LV נתמך על ידי ה-NSF (IOS 1002837)

1. ביצוע Protoplasts <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> הוסף 9 מ&quot;ל של 8% מניטול כדי בצלחת פטרי. </li><li style=";text-align:right;direction:rtl"> בעזרת מרית, את האזוב בן 7 יום 2-3 PPNH4 צלחות של 5 - ב mannitol את צלחת פטרי המכילה. </li><li style=";text-align:right;direction:rtl"> הוסף 3 מ&quot;ל של Driselase 2% (Sigma D9515-25 גרם) כדי בצלחת פטרי. </li><li style=";text-align:right;direction:rtl"> דגירה צלחת פטרי בטמפרטורת החדר עם עדין רועדת במשך שעה 1. </li><li style=";text-align:right;direction:rtl"> סנן את ההשעיה protoplast באמצעות רשת של 100 מיקרומטר (BD פלקון 352,350). </li><li style=";text-align:right;direction:rtl"> ספין ההשעיה מסוננים על 250 גרם במשך 5 דקות. הסר את supernatant. </li><li style=";text-align:right;direction:rtl"> Re-להשעות את protoplasts בעדינות עם μL 500 mannitol של 8%. </li><li style=";text-align:right;direction:rtl"> הוסף 9.5 מ&quot;ל mannitol 8% בצינור תרבות. ודא protoplasts תלויים לגמרי. </li><li style=";text-align:right;direction:rtl"> חזור על סינון מחדש ההשעיה צעדים (1.6, 1.7 ו -1.8) פעמיים נוספות. </li><li style=";text-align:right;direction:rtl"> קח 10 μL הפתרון protoplast ולספור את protoplasts ב hemocytometer. </li><li style=";text-align:right;direction:rtl"> הכפל את מספר protoplast באזור 16 מרובע על ידי 10,000 להשיג את מספר protoplasts לכל מ&quot;ל (ראו תרשים 1). </li></ol> 2. טרנספורמציה <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ספין ההשעיה protoplast ב 250 גר &#39;במשך 5 דקות. הסר את supernatant. </li><li style=";text-align:right;direction:rtl"> Re-להשעות protoplasts בפתרון MMg בריכוז של 1.6 מיליון protoplasts / mL. </li><li style=";text-align:right;direction:rtl"> לדגור על השעיית protoplast בטמפרטורת החדר למשך 20 דקות. </li><li style=";text-align:right;direction:rtl"> הוספת 600 μL ההשעיה protoplast לתוך צינור תרבות המכיל 60 מיקרוגרם DNA. מערבולת הצינור בעדינות. </li><li style=";text-align:right;direction:rtl"> הוספת 700 μL של פתרון PEG / Ca לתוך תערובת protoplast / הדנ&quot;א. מערבולת הצינור בעדינות עד לקבלת תערובת הומוגנית כולה. </li><li style=";text-align:right;direction:rtl"> דגירה את התערובת בטמפרטורת החדר למשך 30 דקות. </li><li style=";text-align:right;direction:rtl"> במהלך תקופה זו המתנה, לכסות פ.ר.מ.-B צלחות עם צלופן (80 עיגולים בקוטר מ&quot;מ, א.א. אריזות בע&quot;מ, בריטניה); לאפשר צלופן כדי מימה על פני צלחת דקות לפחות 5. </li><li style=";text-align:right;direction:rtl"> בעזרת מרית, להסיר בועות אוויר שנלכדו בין צלופן ואת צלחת. </li><li style=";text-align:right;direction:rtl"> לדלל את התערובת עם 3 מ&quot;ל של תמיסת W5. </li><li style=";text-align:right;direction:rtl"> ספין את התערובת על 250 גרם במשך 5 דקות. הסר את supernatant. </li><li style=";text-align:right;direction:rtl"> Re-להשעות protoplasts ב 2 מ&quot;ל נמס של פ.ר.מ.-T (לפני הוספתו protoplasts הפתרון הזה אפשר לשמור נוזלי 42 ° C). צלחת של 1 מ&quot;ל מחדש להשעות protoplasts לכל צלחת פ.ר.מ.-B מכוסה בצלופן. לעטוף את הצלחות עם micropore (3M, בריאות). שמור על צלחות של 25 מעלות צלזיוס בתא הגידול. </li><li style=";text-align:right;direction:rtl"> לחלופין, ניתן protoplasts מחדש תלויה מ&quot;ל 1 של המדיום ציפוי נוזלי מ&quot;ל מצופה 0.5 בצלחת פ.ר.מ.-B מכוסה בצלופן. זה מאפשר התחדשות מהירה, אך מספר צמחים התחדשות נמוך. </li><li style=";text-align:right;direction:rtl"> תא צמיחה מוגדר עבור 16 שעות. אור 8 שעות. מחזור כהה. </li><li style=";text-align:right;direction:rtl"> הזז את הצלופן על הצלחת מבחר טרי 4 ימים לאחר השינוי. </li></ol> 3. נציג תוצאות: דוגמה לשינוי מוצג באיור 2. ה-DNA נעשה שימוש בדוגמה זו היתה supercoiled 7.8 kb פלסמיד שנשא קלטת עמידים hygromycin. כמות protoplasts מצופה הופחת ל -50% עבור הצילום. התמונות צולמו כשבועיים לאחר הצמחים הועברו צלחות הבחירה. יעילות השינוי אינו מושפע ריכוז ה-DNA של עד 60 מיקרוגרם. ריכוזים גבוהים יותר של ה-DNA משפיעים לרעה על יעילות הטרנספורמציה. הנתונים הראו כי שיטה זו מספקת תוצאה עקבית לאורך טווח רחב של כמות ה-DNA. שיטה זו יכולה גם ליצור transformants יציב ביעילות כפי שמוצג בטבלה 1, אנו יכולים לקבל על 4 transformants יציב לכל מיקרוגרם של ה-DNA לינארית, אשר הוא הרבה יותר גבוה ממה שדווח קודם לכן 14. כמו כן, אנו נבחן את ההשפעה של הלם החום על יעילות הטרנספורמציה ידי הפיכת פלסמיד supercoiled נושאת גן חלבון פלואורסצנטי כסמן. הלם החום בוצע 10 דקות לאחר הדגרה בטמפרטורת החדר (בתוך שלב 2.6) על ידי דוגרים את התערובת באמבט מים C ° 45 במשך 3 דקות. התערובת הודגר בתוך אמבט מים בטמפרטורת החדר מיד לאחר הלם החום 17 דקות. תוצאות נרשמו 22 שעות לאחר השינוי. יחס transformants שהושגו עם או בלי הלם החום היו דומים מאוד (~ 25%), אך ראינו השפעה שלילית של הלם החום על הכדאיות של protoplasts (מידע לא מוצג). <img src="/files/ftp_upload/2560/2560fig1.jpg" alt="איור 1" /> באיור 1. מבט protoplasts ב hemocytometer. החלק הדרוש ספירת protoplast הוא הצביע על ידי ריבוע אדום. הערה 16 ​​ריבועים נדרש לספור protoplasts (ראה 1.11); התמונה מראה ספירה של 24 protoplasts (240,000 תאים / מ&quot;ל). <img src="/files/ftp_upload/2560/2560fig2.jpg" alt="איור 2" /> איור 2. תמונות של 18 יום transformants בן חלוף. Protoplasts היו טרנסנוצר עם כמות המצוין של פלסמיד supercoiled. ת: 15μg; B: 30μg: C: 60μg; D: 120μg. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> כמות ה-DNA (מיקרוגרם) </td><td> יעילות (tranformants / DNA מיקרוגרם) </td></tr><tr><td> 15, supercoiled פלסמיד </td><td> 120 ± 24 </td></tr><tr><td> 30, supercoiled פלסמיד </td><td> 145 ± 54 </td></tr><tr><td> 60, supercoiled פלסמיד </td><td> 121 ± 60 </td></tr><tr><td> 120, supercoiled פלסמיד </td><td> 71 ± 38 </td></tr><tr><td> 60, לינארית פלסמיד </td><td> 4 ± 1 </td></tr></tbody></table> 1. טבלת טרנספורמציה יעילות בסכומים DNA שונים.

<ol>
	<li>Abel, S., Theologis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+transformation+of+Arabidopsis+leaf+protoplasts:+a+versatile+experimental+system+to+study+gene+expression.">Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression.</a> Plant J. 5, 421-427 (1994).</li><li>Wood, A. J., Oliver, M. J., Cove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bryophytes+as+model+systems.">Bryophytes as model systems.</a> Bryologist. 103, 128-133 (2000).</li><li>Schaefer, D. G., Zr&#255;d, J. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+targeting+in+the+moss+Physcomitrella+patens.">Efficient gene targeting in the moss Physcomitrella patens.</a> Plant J. 11, 1195-1206 (1997).</li><li>Schaefer, D. 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+in+Physcomitrella+patens.">Gene targeting in Physcomitrella patens.</a> Curr. Opin. Plant Biol. 4, 143-150 (2001).</li><li>Kamisugi, Y., Cuming, A. C., Cove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parameters+determining+the+efficiency+of+gene+targeting+in+the+moss+Physcomitrella+patens.">Parameters determining the efficiency of gene targeting in the moss Physcomitrella patens.</a> Nucleic Acids Res. 33, e173-e173 (2005).</li><li>Cove, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+moss+Physcomitrella+patens.">The moss Physcomitrella patens.</a> Annu. Rev. Genet. 39, 339-358 (2005).</li><li>Kamisugi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+gene+targeting+in+Physcomitrella+patens:+homologous+recombination,+concatenation+and+multiple+integration.">The mechanism of gene targeting in Physcomitrella patens: homologous recombination, concatenation and multiple integration.</a> Nucleic Acids Res. 34, 6205-6215 (2006).</li><li>Vidali, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+formin-mediated+actin-filament+elongation+is+essential+for+polarized+plant+cell+growth.">Rapid formin-mediated actin-filament elongation is essential for polarized plant cell growth.</a> Proc. Natl. Acad. Sci. U.S.A. , (2009).</li><li>Sawahel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+foreign+DNA+into+Physcomitrella+protonemal+tissue+by+using+the+gene+gun.">Transfer of foreign DNA into Physcomitrella protonemal tissue by using the gene gun.</a> Plant Mol. Biol. 10, 315-316 (1992).</li><li>Schaefer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transformation+of+the+moss+Physcomitrella+patens.">Stable transformation of the moss Physcomitrella patens.</a> Mol. Gen. Genet. 226, 418-424 (1991).</li><li>Cho, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+bombardment+mediated+transformation+and+GFP+expression+in+the+moss+Physcomitrella+patens.">Particle bombardment mediated transformation and GFP expression in the moss Physcomitrella patens.</a> Mol. Cells. 9, 14-19 (1999).</li><li>Ashton, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bryophyte+Physcomitrella+patens+replicates+extrachromosomal+transgenic+elements.">The bryophyte Physcomitrella patens replicates extrachromosomal transgenic elements.</a> New Phytol. 146, 391-402 (2000).</li><li>Hohe, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+and+highly+standardized+transformation+procedure+allows+efficient+production+of+single+and+multiple+targeted+gene-knockouts+in+a+moss,+Physcomitrella+patens.">An improved and highly standardized transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens.</a> Curr. Genet. 44, 339-347 (2004).</li><li>Vidali, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profilin+is+essential+for+tip+growth+in+the+moss+Physcomitrella+patens.">Profilin is essential for tip growth in the moss Physcomitrella patens.</a> Plant Cell. 19, 3705-3722 (2007).</li></ol>

אין ניגודי אינטרסים הכריז.

השיטה המוצגת כאן מאפשרת שינוי ה-DNA פשוטה ועקבית לתוך protoplasts Physcomitrella patens. שיטה זו פותחה במקור עבור ארבידופסיס 1, אבל כאן הוכיחה כי היא מבצעת היטב עם protoplasts של Physcomitrella patens, צמח פשוט שונה. לכן, בשיטה זו ניתן להחיל על מיני צמחים אחרים עם שינויים קלים. שינויים אפשריים אופטימיזציה כוללים ריכוז protoplast ב MMg, זמן הדגירה בפתרון MMg PEG ו / Ca. אנו בוחרים להשתמש DNA 60 מיקרוגרם בניסויים שגרתית שלנו, כי מספר transformants מגדילה באופן ליניארי עם ריכוז ה-DNA עד לנקודה זו (טבלה 1). אנחנו יכולים לקבל כ 7000 transformants חולף או 200 transformants יציב על מהפך 60 מיקרוגרם אחד DNA, המאפשר הקרנת ניתוח נוסף של אוכלוסייה גדולה של צמחים. Protoplasts טרנספורמציה יכול להיות או להפיץ בינוני עם ציפוי נוזלי או פ.ר.מ.-T. למרות היעילות התחדשות נמוך כאשר בינוני ציפוי נוזלי משמש, אנו בוחרים בשיטה זו כאשר protoplasts הפכו עם פלסמידים supercoiled. ככל הראה בטבלה 1, טרנספורמציות לעשות עם ה-DNA פלסמיד supercoiled ליצור transformants רבים יותר וכך, אנחנו עדיין יכולים להשיג מספר גדול של צמחים עם בינוני ציפוי נוזלי. יתר על כן, הפצת protoplasts בינוני עם ציפוי נוזלי מאפשר התחדשות מהירה, וזה חשוב עבור ניסויים הקשורים פנוטיפים חולף, כמו RNAi להפיל ניסויים 9,15. בנוסף העדר אגר העליון מאפשר בידוד צמח קל immunostaining ו RT-PCR 9,15.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> שם מגיב </td><td> מרכיב </td></tr><tr><td> PPNH 4 </td><td> 1.84 mM KH 2 PO 4 3.4 mM Ca (NO 3) 2 1 mM MgSO 4 2.72 Diammonium mM tartrate 54 מיקרומטר FeSO 4 0.7 H 2 O 9.93 מיקרומטר H 3 BO 3 1.97 מיקרומטר MnCl 0.4 2 H 2 O 0.23 מיקרומטר CoCl 0.6 2 H 2 O 0.19 מיקרומטר ZnSO 4. 7H 2 O 0.22 מיקרומטר CuSO 4. 5H 2 O 0.10 מיקרומטר Na 2 מו 4 .2 H 2 O 0.168 מיקרומטר KI הוסף אגר 0.7% עבור בינוני מוצק. </td></tr><tr><td> פ.ר.מ.-B </td><td> PPNH 4 עם mannitol 6% ו 0.8% אגר. הוסף 10 מ&quot;ל 1M CaCl 2-1 L לפני שמזג צלחות. </td></tr><tr><td> פ.ר.מ.-T </td><td> PPNH 4 עם mannitol 6% ו -0.6% אגר. הוסף 0.5 מ&quot;ל 1M CaCl 2-50 מ&quot;ל לפני השימוש. </td></tr><tr><td> ציפוי נוזלי בינוני </td><td> PPNH 4 עם mannitol 8.5% ללא אגר. הוסף 0.5 מ&quot;ל 1M CaCl 2-50 מ&quot;ל לפני השימוש. </td></tr><tr><td> 2% Driselase </td><td> הוסף 4 גרם של Driselase (Sigma D9515-25 גרם) עד 200 מ&quot;ל של מניטול 8%. מערבבים בעדינות למשך 30 דקות. בטמפרטורת החדר, דגירה 30 דקות. ב 4 ° C, ומערבבים בעדינות במשך 5 דקות. בטמפרטורת החדר. ספין 2,500 גרם ל 10 דקות, להשליך גלולה. מסנן עם 0.22 מיקרומטר, ולאחסן קפוא כמו aliquots 10 מ&quot;ל. להפשיר בתוך אמבט מים בטמפרטורת החדר לפני השימוש. </td></tr><tr><td> MMg </td><td> 0.4 מ &#39;mannitol 15 מ&quot;מ MgCl 2 4 מ&quot;מ MES (pH 5.7) </td></tr><tr><td> PEG / Ca </td><td> 4 גרם PEG4000 3 מ&quot;ל H 2 O 2.5 מ&quot;ל 0.8 M mannitol 1 מ&quot;ל 1M CaCl 2 </td></tr><tr><td> W5 </td><td> 154 mM NaCl 125 mM CaCl 2 5 מ&quot;מ KCl 2 מ&quot;מ MES (pH 5.7) </td></tr></tbody></table>

efficient polyethylene glycol (peg) mediated transformation of the moss physcomitrella patens

<p class="jove_content"&gt; שיטה פשוטה ויעילה להפוך<em&gt; Physcomitrella pantens</em&gt; Protoplasts מתואר. שיטה זו מותאמת פרוטוקולים<em&gt; Physocmitrella</em&gt; Protonemal protoplast ו<em&gt; ארבידופסיס</em&gt; Protoplast mesophyll שינוי<sup&gt; 1</sup&gt;. בשל יכולתו לעבור mitotic יעיל הומולוגיים רקומבינציה<em&gt;, Physcomitrella patens</em&gt; התפתחה כמערכת מודל חשוב בשנים האחרונות<sup&gt; 2</sup&gt;. יכולת זו מאפשרת תדרים גבוהים של גן מיקוד<sup&gt; 3-9</sup&gt;, אשר לא בא לידי ביטוי במודל צמחים אחרים כגון<em&gt; ארבידופסיס</em&gt;. כדי לנצל את מלוא היתרונות של מערכת זו, אנו צריכים שיטה יעילה וקלה להעביר דנ&quot;א לתוך התאים אזוב. הדרכים הנפוצות ביותר להפוך את זה אזוב הם הפצצה החלקיקים<sup&gt; 10</sup&gt; ו - PEG בתיווך DNA ספיגת<sup&gt; 11</sup&gt;. למרות ההפצצות החלקיקים יכול לייצר שינוי יעילות גבוהה<sup&gt; 12</sup&gt;, אקדחים הגן אינם זמינים למעבדות רבות הפרוטוקול קשה לתקנן. מצד שני, השינוי PEG בתיווך אינה דורשת ציוד מיוחד, והוא יכול להתבצע במעבדה כלשהי עם ברדס סטרילית. כאן, אנו מציגים שיטה פשוטה ויעילה מאוד עבור שינוי של protoplasts אזוב. שיטה זו יכולה לייצר יותר מ 120 transformants חולף לכל מיקרוגרם של ה-DNA, אשר מהווה שיפור של פרוטוקול היעילה ביותר שדווח קודם לכן<sup&gt; 13</sup&gt;. בשל יעילותה, פשטות, שחזור, שיטה זו ניתן ליישם פרויקטים הדורשים מספר רב של transformants כמו גם שינוי השגרה.</p

Watch this Scientific Journal Video about Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens at JoVE.com

Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens

שיטה פשוטה ויעילה להפוך<em&gt; Physcomitrella pantens</em&gt; Protoplasts מתואר. שיטה זו מותאמת פרוטוקולים<em&gt; Physocmitrella</em&gt; Protonemal protoplast ו<em&gt; ארבידופסיס</em&gt; Protoplast mesophyll שינוי<sup&gt; 1</sup&gt;.

פוליאתילן גליקול יעיל (PEG) מתווכת טרנספורמציה של מוס Physcomitrella patens</em

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella ...

efficient-polyethylene-glycol-peg-mediated-transformation-moss

Research

JoVE Journal

Biology

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

40.5K Views.  Worcester Polytechnic Institute- WPI. שיטה פשוטה ויעילה להפוך Physcomitrella pantens Protoplasts מתואר. שיטה זו מותאמת פרוטוקולים Physocmitrella Protonemal protoplast ו ארבידופסיס Protoplast mesophyll שינוי 1.

Video: Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens

Environmentally Induced Heritable Changes in Flax

Some flax varieties respond to nutrient stress by modifying their genome and these modifications can be inherited through many generations. Also associated with these genomic changes are heritable phenotypic variations 1,2. The flax variety Stormont Cirrus (Pl) when grown under three different nutrient conditions can either remain inducible (under the control conditions), or become stably modified to either the large or small genotroph by growth under high or low nutrient conditions respectively. The lines resulting from the initial growth under each of these conditions appear to grow better when grown under the same conditions in subsequent generations, notably the Pl line grows best under the control treatment indicating that the plants growing under both the high and low nutrients are under stress. One of the genomic changes that are associated with the induction of heritable changes is the appearance of an insertion element (LIS-1) 3, 4 while the plants are growing under the nutrient stress. With respect to this insertion event, the flax variety Stormont Cirrus (Pl) when grown under three different nutrient conditions can either remain unchanged (under the control conditions), have the insertion appear in all the plants (under low nutrients) and have this transmitted to the next generation, or have the insertion (or parts of it) appear but not be transmitted through generations (under high nutrients) 4. The frequency of the appearance of this insertion indicates that it is under positive selection, which is also consistent with the growth response in subsequent generations. Leaves or meristems harvested at various stages of growth are used for DNA and RNA isolation. The RNA is used to identify variation in expression associated with the various growth environments and/or t he presence/absence of LIS-1. The isolated DNA is used to identify those plants in which the insertion has occurred.

Growing some flax varieties under nutrient stress results in genomic variation within a subset of the genome and phenotypic variation. A complex insertion at a specific site is associated with growth under various nutrient regimes and with changes in gene expression around this site.

מושרה לסביבה תורשתי שינויים פשתן

Some flax varieties respond to nutrient stress by modifying their genome and these modifications can be inherited through many generations. Also ...

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1, then followed by vitrification methods developed in the late 1980s2. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4&deg;C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4.
Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.

An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.

Cryopreservation של עוברי עכברים ידי Vitrification אתילן גליקול מבוסס

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

שינוי גנטי של Picoeukaryote<em&gt; Ostreococcus tauri</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

Efficient Agroinfiltration of Plants for High-level Transient Expression of Recombinant Proteins

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing worldwide demand for pharmaceuticals due to its limited scalability and high cost. Plants have shown to be one of the most promising alternative pharmaceutical production platforms that are robust, scalable, low-cost and safe. The recent development of virus-based vectors has allowed rapid and high-level transient expression of recombinant proteins in plants. To further optimize the utility of the transient expression system, we demonstrate a simple, efficient and scalable methodology to introduce target-gene containing Agrobacterium into plant tissue in this study. Our results indicate that agroinfiltration with both syringe and vacuum methods have resulted in the efficient introduction of Agrobacterium into leaves and robust production of two fluorescent proteins; GFP and DsRed. Furthermore, we demonstrate the unique advantages offered by both methods. Syringe infiltration is simple and does not need expensive equipment. It also allows the flexibility to either infiltrate the entire leave with one target gene, or to introduce genes of multiple targets on one leaf. Thus, it can be used for laboratory scale expression of recombinant proteins as well as for comparing different proteins or vectors for yield or expression kinetics. The simplicity of syringe infiltration also suggests its utility in high school and college education for the subject of biotechnology. In contrast, vacuum infiltration is more robust and can be scaled-up for commercial manufacture of pharmaceutical proteins. It also offers the advantage of being able to agroinfiltrate plant species that are not amenable for syringe infiltration such as lettuce and Arabidopsis. Overall, the combination of syringe and vacuum agroinfiltration provides researchers and educators a simple, efficient, and robust methodology for transient protein expression. It will greatly facilitate the development of pharmaceutical proteins and promote science education.

Plants offer a novel system for the production of pharmaceutical proteins on a commercial scale that is more scalable, cost-efficient and safe than current expression paradigms. In this study, we report a simple and convenient, yet scalable approach to introduce target-gene containing Agrobacterium tumefaciens into plants for protein transient expression.

Agroinfiltration היעילה של צמחים לביטוי חולף ברמה גבוהה של חלבונים רקומביננטי

Mammalian cell culture is the major platform for commercial production of human vaccines and therapeutic proteins. However, it cannot meet the increasing ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

דור יעיל iPS תא מהדם באמצעות Episomes וHDAC המעכבים

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

Agrobacterium-תיווך המעבר העובר בלתי בוגר של הסרבן מתוך תירס שורות באמצעות גנים מורפולגניים

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

הכנה והזרקה של עוברים של יתושי קולקס כדי ליצור מוטציות Null באמצעות CRISPR /Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2&#8211;3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

This work describes the use of CRISPR-Cas9 genome editing technology to knockout endogenous gene OsABCG15 followed by a modified Agrobacterium-mediated transformation protocol to produce a stable male-sterile line in rice.

אגרובקטריום -תווךטרנספורמציה גנטית, ייצור טרנסגנית, ויישום שלה לחקר התפתחות הרבייה הגברית באורז

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive ...

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic pest management strategies. The most used transposable element in insect transformation is piggyBac, and piggyBac-based germline transformation has been successfully conducted in model insects. However, it is still challenging to employ this technology in non-model insects that include agricultural pests. This paper reports on germline transformation of a global agricultural pest, the fall armyworm (FAW), Spodoptera frugiperda, using the hyperactive piggyBac transposase (hyPBase).
In this work, the hyPBase mRNA was produced and used in place of helper plasmid in embryo microinjections. This change led to the successful generation of transgenic FAW. Furthermore, the methods of screening transgenic animals, PCR-based rapid detection of transgene insertion, and thermal asymmetric interlaced PCR (TAIL-PCR)-based determination of the integration site, are also described. Thus, this paper presents a protocol to produce transgenic FAW, which will facilitate piggyBac-based transgenesis in FAW and other lepidopteran insects.

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Successful germline transformation in the fall armyworm, Spodoptera frugiperda, was achieved using mRNA of hyperactive piggyBac transposase.

piggyBac טרנספוסאז בתיווך טרנספורמציה נבטים בתולעת צבא הסתיו, Spodoptera frugiperda

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic ...

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is a newly isolated filamentous cyanobacterium. This paper describes how new filamentous cyanobacteria can be isolated from marine rockpools. It also describes how DNA can be extracted from filaments and how the genomes can be sequenced. Although transformation is established for many single-celled species, it is less frequently reported for filamentous cyanobacteria. A simplified method for the natural transformation of P. lacuna is described here. P. lacuna is the only member of the order Oscillatoriales for which natural transformation is established. This paper also shows how natural transformation is used to express superfolder green fluorescent protein (sfGFP). An endogenous cpcB promoter induced approximately 5 times stronger expression than cpc560, A2813, or psbA2 promoters from Synechocystis sp. PCC6803. Further, a method for the cryopreservation of P. lacuna and Synechocystis sp. CPP 6803 was established, and methods for assessing motility in a liquid medium and on agar and plastic surfaces are described.

Natural Transformation, Protein Expression, and Cryoconservation of the Filamentous Cyanobacterium Phormidium lacuna

Phormidium lacuna is a filamentous cyanobacterium that was isolated from marine rockpools. This article describes the isolation of filaments from natural sources, DNA extraction, genome sequencing, natural transformation, expression of sfGFP, cryoconservation, and motility methods.

טרנספורמציה טבעית, ביטוי חלבונים ושימור קריו-קונסרבציה של הציאנובקטריום הנימה פורמידיום לקונה

Cyanobacteria are the focus of basic research and biotechnological projects in which solar energy is utilized for biomass production. Phormidium lacuna is ...