Name: hydrodynamic renal pelvis injection for non-viral expression of proteins in the kidney
Uploaded: 2018-01-08T12:30:00.000+00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney at JoVE.com

פרס פיתוח הקריירה של המחלקה ותיקי לענייני [BX002797] תמך L.E.W. מכוני הבריאות הלאומיים [R01-DK095867] ונתמך American Heart Association [15GRNT25700209] ג'יי מכוני הבריאות הלאומיים [DK093660], המחלקה לענייני חיילים משוחררים [BX002190] ואנדרבילט לחקר מחלת כליות תמיכה, M.H.W. חומר זה הוא התוצאה של עבודה נתמכת עם משאבים ושימוש במתקני במערכת הבריאות VA עמק טנסי.

<ol>
	<li>Liu, F., Song, Y., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamics-based+transfection+in+animals+by+systemic+administration+of+plasmid+DNA.">Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA.</a> Gene Ther. 6 (7), 1258-1266 (1999).</li><li>Zhang, G., Budker, V., Wolff, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+levels+of+foreign+gene+expression+in+hepatocytes+after+tail+vein+injections+of+naked+plasmid+DNA.">High levels of foreign gene expression in hepatocytes after tail vein injections of naked plasmid DNA.</a> Human Gene Therapy. 10, 1735-1737 (1999).</li><li>Fumoto, S., Nishimura, K., Nishida, K., Kawakami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+Imaging+of+the+Intracellular+Fate+of+Plasmid+DNA+and+Transgene+Expression:+ZsGreen1+and+Tissue+Clearing+Method+CUBIC+Are+an+Optimal+Combination+for+Multicolor+Deep+Imaging+in+Murine+Tissues.">Three-Dimensional Imaging of the Intracellular Fate of Plasmid DNA and Transgene Expression: ZsGreen1 and Tissue Clearing Method CUBIC Are an Optimal Combination for Multicolor Deep Imaging in Murine Tissues.</a> PLoS One. 11 (1), e0148233 (2016).</li><li>Yoshino, H., Hashizume, K., Kobayashi, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naked+plasmid+DNA+transfer+to+the+porcine+liver+using+rapid+injection+with+large+volume.">Naked plasmid DNA transfer to the porcine liver using rapid injection with large volume.</a> Gene Ther. 13 (24), 1696-1702 (2006).</li><li>Kamimura, K., Suda, T., Xu, W., Zhang, G., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-guided,+lobe-specific+hydrodynamic+gene+delivery+to+swine+liver.">Image-guided, lobe-specific hydrodynamic gene delivery to swine liver.</a> Mol Ther. 17 (3), 491-499 (2009).</li><li>Kamimura, K., Zhang, G., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-guided,intravascular+hydrodynamic+gene+delivery+to+skeletal+muscle+in+pigs.">Image-guided,intravascular hydrodynamic gene delivery to skeletal muscle in pigs.</a> Mol Ther. 18 (1), 93-100 (2010).</li><li>Suda, T., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+gene+delivery:+its+principles+and+applications.">Hydrodynamic gene delivery: its principles and applications.</a> Mol.Ther. 15 (12), 2063-2069 (2007).</li><li>Alino, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naked+DNA+delivery+to+whole+pig+cardiac+tissue+by+coronary+sinus+retrograde+injection+employing+non-invasive+catheterization.">Naked DNA delivery to whole pig cardiac tissue by coronary sinus retrograde injection employing non-invasive catheterization.</a> J Gene Med. 12 (11), 920-926 (2010).</li><li>Suda, T., Gao, X., Stolz, D. B., Liu, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+impact+of+hydrodynamic+injection+on+mouse+liver.">Structural impact of hydrodynamic injection on mouse liver.</a> Gene Ther. 14 (2), 129-137 (2007).</li><li>Skrypnyk, N. I., Harris, R. C., de Caestecker, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ischemia-reperfusion+model+of+acute+kidney+injury+and+post+injury+fibrosis+in+mice.">Ischemia-reperfusion model of acute kidney injury and post injury fibrosis in mice.</a> J Vis Exp. (78), (2013).</li><li>Woodard, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kidney-specific+transposon-mediated+gene+transfer+in+vivo.">Kidney-specific transposon-mediated gene transfer in vivo.</a> Sci Rep. 7, 44904 (2017).</li><li>Liang, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+glutathione+S-transferase+A4+accelerates+obstruction-induced+tubule+damage+and+renal+fibrosis.">Loss of glutathione S-transferase A4 accelerates obstruction-induced tubule damage and renal fibrosis.</a> Journal of Pathology. 228 (4), 448-458 (2012).</li><li>Liang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protective+role+of+insulin-like+growth+factor-1+receptor+in+endothelial+cells+against+unilateral+ureteral+obstruction-induced+renal+fibrosis.">Protective role of insulin-like growth factor-1 receptor in endothelial cells against unilateral ureteral obstruction-induced renal fibrosis.</a> Am J Pathol. 185 (5), 1234-1250 (2015).</li><li>Corridon, P. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+to+facilitate+and+monitor+expression+of+exogenous+genes+in+the+rat+kidney+using+plasmid+and+viral+vectors.">A method to facilitate and monitor expression of exogenous genes in the rat kidney using plasmid and viral vectors.</a> Am J Physiol Renal Physiol. 304 (9), F1217-F1229 (2013).</li><li>Wallace, D. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+kidney+volume+in+mice+with+polycystic+kidney+disease+by+magnetic+resonance+imaging.">Tracking kidney volume in mice with polycystic kidney disease by magnetic resonance imaging.</a> Kidney Int. 73 (6), 778-781 (2008).</li><li>Woodard, L. E., Wilson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=piggyBac-ing+models+and+new+therapeutic+strategies.">piggyBac-ing models and new therapeutic strategies.</a> Trends Biotechnol. 33 (9), 525-533 (2015).</li><li>Yeikilis, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamics+based+transfection+in+normal+and+fibrotic+rats.">Hydrodynamics based transfection in normal and fibrotic rats.</a> World J Gastroenterol. 12 (38), 6149-6155 (2006).</li><li>Thalhofer, C. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+transgenic+Leishmania+parasites+in+a+live+host.">In vivo imaging of transgenic Leishmania parasites in a live host.</a> J Vis Exp. (41), (2010).</li><li>Wooddell, C. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+damage+after+delivery+of+naked+plasmid+DNA+into+skeletal+muscles+is+batch+dependent.">Muscle damage after delivery of naked plasmid DNA into skeletal muscles is batch dependent.</a> Hum Gene Ther. 22 (2), 225-235 (2011).</li><li>Crespo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrodynamic+liver+gene+transfer+mechanism+involves+transient+sinusoidal+blood+stasis+and+massive+hepatocyte+endocytic+vesicles.">Hydrodynamic liver gene transfer mechanism involves transient sinusoidal blood stasis and massive hepatocyte endocytic vesicles.</a> Gene Ther. 12 (11), 927-935 (2005).</li><li>Rocca, C. J., Ur, S. N., Harrison, F., Cherqui, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=rAAV9+combined+with+renal+vein+injection+is+optimal+for+kidney-targeted+gene+delivery:+conclusion+of+a+comparative+study.">rAAV9 combined with renal vein injection is optimal for kidney-targeted gene delivery: conclusion of a comparative study.</a> Gene Ther. 21 (6), 618-628 (2014).</li></ol>

המחברים אין לחשוף ולהצהיר אין ניגוד אינטרסים.

ב פרוטוקול זה מתוארת שיטה חזקה להשגת ביטוי גנים לשחזור באופן ספציפי בתוך הכליה. בידי מנתח מנוסה בינוני מצאנו האחוז של עכברים transfected באמצעות הטכניקה הזו להיות בטווח של 50-100%, בהתאם לגיל העכבר ואת הרגישות של המדידה של transgene. הרמה של ביטוי גנים לוציפראז היה מעל רקע במשך מספר חודשים בעכברים שקיב?...

טכניקת ההזרקה של וריד הזנב hydrodynamic הפך נפוץ דרך כדי להשיג רמות גבוהות של ביטוי גנים העכבר הכבד1,2. הכליות הן גם transfected באמצעות הטכניקה הזו ברמה יותר נמוכה, כ 100-fold פחות3. הזרקת hydrodynamic אגן הכליה המתוארים כאן מספק דרך פשוטה כדי לשלוט יחודיות של איברים ביטוי באמצעים פיזיים, באמצעות אותם עקרונות hydrodynamic שהוקמו בעבר כבד4,5 , שרירים6ו-7,8אחרים איברים. שיטה זו transfects תאים בעלי חיים ויוו באמצעות לחץ ותוקנו מהירות כדי לאלץ את ה-DNA המכיל נוזל לתוך התאים, בו זמנית גרימת נזק לאיבר זה במהירות9. באמצעות טכניקות ניתוחיות ומבוססת להמחיש את הכליה דרך החתך האגף10 יחד עם זריקה אחת על ידי מזרק אינסולין, מצאנו תקנים מוצלחת של סוגים שונים של תאי הכליה, בעיקר interstitial fibroblasts, בקוריאנית, צינור איסוף11. דיסקציה של עכברים אלה הראו כי איברים אחרים הם לא transfected ברמה גבוהה מספיק כדי להמחיש מאת לוציפראז הדמיה טכניקות11. מאז הטכניקה היא נגיפי, השימוש פלסמיד DNA עבור תרביות תאים מאפשרת הכנה קלה ומהירה של ריאגנטים הנדרש להזרקה.
השתמשנו זריקות hydrodynamic מקומי להביע גלוטתיון נוגד חמצון S-טרנספראז A4 הקולטן 1 פקטורי גדילה דמויי אינסולין, את אריתרופויאטין ההורמון בכליה, כולם בעלי השפעות ביולוגיות הצפוי11, 12 , 13. הערכה מפורטת של התוואי של המינהל, נפח הזרקה, מינון ה-DNA, יזם הבחירה היה שבוצעו11. בנוסף, שתי piggyBac transposon מערכת ו/או ציקלופוספמיד הטיפול לדכא את התגובה החיסונית transgene כבר הראו לשפר לטווח ארוך ג'ין ביטוי תוצאות11. חוקרים אחרים יש להשתמש בגישה וריד הכליה לעכבר עם הצלחה, השגת יעילות גבוהה תקנים עבור פרקי זמן של יותר מ חודש14. עם זאת, תיקון גנטי של פנוטיפים מחקה מחלות אנושיות בדרך כלל מבוצעות בעכברים לראשונה כמושג הוכחה-של-מאחר ביותר בתרבית של דגמים גנטיים הם מודלים העכבר. אנו לעומת הזרקה וריד הכליה אל אגן הכליה הזרקה, נמצא כי הזרקה לתוך האגן הכליה סופריור לוריד הכליה על ביטוי גנים (כ כוחנו גבוה) והישרדות11. אגן הכליה הוא מסלול אידיאלי של הערך לתוך הכליה כי זה גמיש מספיק לסבול תנודות ייצור השתן, והוא לעיתים קרובות מסוגל לשמור המבנית שלו גם כאשר התרחבו במהלך hydronephrosis. בנוסף, הזרקה לתוך האגן הכליה איפשרה גישה הכליה ללא פירסינג הקפסולה כליות, המאפשר את הנוזלים מוזרק בעליל יישמרו על ידי הכליות טוב יותר intraparenchymal הזרקת. איברים אחרים העכבר אין מסלול של ערך אחר מאשר להערכת, אבל המרחב השתן של הכליה הוא אתר אידיאלי הזרקה. בנוסף, זריקה לווריד כליות כתוצאה דליפה של דם לתוך חלל הבטן. עוצמת כליות מוחלטת פראי-סוג העכבר הכליות הוערך על ידי תהודה מגנטית יהיה כ 0.2 ס מ3, אז הנפח של כליה אחת שווה בקירוב כמות הנוזלים מוזרק על ידי רופא אגן הכליה hydrodynamic הזרקה (100 µL)15. במסמך זה, עשינו זמין לכל הניואנסים מפורט של פרוטוקול הזרקת hydrodynamic אגן הכליה להשגת תקנים לשחזור של הכליה.

<table><tbody><tr><td>AnaSed Xylazine</td><td>Patterson Veterinary</td><td>07-808-1947</td><td>Anesthetic - Not controlled substance</td></tr><tr><td>BD Insulin Syringe 0.5 mL 29G 1/2 Inch</td><td>Cardinal Health</td><td>309306</td><td>Required syringes</td></tr><tr><td>Buprenex</td><td>Pharmacist/Veterinarian</td><td></td><td>Analgesia - Controlled Substance</td></tr><tr><td>Dynarex Disposable Towel Drape</td><td>Thermo Fisher Scientific</td><td>19-310-671</td><td>Place over heat pad</td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>Qiagen</td><td>12362</td><td>Use only endotoxin-free plasmid DNA</td></tr><tr><td>Endosafe Gel-Clot LAL Rapid Positive Control</td><td>Charles River</td><td>PC200</td><td>Positive control for endotoxin test</td></tr><tr><td>Endosafe Gel-Clot LAL Rapid Single Test Vial</td><td>Charles River</td><td>R13500</td><td>Endotoxin test</td></tr><tr><td>Extra Fine Micro Dissecting Scissors</td><td>Roboz Surgical Instrument</td><td>RS-5882</td><td>Surgical tool</td></tr><tr><td>Fisherbrand Instant Sealing Sterilization Pouch - 9&quot;</td><td>Thermo Fisher Scientific</td><td>01-812-51</td><td>For autoclaving surgical tools</td></tr><tr><td>Gaymar Heat Pump</td><td>Paragon Medical</td><td>TP-700</td><td>Water-circulating heat pump</td></tr><tr><td>Germinator 500</td><td>Roboz Surgical Instrument</td><td>DS-401</td><td>To reuse surgical tools during surgery</td></tr><tr><td>Graefe Forceps</td><td>Roboz Surgical Instrument</td><td>RS-5136</td><td>Surgical tool</td></tr><tr><td>Graefe Tissue Forceps</td><td>Roboz Surgical Instrument</td><td>RS-5153</td><td>Surgical tool</td></tr><tr><td>Halsey Needle Holder, 5&quot; Length</td><td>Roboz Surgical Instrument</td><td>RS-7841</td><td>Surgical tool</td></tr><tr><td>Heat pads - 15&quot; x 21&quot; - need at least 3</td><td>Paragon Medical</td><td>TP22G</td><td>For use with Gaymar Heat Pump</td></tr><tr><td>IsoFlo (Isoflurane, USP)</td><td>Abbott Animal Health</td><td>5260-04-05</td><td>For imaging and euthanasia</td></tr><tr><td>Isotec Isoflurane Delivery System Vaporizor</td><td>Smiths Medical</td><td>VCT3K2</td><td>For imaging and euthanasia</td></tr><tr><td>Ketamine</td><td>Pharmacist/Veterinarian</td><td></td><td>Anesthetic - Controlled Substance</td></tr><tr><td>Kimwipes</td><td>Kimberly-Clark Professional</td><td>34120</td><td>Laboratory tissues</td></tr><tr><td>Living Image software</td><td>Caliper Life Sciences</td><td></td><td>For live animal imaging</td></tr><tr><td>Luciferin</td><td>Perkin Elmer</td><td>122796</td><td>For live animal imaging</td></tr><tr><td>Nanodrop 2000</td><td>Thermo Scientific</td><td>ND-2000-US-CAN</td><td>Spectrophotometer for DNA measurement</td></tr><tr><td>Prevantics Swabs</td><td>Thermo Fisher Scientific</td><td>23-100-110</td><td>For skin surgery prep</td></tr><tr><td>Prolene 5-0 sutures Taper 30&quot;</td><td>Thermo Fisher Scientific</td><td>NC0256891</td><td>Non-absorbable sutures for skin</td></tr><tr><td>Puralube Brand Opthalmic Ointment</td><td>Patterson Veterinary</td><td>07-888-2572</td><td>To keep eyes moist during surgery</td></tr><tr><td>Trans IT - QR Hydrodynamic Delivery Solution</td><td>Mirus Bio</td><td>MIR-5240</td><td>Hydrodynamic delivery buffer for diluting DNA</td></tr><tr><td>Vicryl 5-0 Sutures J303H</td><td>Thermo Fisher Scientific</td><td>NC9816710</td><td>Absorbable sutures for muscle layer</td></tr><tr><td>Wahl Mini Arco Clipper</td><td>Med-Vet International</td><td>8787-1550</td><td>Shaver for skin prep</td></tr><tr><td>Xenogen IVIS 200</td><td>Caliper Life Sciences</td><td></td><td>For live animal imaging</td></tr></tbody></table>

hydrodynamic renal pelvis injection for non-viral expression of proteins in the kidney

הזרקת hydrodynamic יוצר סביבה מקומיים, בלחץ גבוה כדי transfect רקמות שונות עם דנ א פלסמיד וחומרים אחרים. הזרקת וריד הזנב hydrodynamic, לדוגמה, היא שיטה ומבוססת שבאמצעותו יכול להיות transfected הכבד. כתב יד זה מתאר יישום של עקרונות hydrodynamic על ידי הזרקה של הכליה העכבר ישירות עם פלסמיד הדנ א על ביטוי גנים ספציפיים כליות. עכברים הם מרדימים, הכליה נחשף על ידי חתך האגף ולאחריה זריקה מהירה של פלסמיד פתרון ה-DNA המכיל ישירות לתוך אגן הכליה. המחט הוא שמר על המקום לעשר שניות, מקום החתך מאוחה. למחרת, לחיות הדמיה בעלי חיים, תספיג או אימונוהיסטוכימיה עשוי לשמש assay ביטוי גנים או אחרים מבחני מתאים את transgene של בחירה משמשים לגילוי של החלבון עניין. שיטות שפורסמו להאריך ביטוי גנים כוללים אינטגרציה transgene בתיווך transposon וטיפול ציקלופוספמיד כדי לעכב את התגובה החיסונית כדי transgene.

טכניקת הניתוח, הזרקת פשוטים לביצוע נשכחו, הדורשים לא הציוד העיקרי או בחומרים יקרים. אם חדש האגף-חתך ניתוח בכליה, יום הדרכה אחד על מספר עכברים מתוכננת המתת חסד אסור שבו העכברים אינם התאושש הניתוח הבא כי הניסיון הראשון-ניתוח זה עשוי לארוך זמן רב יותר מהרגיל. לחלופין, חוקרים ?...

Watch this Scientific Journal Video about Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney at JoVE.com

Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney

הזרקת hydrodynamic אגן הכליה נגיפי ביטוי של חלבונים בתוך הכליה.

פרוטוקול זה מתאר שיטה להחדיר פלסמיד DNA לתוך הכליה העכבר באמצעות אגן הכליה כדי לייצר transgene ביטוי במיוחד בתוך הכליה.

Hydrodynamic injection creates a local, high-pressure environment to transfect various tissues with plasmid DNA and other substances. Hydrodynamic tail ...

hydrodynamic-renal-pelvis-injection-for-non-viral-expression-proteins

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

18.4K Views.  Tennessee Valley Healthcare System. פרוטוקול זה מתאר שיטה להחדיר פלסמיד DNA לתוך הכליה העכבר באמצעות אגן הכליה כדי לייצר transgene ביטוי במיוחד בתוך הכליה.

Video: Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney

Technique to Target Microinjection to the Developing Xenopus Kidney

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 embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

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 ...

Developmental Biology

Intravenous Microinjections of Zebrafish Larvae to Study Acute Kidney Injury

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections on 2 dpf zebrafish. This technique represents an efficient and rapid method to deliver soluble substances into the bloodstream of zebrafish larvae, allowing for the injection of 15-20 fish per hour. In addition to AKI studies, this microinjection technique can also be used for other types of experimental studies such as angiography.  We provide a detailed protocol of the technique from equipment required to visual measures of decreased kidney function. In addition, we also demonstrate the process of fixation, whole mount immunohistochemistry with a kidney tubule marker, plastic embedding and sectioning of the larval zebrafish. We demonstrate that zebrafish larvae injected with gentamicin show morphological features consistent with AKI: edema, loss of cell polarity in proximal tubular epithelial cells, and morphological disruption of the tubule.

We describe a technique of microinjecting the aminoglycoside, gentamicin, into 2 days post-fetilization (dpf) zebrafish larvae to induce acute kidney injury (AKI). We also describe a method for whole mount immunohistochemistry, plastic embedding and sectioning of zebrafish larvae to visualize the AKI mediated damage.

Microinjections תוך ורידי של הזחלים דג הזברה לחקר פגיעה כליות חריפה

In this video article we describe a zebrafish model of AKI using gentamicin as the nephrotoxicant.  The technique consists of intravenous microinjections ...

Intravascular Delivery of Biologics to the Rat Kidney

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage Renal Disease with high risk of death for cardiovascular events. Moreover, recent clinical studies have shown that renovascular structure and function may have a great impact on functional renal recovery after surgery. Here, we describe a protocol for the delivery of drugs into the renal artery of rats. This procedure offers significant advantages over the frequently used systemic administration as it may allow a more localized therapeutic effect. In addition, the use of rodents in pharmacodynamic analysis of preclinical studies may be cost effective, paving the way for the design of translational experiments in larger animal models. Using this technique, infusion of rat recombinant Vascular Endothelial Growth Factor (VEGF) protein in rats has induced activation of VEGF signaling as shown by increased expression of FLK1, pAKT/AKT, pERK/ERK. In summary, we established a protocol for the intrarenal delivery of drugs in rats, which is simple and highly reproducible.

The administration of drugs for recovery of kidney function requires control of the localization and distribution of the therapeutic compound. Here, we describe in detail a simple technique for intrarenal delivery of drugs in rats. This procedure may be easily performed with no mortality and high reproducibility.

משלוח intravascular של מוצרים ביולוגיים לכליה עכברוש

The renal microvascular compartment plays an important role in the progression of kidney disease and hypertension, leading to the development of End Stage ...

Medicine

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal inflammation and fibrosis ultimately resulting in declining kidney function. Before improved clinical management and prevention of pyelonephritis can be instituted, a reliable animal model must be established in order to study the mechanisms of progression, recurrence, and therapeutic efficacy. The transurethral infection model closely mimics human pyelonephritis but exhibits considerable variation due to its reliance on urethral reflux to transport the bacteria to the kidney. Herein, a detailed surgical protocol for performing bacterial injections into the rat renal pelvis is provided and confirmed by non-invasive Magnetic Resonance Imaging (MRI). Using this protocol, animals receive direct exposure to a desired concentration of E. coli bacteria and can fully recover from the surgical procedure with adequate post-operative care. This facilitates subsequent longitudinal MRI assessments of the experimental animal models for comparison with saline (sham) controls. Using this direct delivery approach, the severity of infection is controllable and applicable for mechanistic studies of progression as well as development of novel treatment strategies.

Intrarenal Injection of Escherichia coli in a Rat Model of Pyelonephritis

This manuscript describes a rat surgical model of pyelonephritis using direct intra-renal infection by Escherichia coli into the renal pelvis. The experimental procedure can be utilized to study the pathogenesis of pyelonephritis as well as the associated inflammation and fibrosis.

הזרקת Intranenal של<em&gt; Escherichia coli</em&gt; במודל עכברוש של Pyelonephritis

Pyelonephritis is a bacterial infection of the kidney and is most commonly caused by Escherichia coli. Recurrent infections can cause significant renal ...

Transient Expression of Proteins by Hydrodynamic Gene Delivery in Mice

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past years, hydrodynamic gene delivery (HGD) has emerged as a simple, fast, safe and effective method for delivering transgenes into rodents. This technique relies on the force generated by the rapid injection of a large volume of physiological solution to increase the permeability of cell membranes of perfused organs and thus deliver DNA into cells. One of the main advantages of HGD is the ability to introduce transgenes into mammalian cells using naked plasmid DNA (pDNA). Introducing an exogenous gene using a plasmid is minimally laborious, highly efficient and, contrary to viral carriers, remarkably safe. HGD was initially used to deliver genes into mice, it is now used to deliver a wide range of substances, including oligonucleotides, artificial chromosomes, RNA, proteins and small molecules into mice, rats and, to a limited degree, other animals. This protocol describes HGD in mice and focuses on three key aspects of the method that are critical to performing the procedure successfully: correct insertion of the needle into the vein, the volume of injection and the speed of delivery. Examples are given to show the application of this method to the transient expression of two genes that encode secreted, primate-specific proteins, apolipoprotein L-I (APOL-I) and haptoglobin-related protein (HPR).

In vivo transfection of naked DNA by hydrodynamic gene delivery introduces genes into the tissue of an animal with minimal inflammatory response. Sufficient amounts of gene product are generated such that gene function and regulation as well as protein structure and function can be analyzed.

ביטוי חולף של חלבונים על ידי הידרודינמית ג&#39;ין משלוח בעכברים

Efficient expression of transgenes in vivo is of critical importance in studying gene function and developing treatments for diseases. Over the past ...

Direct Drug Delivery to Kidney via the Renal Artery

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research field. Accumulation of drugs in certain organs may give rise to adverse and unwanted effects, depending on the nature of injectate. To minimize spillover and/or accumulation in other tissues, the herein described method directs the formulation into the renal artery bloodstream by inserting a catheter in the infra renal aorta, just below where it branches into the renal artery, resulting in the kidney as first reached organ and distributing of formulation throughout the kidney.
This manuscript provides a detailed description of the method, as well as its challenges and difficulties. It guides the experimenter to become skillful with this type of microsurgery that requires accuracy under sterile conditions. Speed is crucial for minimizing the ischemia and practicing the procedure will increase the chance of successful injections without adverse effects. By modulating the time between injection and reperfusion as well as the injected volume, the risk of spillover to other organs is mitigated. 
Note that this technique is suitable for single dosing strategies.

This manuscript describes a method for targeted delivery to a single kidney via a catheter placed in the infrarenal abdominal aorta in the mouse.

משלוח תרופות ישיר לכליות דרך עורק הכליה

There is a need for directed injections to enable increased and specific renal exposure for efficient evaluation of drug targets in the renal research ...

Delivery of Exogenous Artificially Synthesized miRNA Mimic to the Kidney Using Polyethylenimine Nanoparticles in Several Kidney Disease Mouse Models

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by destabilizing and inhibiting their translation in various kidney diseases. Therefore, alternation of miRNA expression by exogenous artificially synthesized miRNA mimics is a potentially useful treatment option for inhibiting the development of many kidney diseases. However, because serum RNAase immediately degrades systematically administered exogenous miRNA mimics in vivo, delivery of miRNA to the kidney remains a challenge. Therefore, vectors that can protect exogenous miRNA mimics from degradation by RNAase and significantly deliver them to the kidney are necessary. Many studies have used viral vectors to deliver exogenous miRNA mimics or inhibitors to the kidney. However, viral vectors may cause an interferon response and/or genetic instability. Therefore, the development of viral vectors is also a hurdle for the clinical use of exogenous miRNA mimics or inhibitors. To overcome these concerns regarding viral vectors, we developed a nonviral vector method to deliver miRNA mimics to the kidney using tail vein injection of polyethylenimine nanoparticles (PEI-NPs), which led to significant overexpression of target miRNAs in several mouse models of kidney disease.

Here, we deliver exogenous artificially synthesized miRNA mimics to the kidney via tail vein injection of a nonviral vector and polyethylenimine nanoparticles in several kidney disease mouse models. This led to significant overexpression of target miRNA in the kidney, resulting in inhibited progression of kidney disease in several mouse models.

העברה של miRNA אקסוגני מסונתז באופן מלאכותי לחקות לכליה באמצעות ננו-חלקיקי פוליאתילנימין במספר מודלים של עכברים למחלות כליות

microRNAs&#160;(miRNAs), small noncoding RNAs (21-25 bases) that are not translated into proteins, inhibit lots of target messenger RNAs (mRNAs) by ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nephrotoxin Microinjection דג הזברה למודל אי-ספיקת כליות חריפה

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

חקר ביטוי חלבונים במהלך התפתחות עדשת עוף

Microinjection of Recombinant RCAS(A) Retrovirus into Embryonic Chicken Lens

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of similarity with the human lens. RCAS(A) is a replication-competent chicken retrovirus that infects dividing cells, which serves as a powerful tool to study the in situ expression and function of wild-type and mutant proteins during lens development by microinjection into the empty lumen of lens vesicle at early developmental stages, restricting its action to surrounding proliferating lens cells. Compared to other approaches, such as transgenic models and ex vivo cultures, the use of an RCAS(A) replication-competent avian retrovirus provides a highly effective, rapid, and customizable system to express exogenous proteins in chick embryos. Specifically, targeted gene transfer can be confined to proliferative lens fiber cells without the need for tissue-specific promoters. In this article, we will briefly overview the steps needed for recombinant retrovirus RCAS(A) preparation, provide a detailed, comprehensive overview of the microinjection procedure, and provide sample results of the technique.

מחבר זרקור: חקירת התפתחות ותפקוד עדשה באמצעות מיקרו-הזרקה של רטרו-וירוס RCAS(A) בעדשת עוף עוברית 

This protocol paper describes the methodology of embryonic chicken lens microinjection of an RCAS(A) retrovirus as a tool for studying in situ function and expression of proteins during lens development.

מיקרו-הזרקה של רטרו-וירוס רקומביננטי RCAS(A) לעדשת עוף עוברית

Embryonic chicken (Gallus domesticus) is a well-established animal model for the study of lens development and physiology, given its high degree of ...

הזרקת hydrodynamic אגן הכליה נגיפי ביטוי של חלבונים בתוך הכליה.

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Chapters in this video

טכניקה לכוון Microinjection אל פיתוח

Microinjections תוך ורידי של הזחלים דג הזברה לחקר פגיעה כליות חריפה

משלוח intravascular של מוצרים ביולוגיים לכליה עכברוש

הזרקת Intranenal של

ביטוי חולף של חלבונים על ידי הידרודינמית ג'ין משלוח בעכברים

משלוח תרופות ישיר לכליות דרך עורק הכליה

העברה של miRNA אקסוגני מסונתז באופן מלאכותי לחקות לכליה באמצעות ננו-חלקיקי פוליאתילנימין במספר מודלים של עכברים למחלות כליות

Nephrotoxin Microinjection דג הזברה למודל אי-ספיקת כליות חריפה

מיקרו-הזרקה של רטרו-וירוס רקומביננטי RCAS(A) לעדשת עוף עוברית

מיקרו-הזרקה של רטרו-וירוס רקומביננטי RCAS(A) לעדשת עוף עוברית