ברצוננו להודות לוגו Bellen ומרכז מאגר בלומינגטון לחומרים כימיים. בנוסף, אנו מודים כהן Venken, הוגו Bellen וכל חברי Buszczak וHiesinger המעבדות לדיונים מועילים. עבודה זו נתמכה על ידי מענקים מהמכון הלאומי לבריאות לACR (T32GM083831), PRH (RO1EY018884) ומגה (RO1GM086647), מענק על ידי מניעת סרטן מכון המחקר של טקסס למגה וPRH (RP100516), וולץ&#39; קרן (I-1657) לPRH. מגה הוא א &#39;א&#39; וגריר גרסון Fogelson Scholar במחקר ביו וPRH הוא חוקר יוג&#39;ין מק &#39;דרמוט במחקר ביו במרכז רפואי UT Southwestern.

<ol>
	<li>Rong, Y. S., Golic, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+targeted+gene+knockout+in+Drosophila.">A targeted gene knockout in Drosophila.</a> Genetics. 157, 1307-1312 (2001).</li><li>Rong, Y. S., Golic, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+targeting+by+homologous+recombination+in+Drosophila.">Gene targeting by homologous recombination in Drosophila.</a> Science. 288, 2013-2018 (2000).</li><li>Gong, W. J., Golic, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ends-out,+or+replacement,+gene+targeting+in+Drosophila.">Ends-out, or replacement, gene targeting in Drosophila.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 2556-2561 (1073).</li><li>Warming, S., Costantino, N., Court, D. L., Jenkins, N. A., Copeland, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+and+highly+efficient+BAC+recombineering+using+galK+selection.">Simple and highly efficient BAC recombineering using galK selection.</a> Nucleic Acids Res. 33, e36 (2005).</li><li>Copeland, N. G., Jenkins, N. A., Court, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombineering:+a+powerful+new+tool+for+mouse+functional+genomics.">Recombineering: a powerful new tool for mouse functional genomics.</a> Nat. Rev. Genet. 2, 769-779 (2001).</li><li>Venken, K. J., He, Y., Hoskins, R. A., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P[acman]:+a+BAC+transgenic+platform+for+targeted+insertion+of+large+DNA+fragments+in+D.+melanogaster.">P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster.</a> Science. 314, 1747-1751 (2006).</li><li>Venken, K. 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=Versatile+P[acman]+BAC+libraries+for+transgenesis+studies+in+Drosophila+melanogaster.">Versatile P[acman] BAC libraries for transgenesis studies in Drosophila melanogaster.</a> Nat. Methods. 6, 431-434 (2009).</li><li>Groth, A. C., Fish, M., Nusse, R., Calos, 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=Construction+of+transgenic+Drosophila+by+using+the+site-specific+integrase+from+phage+phiC31.">Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31.</a> Genetics. 166, 1775-1782 (2004).</li><li>Bischof, J., Maeda, R. K., Hediger, M., Karch, F., Basler, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+transgenesis+system+for+Drosophila+using+germ-line-specific+phiC31+integrases.">An optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 3312-3317 (2007).</li><li>Chan, C. C., 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=Systematic+discovery+of+Rab+GTPases+with+synaptic+functions+in+Drosophila.">Systematic discovery of Rab GTPases with synaptic functions in Drosophila.</a> Current Biology: CB. 21, 1704-1715 (2011).</li><li>Chan, C. C., Scoggin, S., Hiesinger, P. R., Buszczak, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combining+recombineering+and+ends-out+homologous+recombination+to+systematically+characterize+Drosophila+gene+families:+Rab+GTPases+as+a+case+study.">Combining recombineering and ends-out homologous recombination to systematically characterize Drosophila gene families: Rab GTPases as a case study.</a> Commun. Integr. Biol. 5, 179-183 (2012).</li><li>Wu, N., Matand, K., Kebede, B., Acquaah, G., Williams, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhancing+DNA+electrotransformation+efficiency+in+Escherichia+coli+DH10B+electrocompetent+cells.">Enhancing DNA electrotransformation efficiency in Escherichia coli DH10B electrocompetent cells.</a> Electronic Journal of Biotechnology. 13, (2010).</li><li>Wang, S., Zhao, Y., Leiby, M., Zhu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+positive/negative+selection+scheme+for+precise+BAC+recombineering.">A new positive/negative selection scheme for precise BAC recombineering.</a> Mol. Biotechnol. 42, 110-116 (2009).</li><li>Venken, K. J., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenesis+upgrades+for+Drosophila+melanogaster.">Transgenesis upgrades for Drosophila melanogaster.</a> Development. 134, 3571-3584 (2007).</li><li>Dietzl, G., 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+genome-wide+transgenic+RNAi+library+for+conditional+gene+inactivation+in+Drosophila.">A genome-wide transgenic RNAi library for conditional gene inactivation in Drosophila.</a> Nature. 448, 151-156 (2007).</li></ol>

אין לי המחברים כל אינטרסים מתחרים בכל הקשור לטכניקות שתוארו כאן.

כוחו של אורגניזמים מודל גנטיים במחקר ביו מבוסס במידה רבה על הכלים הזמינים למניפולציה גנטית. הדגמים הקטנים ג elegans ותסיסנית בפרט יאפשר לניתוח גנטי מולקולרי זול ומהיר של מסלולים שלמים ומשפחות הגן מעורבים בהתפתחות או תפקוד רב תאיים. בשנים אחרונות חלו התקדמות משמעו?...

נקה מניפולציות מולקולריות מוגדרות של גנים בודדים בלוקוסים אנדוגניים מציעים כלי רב ערך כדי ללמוד מספר עצום של שאלות רלוונטיות לביולוגיה אוקריוטים. תסיסנית להפוך טכניקות גנטיות ליצירת אללים אובדן של פונקציה שהוכיחה להיות מאתגרים עד Golic ועמיתיו הציגו בשנת מיקוד גן vivo באמצעות הומולוגי רקומבינציה לדרוזופילה 1-3. הם הוכיחו כי לוקוסים הגנומי ספציפיים יכולים להיות ממוקדים באמצעות שבר ליניארי של DNA ממבנה מהונדס משולב. דנ&quot;א זה ליניארי &quot;תורם&quot; נוצר in vivo באמצעות רקומבינציה FRT בתיווך (לבלו את ה-DNA מכרומוזום כמולקולה מעגלית) ואחריו ינאריזציה עם meganuclease I-SCEI. למרות שמתודולוגיה זו שימש בהצלחה ליצירת מגוון רחב של נגעים מוגדרים, הטכניקה לא הייתה להרחבה בקלות למניפולציה של גנים רבים במקביל כי כל indiviדורש מבנה נוקאאוט כפול עיצוב ייחודי ומותאם אישית. לדוגמה, קשיים בצורה חלקה מניפולציה שברים גדולים של ה-DNA (&gt; 5 קילו) במבחנה באמצעות אנזים הגבלה קלסית / קשירה או שיבוט PCR, כמו גם את מגבלות הגודל של מסורתי בוקטורי שינוי vivo לעתים קרובות מפריעים ליצירה המהירה של הומולוגי רקומבינציה מיקוד וקטורים. כדי להתגבר על המגבלות האלה, אנחנו בשילוב P [acman] מערכת recombineering / transgenesis, המאפשר תת השיבוט וtransgenesis של עד 100 קילו של ה-DNA, עם קצוות החוצה גן מיקוד המתודולוגיה להקים פלטפורמה יעילה ומהירה יחסית כי הגן מאפשר תסיסנית מיקוד. רקומבינציה בתיווך הנדסה גנטית (recombineering) היא הומולוגי רקומבינציה מבוססת טכנולוגיית שיבוט עוצמה 4,5. בניגוד לשיבוט אנזים / האנזים הגבלה קונבנציונלי, recombineering אינו מוגבל על ידי הרצף או sizדואר של ה-DNA מניפולציות. Recombineering משתמש א &#39;מיוחד זן חיידק שמטפח מכונות רקומבינציה מסופקות על ידי λ פגום prophage 4. טכניקה זו אומצה לאחרונה לשימוש בדרוזופילה 6,7. Recombineering בדרוזופילה מסתמך על כרומוזום שונה מותנה amplifiable קטריאלי מלאכותי (BAC) וקטור נקרא P [acman] 6,7. וקטור זה נושא שני מקורותיה של שכפול: OriV, אשר מייצר מספר גבוה עותק על זירוז כימי לטיהור כמויות גדולות של DNA הנדרשות לריצוף והזרקת עובר וOris, אשר שומר על מספר נמוך עותק בתנאים בסיסיים. בנוסף, P [acman] הווקטור מצויד באתר מצורף חיידקים (attB). אתר attB משמש כמצע לΦC31 transgenesis integrase בתיווך המאפשר שילוב של קטעי דנ&quot;א גדולים לתוך אתר נחיתה קבוע מראש בתוך הגנום תסיסנית 8,9. יש לנו שנוצרו P [acman] וקטור (המכונה P [acman]-KO 1.0) שיכול לשמש כוקטור למיקוד מסתיים החוצה הומולוגי רקומבינציה 10,11. לשלב קצוות החוצה גן מיקוד טכנולוגיה למערכת, הוספנו שניים FRT ושני אתרי I-SCEI. יש לנו גם כללו קלטת שער בתוך וקטור שונה זה כדי לייעל את התהליך של שילוב זרועות ההומולוגיה לP [acman]-KO 1.0. זה מספק דרך מהירה ופשוטה כדי להציג כמעט כל אזור גנומי עניין לתוך וקטור המיקוד. בפרוטוקול זה נתאר כיצד להנדס וקטור באמצעות מיקוד P [acman]-KO 1.0, ואיך לגייס את הווקטור הזה in vivo למקד את לוקוס אנדוגני. לצורך הפרוטוקול זה אנו נשתמש בקלטת RFP / קאן להחליף גן של עניין, אלא מגוון רחב של קלטות המכילות סמן בחירת אנטיביוטיקה ניתן להשתמש בפרוטוקול זה. יש לנו תוכננו ושימש בהצלחה סדרה של קלטות עבורהחלפת גן ותיוג 10,11.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>SW102 Recombination competent bacteria</td>
			<td>NCI-Frederick</td>
			<td>Recombination Bacteria (SW102, SW105 and SW106)</td>
			<td><a href="http://ncifrederick.cancer.gov/research/brb/logon.aspx" target="_blank">http://ncifrederick.cancer.gov/research/brb/logon.aspx</a></td>
		</tr>
		<tr>
			<td>TransforMax EPI300 electrocopmpetent E. coli</td>
			<td>Epicentre</td>
			<td>EC300110</td>
			<td>Includes CopyControl induction solution</td>
		</tr>
		<tr>
			<td>PfuUltra II Fusion HS DNA Polymerase</td>
			<td>Aligent Technology Inc.</td>
			<td>600670</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>BamHI-HF</td>
			<td>New England Biolabs</td>
			<td>R3136S</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Zymoclean Gel DNA Recovery Kit</td>
			<td>Zymo Research</td>
			<td>D4001</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Use Gateway BP ClonaseII Enzyme kit</td>
			<td>Invitrogen</td>
			<td>11789-020</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>P[acman]KO1.010</td>
			<td>Buszczak and Hiesinger Labs</td>
			<td>Upon request</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>pENTR RFP-Kan11</td>
			<td>Buszczak and Hiesinger Labs</td>
			<td>Upon request</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Flystocks</td>
			<td>Bloomington stock center</td>
			<td>Stock numbers: 25680, 25679</td>
			<td>y1 w*/Dp(2;Y)G, P{hs-hid}Y; P{70FLP}11 P{70I-SceI}2B snaSco/CyO, P{hs-hid}4 
			y1 w*/Dp(2;Y)G, P{hs-hid}Y; P{70FLP}23 P{70I-SceI}4A/TM3, P{hs-hid}14, Sb1</td>
		</tr>
		<tr>
			<td>Electroporation machine</td>
			<td>Biorad GenePulser Xcell with PC module</td>
			<td>165-2662</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cuvettes</td>
			<td>Fisher Brand</td>
			<td>#FB101</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

recombineering homologous recombination constructs in drosophila

המשך הפיתוח של טכניקות למניפולציה מהירה, בקנה מידה גדולה של לוקוסי גנים אנדוגניים יהיה להרחיב את השימוש בתסיסנית כאורגניזם מודל גנטי למחקר הקשור לאדם מחלה. בשנים אחרונות חלו התקדמות טכנולוגית כמו הומולוגי רקומבינציה וrecombineering. עם זאת, יצירת מוטציות null חד משמעיות או חלבונים אנדוגניים תיוג נשארה מאמץ משמעותי עבור רוב הגנים. כאן, אנו מתארים ומדגימים טכניקות לשימוש בשיטות שיבוט מבוססות recombineering לייצר וקטורים שניתן להשתמש בם כדי למקד ולטפל לוקוסים אנדוגניים in vivo באופן ספציפי, הקמנו שילוב של שלוש טכנולוגיות:. (1) transgenesis BAC / recombineering, ( 2) (3) טכנולוגית Gateway הומולוגית רקומבינציה קצוות החוצה וכדי לספק שיטה יציבה, יעילה וגמישה למניפולצית לוקוסים הגנומי אנדוגניים. בפרוטוקול זה, אנו מספקים פרטי צעד אחר צעד על איך (1) העיצוב individuaוקטורי l, (2) איך לשכפל ברים גדולים של הדנ&quot;א הגנומי לתוך וקטור רקומבינציה הומולוגית באמצעות תיקון פער, ו (3) איך להחליף או לתייג את הגנים של עניין בתוך וקטורים אלה באמצעות סיבוב שני של recombineering. לבסוף, אנו גם מספקים פרוטוקול לאיך לגייס את הקלטות הללו in vivo כדי לייצר פצצה, או גן מתויג באמצעות דפיקה ב. בקלות ניתן לאמץ שיטות אלה למטרות מרובות במקביל ולספק אמצעי למניפולציה של הגנום דרוזופילה במועד ויעיל.

הגברה של לוס אנג&#39;לס וזרועות הומולוגיה RA צריכה לייצר 500 מוצרי BP ותגובת PCR-SOE אמורה להניב 1.0 קילו מוצר (סעיפים 2.1-2.4; איור 2). תגובת BP ביצע בסעיף 2.5 היא בדרך כלל שינוי מאוד יעיל וחיידקים של תשואות מוצר 5-100 מושבות בממוצע. כמעט כל המושבות שנבדקו עם PCR לבדוק להראות את המ...

Watch this Scientific Journal Video about Recombineering Homologous Recombination Constructs in Drosophila at JoVE.com

Recombineering Homologous Recombination Constructs in Drosophila

הומולוגי רקומבינציה טכניקות מאוד לקדם<em&gt; דרוזופילה</em&gt; גנטיקה כך שהיא מאפשרת יצירת מוטציות מולקולריות מדויקות. האימוץ האחרון של recombineering מאפשר לתפעל את החתיכות גדולות של ה-DNA ולהפוך אותם<em&gt; דרוזופילה</em<sup&gt; 6</sup&gt;. השיטות שהוצגו כאן משלבים טכניקות אלה כדי ליצור במהירות הומולוגי רקומבינציה וקטורים גדולים.

Recombineering הומולוגי רקומבינציה בבונה<em&gt; דרוזופילה</em

The continued development of techniques for fast, large-scale manipulation of endogenous gene loci will broaden the use of Drosophila melanogaster as a ...

recombineering-homologous-recombination-constructs-in-drosophila

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JoVE Journal

Biology

Recombineering Homologous Recombination Constructs in Drosophila

19.1K Views.  University of Texas Southwestern Medical Center at Dallas. הומולוגי רקומבינציה טכניקות מאוד לקדם דרוזופילה גנטיקה כך שהיא מאפשרת יצירת מוטציות מולקולריות מדויקות. האימוץ האחרון של recombineering מאפשר לתפעל את החתיכות גדולות של ה-DNA ולהפוך אותם דרוזופילה

Video: Recombineering Homologous Recombination Constructs in Drosophila

Wolbachia Bacterial Infection in Drosophila

Wolbachia זיהום חיידקי ב תסיסנית

Fabrication of Myogenic Engineered Tissue Constructs

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to the restrictions imposed by a child's small size and their inevitable growth. Consequently, there is a genuine need for innovative therapies designed specifically for pediatric patients with cardiac rhythm disorders. We propose that a conductive biological alternative consisting of a collagen-based matrix containing autologously-derived cells could better adapt to growth, reduce the need for recurrent surgeries, and greatly improve the quality of life for these patients. In the present study, we describe a procedure for incorporating primary skeletal myoblast cell cultures within a hydrogel matrix to fashion a surgically-implantable tissue construct that will serve as an electrical conduit between the upper and lower chambers of the heart. Ultimately, we anticipate using this type of engineered tissue to restore atrioventricular electrical conduction in children with complete heart block. In view of that, we isolate myoblasts from the skeletal muscles of neonatal Lewis rats and plate them onto laminin-coated tissue culture dishes using a modified version of established protocols[2, 3]. After one to two days, cultured cells are collected and mixed with antibiotics, type 1 collagen, Matrigel&trade;, and NaHCO3. The result is a viscous, uniform solution that can be cast into a mold of nearly any shape and size[1, 4, 5]. For our tissue constructs, we employ type 1 collagen isolated from fetal lamb skin using standard procedures[6]. Once the tissue has solidified at 37&deg;C, culture media is carefully added to the plate until the construct is submerged. The engineered tissue is then allowed to further condense through dehydration for 2 more days, at which point it is ready for in vitro assessment or surgical-implantation.

Here, we demonstrate fabrication of collagen-based, tissue constructs containing skeletal myoblasts. These 3-D engineered constructs may be used to replace or repair tissues in vivo. For our purposes, we have designed these as an atrioventricular electrical conduit for the repair of complete heart block[1].

המצאה של Myogenic בונה רקמות מהונדסות

Despite the fact that electronic pacemakers are life-saving medical devices, their long-term performance in pediatric patients can be problematic owing to ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

הייצור של ג elegans Transgenes דרך Recombineering עם GalK לבחירה מרקר

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

זיהוי השפעות של מוטציות BRCA1 על רקומבינציה הומולוגיים באמצעות תאים אקספרס BRCA1 אנדוגני wild-type

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

צורה הדמיה תא שינוי חיים תסיסנית עוברים

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

שינוי ורקומבינציה גנטי של תרבויות איברי בלוטות רוק

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

חי הדמיה של חיסול תאים אפופטוטיים במהלך<em&gt; דרוזופילה</em&gt; עובר

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Preparation of the Mgm101 Recombination Protein by MBP-based Tagging Strategy

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the Mgm101 protein is involved in the recombinational repair of mitochondrial DNA. Recent investigations have indicated that Mgm101 is related to the Rad52-type recombination protein family. These proteins form large oligomeric rings and promote the annealing of homologous single stranded DNA molecules. However, the characterization of Mgm101 has been hindered by the difficulty in producing the recombinant protein. Here, a reliable procedure for the preparation of recombinant Mgm101 is described. Maltose Binding Protein (MBP)-tagged Mgm101 is first expressed in Escherichia coli. The fusion protein is initially purified by amylose affinity chromatography. After being released by proteolytic cleavage, Mgm101 is separated from MBP by cationic exchange chromatography. Monodispersed Mgm101 is then obtained by size exclusion chromatography. A yield of ~0.87 mg of Mgm101 per liter of bacterial culture can be routinely obtained. The recombinant Mgm101 has minimal contamination of DNA. The prepared samples are successfully used for biochemical, structural and single particle image analyses of Mgm101. This protocol may also be used for the preparation of other large oligomeric DNA-binding proteins that may be misfolded and toxic to bacterial cells.

The yeast mitochondrial nucleoid protein, Mgm101, is a Rad52-type recombination protein that forms large oligomeric rings. A protocol is described to prepare soluble recombinant Mgm101 using the Maltose Binding Protein (MBP)-tagging strategy coupled with cation exchange and size exclusion chromatography.

הכנה של חלבון רקומבינציה Mgm101 ידי אסטרטגיה תיוג מבוססת MBP

The MGM101 gene was identified 20 years ago for its role in the maintenance of mitochondrial DNA. Studies from several groups have suggested that the ...

Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic mouse models using this method necessitates the construction of a &#8216;targeting&#8217; vector, which contains homologous DNA sequences to the target gene, and has for many years been a limiting step in the process. Vector construction can be performed in vivo in Escherichia coli cells using homologous recombination mediated by phage recombinases using a technique termed recombineering. Recombineering is the preferred technique to subclone the long homology sequences (>4kb) and various targeting elements including selection markers that are required to mediate efficient allelic exchange between a targeting vector and its cognate genomic locus. Typical recombineering protocols follow an iterative scheme of step-wise integration of the targeting elements and require intermediate purification and transformation steps. Here, we present a novel recombineering methodology of vector assembly using a multiplex approach. Plasmid gap repair is performed by the simultaneous capture of genomic sequence from mouse Bacterial Artificial Chromosome libraries and the insertion of dual bacterial and mammalian selection markers. This subcloning plus insertion method is highly efficient and yields a majority of correct recombinants. We present data for the construction of different types of conditional gene knockout, or knock-in, vectors and BAC reporter vectors that have been constructed using this method. SPI vector construction greatly extends the repertoire of the recombineering toolbox and provides a simple, rapid and cost-effective method of constructing these highly complex vectors.

Subcloning Plus Insertion SPI - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors

Gene targeting methodologies can be used to generate transgenic mice with knockout, knock-in and tagged alleles. Here, we describe an improved method of recombineering in E. coli, that we term &#8216;subcloning plus insertion&#8217;, which can be used to generate custom gene targeting vectors rapidly.

Subcloning Plus ההכנסה (SPI) - Recombineering שיטה חדשה לבנייה המהירה של וקטורי גן המיקוד

Gene targeting refers to the precise modification of a genetic locus using homologous recombination. The generation of novel cell lines and transgenic ...

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

RNA-mediated knockdowns are widely used to control gene expression. This versatile family of techniques makes use of short RNA (sRNA) that can be synthesized with any sequence and designed to complement any gene targeted for silencing. Because sRNA constructs can be introduced to many cell types directly or using a variety of vectors, gene expression can be repressed in living cells without laborious genetic modification. The most common RNA knockdown technology, RNA interference (RNAi), makes use of the endogenous RNA-induced silencing complex (RISC) to mediate sequence recognition and cleavage of the target mRNA. Applications of this technique are therefore limited to RISC-expressing organisms, primarily eukaryotes. Recently, a new generation of RNA biotechnologists have developed alternative mechanisms for controlling gene expression through RNA, and so made possible RNA-mediated gene knockdowns in bacteria. Here we describe a method for silencing gene expression in E. coli that functionally resembles RNAi. In this system a synthetic phagemid is designed to express sRNA, which may designed to target any sequence. The expression construct is delivered to a population of E. coli cells with non-lytic M13 phage, after which it is able to stably replicate as a plasmid. Antisense recognition and silencing of the target mRNA is mediated by the Hfq protein, endogenous to E. coli. This protocol includes methods for designing the antisense sRNA, constructing the phagemid vector, packaging the phagemid into M13 bacteriophage, preparing a live cell population for infection, and performing the infection itself. The fluorescent protein mKate2 and the antibiotic resistance gene chloramphenicol acetyltransferase (CAT) are targeted to generate representative data and to quantify knockdown effectiveness.

Phage-mediated Delivery of Targeted sRNA Constructs to Knock Down Gene Expression in E. coli

We describe a method to knock down gene expression in a growing population of E. coli cells using sequence-targeted sRNA expression cassettes delivered by an M13 phagemid vector.