכים כירורגיים. בנוסף, הגנים לפיקוח על בניית כליות אצל יונקים ודו-חיים הם משומר. כליות יונקות להתקדם דרך שלושה שלבים: כִּליַת רֵאשִׁית, הכליה, ו metanephros 1, בעוד דו חיים עובריים כִּליַת רֵאשִׁית ודו-חי בוגרים יש metanephros. יחידת הסינון הבסיסית של צורות כליות אלה היא נפרון, ושניהם יונקים ודו-חיים דורשים את אותה מפלי איתות ואירועים אינדוקטיביים לעבור nephrogenesis 2, 3. כִּליַת רֵאשִׁית Xenopus מכילה נפרון בודד מורכב הפרוקסימלי, ביניים, דיסטלי וחיבור tubules, וכן glomus (מקביל ל glomerulus היונק) 1, 4-6 (איור 1 </strאונג&gt;). הווה נפרון הבודד, הגדול Xenopus כִּליַת רֵאשִׁית הופך אותו מתאים כמודל פשוט לחקר הגנים המעורבים בתהליכי פיתוח כליות ומחלות. מפות גורל התא הוקמו עבור עוברי Xenopus מוקדם, והם זמינים באופן חופשי באינטרנט ב Xenbase 7-11. כאן אנו מתארים טכניקה microinjection של קליעים נותבים השושלת למקד את כִּליַת רֵאשִׁית Xenopus פיתוח, אם כי באותה טכניקה ניתן להתאים למקד ברקמות אחרות כגון הלב או העיניים. קליעים נותבים השושלת הם תוויות (כולל צבעים חיוניים, dextrans שכותרתו fluorescently, אנזימים לגילוי histochemically, ו mRNA קידוד חלבוני ניאון) כי ניתן להזריק לתוך blastomere מוקדם, המאפשר הדמיה של הצאצאים של התא במהלך ההתפתחות. פרוטוקול זה מנצל mRNA MEM-RFP, קרום קידוד ממוקד חלבון פלואורסצנטי אדום 12, כמו נותב השושלת. טק microinjection הממוקדniques עבור בלסטומרים בודדים עוברים 4 ו 8 תאים המתואר כאן יכול להיות מנוצל להזרקה עם morpholinos להפיל ביטוי גנים, או עם RNA אקסוגני overexpress הגן של עניין. באמצעות הזרקה לתוך blastomere הגחון, הצמחי, בעיקר כִּליַת רֵאשִׁית של העובר תהיה ממוקדת, עוזבת את כִּליַת רֵאשִׁית הנגדית כביקורת התפתחותית. שיתוף ההזרקה נותב מוודאת כי blastomere הנכון הוזרק, ומופעים אשר רקמות בעובר נבעו blastomere המוזרק, אימות מיקוד של כִּליַת רֵאשִׁית. Immunostaining של כִּליַת רֵאשִׁית מאפשר הדמיה של מידת הצלחה של tubules pronephric היה ממוקד. תופעות התבטאות יתר ו מציאה אז יכול להיות הבקיע נגד הצד הנגדי של העובר, אשר משמש כביקורת התפתחותי, והוא יכול לשמש כדי לחשב את מדד pronephric 13. הזמינות של מפות גורל התא מאפשרת טכניקת microinjection ממוקדת זה כדי לשמש יעד רקמות othאה מאשר כִּליַת רֵאשִׁית, ואת שיתוף ההזרקה נותב ניאון מאפשר microinjection המתמקדת בכל רקמות להיות מאומתת לפני הניתוח. במהלך microinjection העובר, טמפרטורה התפתחותי צריכה להיות מוסדרת בחוזקה, בהתחשב בכך את קצב התפתחות Xenopus תלוי מאוד עליו 14. עובר צריך להיות מודגרות בטמפרטורות קרירות (14 - 16 מעלות צלזיוס) במשך 4 ו זריקות 8 תאים כי זמן הפיתוח הוא האטה. בגיל 22 ° C, זמן הפיתוח מ -1 הבמה (תא 1) לביים 3 (4 תאים) הוא כ 2 שעות, תוך 16 ° C זמן הפיתוח לשלב 3 הוא כ 4 שעות. זה לוקח בערך 15 דקות לסיום מעובר 4 תאים לעובר 8 תאים (שלב 4) על 22 מעלות צלזיוס, אבל לוקח בערך 30 דקות על 16 מעלות צלזיוס. באופן דומה, על 22 מעלות צלזיוס, זה לוקח רק 30 דקות עבור עובר 8 תאים כדי להתקדם עובר 16 תאים (שלב 5). הפעם הוא עלה ל 45 דקות ב 16 מעלות צלזיוס. הrefore, כדאי להאט את קצב הפיתוח של עוברים כדי לאפשר מספיק זמן עבור זריקות בשלב 8 תאים לפני העוברים להתקדם לשלב 16 תאים. בנוסף, בטמפרטורות צמיחה יכולות להיות מווסתות כדי להאיץ או להאט התפתחות עוברית עד הכליות פתחו באופן מלא. האפידרמיס של עובר ראשן שלבית Xenopus הוא יחסית שקוף, המאפשר הדמיה קלה של כִּליַת רֵאשִׁית בפיתוח בלי דיסקציה או סליקה של הרקמה 15. בשל השקיפות היחסית של עוברי Xenopus, הדמיה תא חי הוא גם אפשרי 16,17. כל הר immunostaining לדמיין את כִּליַת רֵאשִׁית אפשרי עם נוגדנים נקבע כי התווית הפרוקסימלי, ביניים, דיסטלי ו tubules חיבור של שלב 38 - 40 עוברים המאפשרים הערכת התפתחות pronephric ממוקד לאחר מניפולציה של ביטוי גנים העוברים Xenopus 18-20.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher</td>
			<td>S271-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Fisher</td>
			<td>P217-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate&#160;</td>
			<td>Fisher</td>
			<td>M63-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Fisher</td>
			<td>C79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Fisher</td>
			<td>S311-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin solution, 50 mg/mL</td>
			<td>Amresco</td>
			<td>E737-20ML</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine, 99%+</td>
			<td>Acros Organics</td>
			<td>52-90-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll</td>
			<td>Fisher</td>
			<td>BP525-100</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm x 15 mm Petri dish</td>
			<td>Fisher</td>
			<td>FB0875712</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Fridge II</td>
			<td>Boekel</td>
			<td>260009</td>
			<td>Benchtop incubator for embryos.</td>
		</tr>
		<tr>
			<td>Gap43-RFP plasmid</td>
			<td></td>
			<td></td>
			<td>For making tracer RNA. Ref: Davidson et al., 2006.</td>
		</tr>
		<tr>
			<td>Rhodamine dextran, 10,000 M.W.</td>
			<td>Invitrogen</td>
			<td>D1817</td>
			<td>Tracer.</td>
		</tr>
		<tr>
			<td>Molecular biology grade, USP sterile purified water</td>
			<td>Corning</td>
			<td>46-000-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>7&#34; Drummond replacement tubes</td>
			<td>Drummond</td>
			<td>3-000-203-G/XL</td>
			<td>Microinjection needles.</td>
		</tr>
		<tr>
			<td>Needle puller</td>
			<td>Sutter Instruments</td>
			<td>P-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Dumont</td>
			<td>11252-30</td>
			<td>For breaking microinjection needle tip.</td>
		</tr>
		<tr>
			<td>Nanoject II</td>
			<td>Drummond</td>
			<td>3-000-204</td>
			<td>Microinjector.</td>
		</tr>
		<tr>
			<td>Mineral oil, heavy</td>
			<td>Fisher</td>
			<td>CAS 8042-47-5</td>
			<td>Oil for needles.</td>
		</tr>
		<tr>
			<td>27G monoject hypodermic needle</td>
			<td>Covidien</td>
			<td>8881200508</td>
			<td>For loading mineral oil into microinjection needle.</td>
		</tr>
		<tr>
			<td>5 mL Luer-Lok syringe</td>
			<td>BD</td>
			<td>309646</td>
			<td>For loading mineral oil into microinjection needle.</td>
		</tr>
		<tr>
			<td>60 mm x 15 mm Petri dish</td>
			<td>Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>800 micron polyester mesh</td>
			<td>Small Parts</td>
			<td>CMY-0800-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Transfer pipets</td>
			<td>Fisher</td>
			<td>13-711-7M</td>
			<td>For transferring embryos. Cut off the tip so that the embryos are easily taken up by the pipette.</td>
		</tr>
		<tr>
			<td>6-well cell culture plate</td>
			<td>Nest Biotechnology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Fisher</td>
			<td>BP308-500</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Acros Organics</td>
			<td>67-42-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Fisher</td>
			<td>BP531-500</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mm x 45 mm screw thread vial</td>
			<td>Fisher</td>
			<td>03-339-25B</td>
			<td>For fixing, staining, and storing embryos.</td>
		</tr>
		<tr>
			<td>24-well cell culture plate</td>
			<td>Nest Biotechnology</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Benzocaine</td>
			<td>Spectrum</td>
			<td>BE130</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Fisher</td>
			<td>BP2818-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Fisher</td>
			<td>A412-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline (PBS) 1X powder</td>
			<td>Fisher</td>
			<td>BP661-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumen</td>
			<td>Fisher</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher</td>
			<td>BP151-500</td>
			<td></td>
		</tr>
		<tr>
			<td>3D mini rocker, model 135</td>
			<td>Denville Scientific</td>
			<td>57281</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum, New Zealand origin</td>
			<td>Invitrogen</td>
			<td>16210064</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium azide</td>
			<td>Fisher</td>
			<td>S227I-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal 3G8 antibody</td>
			<td>European Xenopus Resource Centre</td>
			<td></td>
			<td>Primary antibody to label proximal tubules.</td>
		</tr>
		<tr>
			<td>Monoclonal 4A6 antibody</td>
			<td>European Xenopus Resource Centre</td>
			<td></td>
			<td>Primary antibody to label distal tubule and duct.</td>
		</tr>
		<tr>
			<td>anti-RFP pAb, purified IgG/rabbit</td>
			<td>MBL</td>
			<td>PM005</td>
			<td>Primary antibody to label Gap43-RFP tracer.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 goat anti-mouse IgG</td>
			<td>Life Technologies</td>
			<td>A11001</td>
			<td>Secondary antibody to label distal tubule and duct.</td>
		</tr>
		<tr>
			<td>Alexa Fluor 555 goat anti-rabbit IgG</td>
			<td>Life Technologies</td>
			<td>A21428</td>
			<td>Secondary antibody to label Gap43-RFP tracer.</td>
		</tr>
		<tr>
			<td>9 cavity spot plate</td>
			<td>Corning</td>
			<td>7220-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Benzyl benzoate</td>
			<td>Fisher</td>
			<td>105862500</td>
			<td>Optional - for clearing embryos</td>
		</tr>
		<tr>
			<td>Benzyl alcohol</td>
			<td>Fisher</td>
			<td>A396-500</td>
			<td>Optional - for clearing embryos</td>
		</tr>
	</tbody>
</table>

technique to target microinjection to the developing xenopus kidney

הכליה העוברית של Xenopus laevis (צפרדע), את כִּליַת רֵאשִׁית, מורכב נפרון יחיד, והוא יכול לשמש כמודל למחלת כליות. עוברי Xenopus הם גדולים, לפתח חיצונית, ניתן להשפיע בקלות על ידי microinjection או הליכים כירורגיים. בנוסף, מפות גורל הוקמו עבור עוברי Xenopus מוקדם. microinjection ממוקד לתוך blastomere הפרט כי בסופו של דבר יהיה להצמיח איבר או רקמה של עניין ניתן להשתמש כדי overexpress או להפיל ביטוי גנים באופן סלקטיבי בתוך האזור המוגבל הזאת, הפחתת השפעות משניות בשאר העובר המתפתח. בפרוטוקול זה, אנו מתארים כיצד לנצל מפות גורל Xenopus הוקמה כדי למקד את הכליה Xenopus פיתוח (להלן כִּליַת רֵאשִׁית), באמצעות microinjection לתוך blastomere ספציפיים של עוברים תאים 8 4 ו. הזרקה של קליעי נותבים שושלת מאפשרת אימות של המיקוד הספציפי של הזריקה.לאחר עוברים פתחו לשלב 38 - 40, כל הר immunostaining משמש כדי לחזות התפתחות pronephric, והתרומה ידי תאים ממוקדים אל כִּליַת רֵאשִׁית ניתן להעריך. באותה טכניקה ניתן להתאים למקד סוגי רקמות אחרות בנוסף כִּליַת רֵאשִׁית.

Microinjections של 4 ו-תאים 8 עוברי Xenopus עם mRNA MEM-RFP להראות רמות שונות של מיקוד אל כִּליַת רֵאשִׁית. איור 4 מראה הבמה 40 עוברים עם דפוסי ביטוי mRNA MEM-RFP הנכון. עובר הוזרקו ב blastomere הגחון השמאלי (איור 4 א), מיון עבור דפוס הביטוי ההולם של mRNA MEM-RFP. בנ...

Watch this Scientific Journal Video about Technique to Target Microinjection to the Developing Xenopus Kidney at JoVE.com

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

טכניקה לכוון Microinjection אל פיתוח<em&gt; Xenopus</em&gt; כליה

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

The overall goal of this procedure is to target microinjections to the developing xenopus kidney. This method can help address key questions in the field of developmental biology such as how the kidney forms and what genes are important for this process. The main advantage of this technique is that it maximizes the delivery of microinjected reagents to a targeted tissue.Visualization of this method is important because identification of the correct blastomere for targeted microinjection is critical. To begin this procedure both testes were isolated from a single male frog and placed in a 60 millimeter petri dish filled with 10 millileters of testes storage solution. Then store the testes at four degress Celsius.Next, eggs were collected from a female frog in a 100 millimeter petri dish. Pour off excess water from the petri dish. After that cut off a quarter of a testis in Testes Storage Solution.Transfer the piece of testis to the petri dish containing the eggs. Subsequently, cut the testis portion into small pieces. Add MMR plus Gentamycin to the petri dish to cover the eggs.Mix them by swirling the dish and then wait approximately 30 minutes for fertilization to take place at room temperature. After fertilization the pigmented side of the embryos should be facing up. Remove MMR from the petri dish using a transfer pipette and add enough de-jelly solution to cover the embryos.Afterward, swirl the dish over the next few minutes to disolve the jelly coat on the embryos. Once the embryos are closely touching each other in the center of the dish, the jelly coat has been removed. Remove the de-jelly solution with a transfer pipette.Next, wash the de-jellied embryos 3 to 5 times in MMR plus Gentamycin by carefully pouring off the MMR and filling the dish with new MMR. Do not remove all of the MMR from the dish, which may damage the embryos. After that, remove any unfertilized eggs or pieces of testis from the petri dish.To stagger their development, place half of the embryos from a single fertilization in a petri dish kept at 14 degrees Celsius, and the other half of the embryos in a petri dish kept at 18 degrees Celsius. Embryos will develop more slowly at 14 degress Celsius than at 18 degress Celsius. Allowing for multiple sets of injections from a single fertilized clutch.Prepare the injection solution containing point zero one nanogram per nanoliter Membrane-bound Red Fluorescent Protein mRNA. While the embryos are developing to the 4-cell or 8-cell stage. Keep the injection solution on ice until use.Then, load a seven inch replacement glass capillary tube into a needle puller with the top of the replacement tube aligned with the top of the needle puller case. Set the heat number two value to 800 and the pull value to 650 and press the pull button to pull the needle. After that, snip off the tip of the pulled needle with a pair of Dumont forceps.Slip the micropipette collet onto the back of the needle. Next, slip the large hole O-ring onto the back of the needle behind the collet. Now, fill the needle with mineral oil using a 27 gauge hyperdermic needle, being careful not to get air bubbles into the needle.Slip the needle onto the plunger of the microinjector, seating the needle into the large hole of the white plastic spacer installed on the plunger. Secure the needle by tightening the collet. Next, press and hold the Empty"button on the microinjector control box until there are two beeps.Afterward, pipette three microliters of injection solution onto a piece of parafilm. Then, insert the tip of the needle into the bead of injection solution on the parafilm. Press and hold the Fill"button on the microinjector control box to draw the injection solution into the needle.Prior to microinjection, it is important to understand the early cell divisions of the xenopus embryo. The single cell embryo has a darkly pigmented, animal pole and a vegetal pole which is white and yoky. In the two cell embryo the first cleavage typically occurs between the left and right sides of the embryo.These cells contribute equally to the pronephric lineage. The second cleavage divides the dorsal and ventral halves of the embryo leading to a 4-cell embryo. The dorsal cells are smaller and have less pigment than the ventral cells.If injecting into a 4-cell embryo, inject the left ventral blastomere to target the left kidney. The third cleavage bi-sects the animal and vegetal sides. Resulting in an 8-cell embryo.At this point there are four animal blastomeres and four vegetal blastomeres. To target the left kidney of an 8-cell embryo inject into the left ventral vegetal blastomere. Subsequently, fill a 60 millimeter petri dish lined with 500 micron polyester mesh with five percent fycol in MMR plus Gentamycin.Carefully pipette 20 to 30 8-cell embryos into the dish. Using a hair loop manipulate the embryos so that the blastomere to be injected is facing the needle. To target the left kidney, line up the embryos so that the left ventral vegetal blastomeres of 8-cell embryos face the needle.Inject 10 nanoliters of injection solution into the selected blastomere of each embryo in the dish. Then, transfer the injected embryos into the wells of a culture plate with five percent fycol in MMR plus Gentamycin. Incubate the injected embryos at 16 degrees Celsius for approximately one to two hours to allow the injected blastomeres to heal.After that, transfer the microinjected embryos into new wells in the culture plate filled with MMR plus Gentamycin. Incubate the embryos at 14 to 22 degress Celsius until they reach stage 38 to 40. In this procedure transfer 10 to 20 stage 38 to 40 embryos into a glass vial.Add 10 microliters of five percent benzocaine in 100 percent ethanol to the vial. Mix by inverting the vial and wait 10 minutes to anesthetize the embryos. Next, remove MMR from the vial and fill it with MEMFA.Then, place it on a three dimensional, rocking platform for one hour at room temperature. After one hour remove MEMFA from the vial and fill it with 100 percent methanol. Then, place the vial on a three dimensional, rocking platform for ten minutes at room temperature.Repeat this wash step one more time and store the embryos in 100 percent methanol overnight at minus 20 degrees Celsius. Embryos are now fixed and ready for immunostaining. This schematic shows which blastomere to inject for targeting of the left pronephros of xenopus embryos at the 8-cell stage using Membrane-bound Red Fluorescent Protein mRNA as a tracer.Tracer locatlization after injection in the left ventral vegetal blastomere is shown in red while the kidney is labeled in green. This image shows that the kidney was stained green with antibodies 3G8 and 4A6. Here is a close up view of the kidney region showing tracer loacalization and here is the merged image showing the co-localization of the tracer with the kidney.Shown here is the confocal image of the kidney of a second embryo stained with 3G8 and 4A6. This is the localization of the red tracer in the kidney and surrounding tissue and this is the merged image showing the co-localization of the tracer 3G8 and 4A6 in the kidney. Prior to performing this procedure it's important to become familiar with the early xenopus embryo.This will allow you to properly target microinjections through the established xenopus fade map. Once mastered, this technique can be adapted to target other tissues such as the neural tube or heart.

technique-to-target-microinjection-to-the-developing-xenopus-kidney

Research

JoVE Journal

Developmental Biology

Technique to Target Microinjection to the Developing Xenopus Kidney

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

ומניפולציה<em&gt; במבחנה</em&gt; ההבשלה של<em&gt; Laevis Xenopus</em&gt; ביציות, ואחריו הזרקת Intracytoplasmic זרע, לחקר התפתחות העוברית

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

באמצעות ניתוח של Confocal<em&gt; Laevis Xenopus</em&gt; לחקירת מאפננים של עירובים מורפוגן Wnt וששש

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

כמו תא גזע<em&gt; Xenopus</em&gt; Explants העובריים לחקר תכונות המוקדמות עצבי התפתחותית<em&gt; במבחנה</em&gt; ו<em&gt; In vivo</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global DNA methylation level of chondrocytes is considered to be a suitable biomarker for the loss of the chondrocyte phenotype. However, results based on different experimental methods can be inconsistent. Therefore, it is important to establish a precise, simple, and rapid method to quantify global DNA methylation levels during chondrocyte dedifferentiation.
Current genome-wide methylation analysis techniques largely rely on bisulfite genomic sequencing. Due to DNA degradation during bisulfite conversion, these methods typically require a large sample volume. Other methods used to quantify global DNA methylation levels include high-performance liquid chromatography (HPLC). However, HPLC requires complete digestion of genomic DNA. Additionally, the prohibitively high cost of HPLC instruments limits HPLC&#39;s wider application.
In this study, genomic DNA (gDNA) was extracted from human chondrocytes cultured with varying number of passages. The gDNA methylation level was detected using a methylation-specific dot blot assay. In this dot blot approach, a gDNA mixture containing the methylated DNA to be detected was spotted directly onto an N+ membrane as a dot inside a previously drawn circular template pattern. Compared with other gel electrophoresis-based blotting approaches and other complex blotting procedures, the dot blot method saves significant time. In addition, dot blots can detect overall DNA methylation level using a commercially available 5-mC antibody. We found that the DNA methylation level differed between the monolayer subcultures, and therefore could play a key role in chondrocyte dedifferentiation. The 5-mC dot blot is a reliable, simple, and rapid method to detect the general DNA methylation level to evaluate chondrocyte phenotype.

A 5-mC Dot Blot Assay Quantifying the DNA Methylation Level of Chondrocyte Dedifferentiation In Vitro

We present a method to quantify DNA methylation based on the 5-methylcytosine (5-mC) dot blot. We determined the 5-mC levels during chondrocyte dedifferentiation. This simple technique could be used to quickly determine the chondrocyte phenotype in ACI treatment.

5-mc נקודה כתם Assay לכמת את מתילציה DNA רמת Chondrocyte Dedifiationiation<em&gt; במבחנה</em

The dedifferentiation of hyaline chondrocytes into fibroblastic chondrocytes often accompanies monolayer expansion of chondrocytes in vitro. The global ...

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

גישה אבולרית מדויק עבור דוגמנות פגיעה בכליות חריפה בפיתוח דג הזברה

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Generation of Genetically Modified Mice through the Microinjection of Oocytes

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear injection of DNA expression constructs into fertilized oocytes remains the most commonly used technique to generate transgenic mice for overexpression. With the introduction of CRISPR technology for gene targeting, pronuclear injection into fertilized oocytes has been extended to the generation of both knockout and knockin mice. This work describes the preparation of DNA for injection and the generation of CRISPR guides for gene targeting, with a particular emphasis on quality control. The genotyping procedures required for the identification of potential founders are critical. Innovative genotyping strategies that take advantage of the "multiplexing" capabilities of CRISPR are presented herein. Surgical procedures are also outlined. Together, the steps of the protocol will allow for the generation of genetically modified mice and for the subsequent establishment of mouse colonies for a plethora of research fields, including immunology, neuroscience, cancer, physiology, development, and others.

The microinjection of mouse oocytes is commonly used for both classic transgenesis (i.e., the random integration of transgenes) and CRISPR-mediated gene targeting. This protocol reviews the latest developments in microinjection, with a particular emphasis on quality control and genotyping strategies.

הדור של עכברים השתנה גנטית דרך Microinjection של ביציות

The use of genetically modified mice has significantly contributed to studies on both physiological and pathological in vivo processes. The pronuclear ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

הערכה של נזק חמצוני בעכבר הראשי פני השטח העינית/תאים גזע בתגובה אולטרה סגול (UV-C) נזק

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

הזרקת דנ א לתוך eyebuds ב xenopus זריזה העוברים והדמיה של gfp המבטא האופטי axonal הביע בשלמותו, חי xenopus tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

הר שלם בתוך היברידיזציה ב-Zebrafish עוברים ושיטת היווצרות צינור ב-iPSC-ECs ללמוד את התפקיד של אנגלין בפיתוח כלי דם

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of cardiovascular disease. ADSCs derived from different adipose tissues show distinct features, and those from the PVAT have not been well characterized. In a recent study, we reported that some ADSCs in the periaortic arch adipose tissue (PAAT) descend from the neural crest cells (NCCs), a transient population of migratory cells originating from the ectoderm.
In this paper, we describe a protocol for isolating red fluorescent protein (RFP)-labeled NCCs from the PAAT of Wnt-1 Cre+/-;Rosa26RFP/+ mice and inducing their adipogenic differentiation in vitro. Briefly, the stromal vascular fraction (SVF) is enzymatically dissociated from the PAAT, and the RFP+ neural crest derived ADSCs (NCADSCs) are isolated by fluorescence activated cell sorting (FACS). The NCADSCs differentiate into both brown and white adipocytes, can be cryopreserved, and retain their adipogenic potential for ~3&#8211;5 passages. Our protocol can generate abundant ADSCs from the PVAT for modeling PVAT adipogenesis or lipogenesis in vitro. Thus, these NCADSCs can provide a valuable system for studying the molecular switches involved in PVAT differentiation.

We present a protocol for the isolation, culture, and adipogenic induction of neural crest derived adipose-derived stem cells (NCADSCs) from the periaortic adipose tissue of Wnt-1 Cre+/-;Rosa26RFP/+ mice. The NCADSCs can be an easily accessible source of ADSCs for modeling adipogenesis or lipogenesis in vitro.

בידוד, תרבות, והשראה של הרכס העצבי המקורי אדיפוז-תאי גזע שמקורם ברקמת אדיפוז

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...