LV wird durch NSF (IOS 1002837) unterstützt

1. Making the Protoplasten <ol><li> Add 9 ml 8% Mannitol in eine Petrischale. </li><li> Mit einem Spatel, legte 7 Tage alt Moos 2 bis 3 PPNH4 Platten von 5 - in der Petrischale mit Mannitol. </li><li> 3 ml 2% Driselase (Sigma D9515-25G) auf der Petrischale. </li><li> Inkubieren Sie die Petrischale bei Raumtemperatur unter leichtem Schütteln für 1 Stunde. </li><li> Filter der Protoplastensuspension durch ein 100 um Sieb (BD Falcon 352350). </li><li> Drehen Sie das gefilterte Suspension bei 250 g für 5 Minuten. Entfernen Sie den Überstand. </li><li> Re-suspend die Protoplasten sehr sanft mit 500 ul 8% Mannitol. </li><li> Fügen Sie 9,5 ml 8% Mannitol in die Kultur Röhre. Stellen Sie sicher, Protoplasten vollständig ausgesetzt. </li><li> Wiederholen Sie die Filtration und Resuspension Schritten (1,6, 1,7 und 1,8) zwei weitere Male. </li><li> Nehmen Sie sich 10 ul der Protoplasten-Lösung und zählen die Protoplasten in einer Zählkammer. </li><li> Multiplizieren Sie die Anzahl der Protoplasten in einem 16 Quadratmeter Fläche um 10.000, die Zahl der Protoplasten pro ml zu erhalten (siehe Abbildung 1). </li></ol> 2. Transformation <ol><li> Spin der Protoplastensuspension bei 250 g für 5 Minuten. Entfernen Sie den Überstand. </li><li> Re-suspend Protoplasten in der MMG-Lösung bei einer Konzentration von 1,6 Millionen Protoplasten / ml. </li><li> Inkubieren Sie die Protoplastensuspension bei Raumtemperatur für 20 Minuten. </li><li> Add 600 ul der Protoplastensuspension in eine Kultur Röhrchen mit 60 ug DNA. Swirl das Rohr vorsichtig. </li><li> Add 700 ul PEG / Ca-Lösung in die Protoplasten / DNA-Mischung. Swirl das Rohr vorsichtig, bis das gesamte Mischung homogen ist. </li><li> Inkubieren Sie die Mischung bei Raumtemperatur für 30 Minuten. </li><li> Während dieser Wartezeit, decken PRM-B-Platten mit Zellophan (80 mm Durchmesser Kreise, AA Packaging Limited, UK); ermöglichen Zellophan zu Hydrat auf der Plattenoberfläche für mindestens 5 min. </li><li> Mit einem Spatel, entfernen Sie alle Luftblasen zwischen dem Cellophan und der Platte. </li><li> Verdünnen Sie die Mischung mit 3 ml W5-Lösung. </li><li> Drehen Sie das Gemisch bei 250 g für 5 Minuten. Entfernen Sie den Überstand. </li><li> Re-suspend Protoplasten in geschmolzenem 2 mL der PRM-T (vor der Zugabe zu den Protoplasten dieser Lösung flüssig gehalten bei 42 ° C sein kann). Plate 1 ml resuspendieren Protoplasten pro PRM-B Platte durch Cellophan abgedeckt. Wickeln Sie die Platten mit Micropore (3M, Health Care). Halten Sie die Platten bei 25 ° C in einer Klimakammer. </li><li> Alternativ können Protoplasten in 1 ml Flüssigkeit plating mittel-und vernickelt 0,5 ml auf einem PRM-B-Platte mit Cellophan abgedeckt resuspendiert werden. Dies ermöglicht eine schnellere Regeneration, aber die Zahl der Regeneration von Pflanzen ist geringer. </li><li> Growth Kammer ist für einen 16 h eingestellt. Licht und 8 Stunden. Dunkel-Zyklus. </li><li> Bewegen Sie das Zellophan auf eine frische Auswahl Platte 4 Tage nach der Transformation. </li></ol> 3. Repräsentative Ergebnisse: Ein Beispiel der Transformation ist in Abbildung 2 dargestellt. Die DNA in diesem Beispiel wurde ein supercoiled 7,8 kb Plasmid, ein Hygromycin resistent Kassette. Die Höhe der Protoplasten vergoldet wurde auf 50% für die Fotografie reduziert. Die Bilder wurden zwei Wochen nach dem Pflanzen, um die Auswahl Platten wurden verlegt. Die Transformationseffizienz wird nicht durch die DNA-Konzentration von bis zu 60 ug betroffen. Höhere Konzentrationen von DNA wirken sich nachteilig auf die Effizienz der Transformation. Die Daten zeigten, dass diese Methode konsistentes Ergebnis über einen weiten Bereich von DNA-Mengen liefert. Diese Methode kann auch zu generieren stabile Transformanten effektiv wie in Tabelle 1 dargestellt, können wir über 4 stabile Transformanten pro Mikrogramm linearisierte DNA, die viel höher sind als das, was bisher 14 gemeldet wird erhalten. Wir testeten auch die Wirkung von Hitzeschock auf die Transformationseffizienz durch Umwandlung eines supercoiled Plasmid ein Gen für ein fluoreszierendes Protein als Marker. Der Hitzeschock erfolgte 10 Minuten nach Inkubation bei Raumtemperatur durchgeführt (im Schritt 2,6) durch Inkubation der Mischung in einem 45 ° C Wasserbad für 3 Minuten. Die Mischung wurde in einer Raumtemperatur Wasserbad unmittelbar nach Hitzeschock für 17 min inkubiert. Die Ergebnisse wurden 22 Stunden nach der Transformation aufgenommen. Die Verhältnisse der erhaltenen Transformanten mit und ohne Hitzeschock sehr ähnlich waren (~ 25%), aber wir beobachten einen negativen Effekt von Hitzeschock auf die Lebensfähigkeit der Protoplasten (Daten nicht gezeigt). <img src="/files/ftp_upload/2560/2560fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Blick auf Protoplasten in Hämozytometer. Der Abschnitt für Protoplasten Zählen gebraucht wird durch ein rotes Quadrat angezeigt. Beachten Sie die 16 Quadrate zu zählen Protoplasten (vgl. 1,11) erforderlich, das Bild zeigt eine Anzahl von 24 Protoplasten (240.000 Zellen / ml). <img src="/files/ftp_upload/2560/2560fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Bilder von 18 Tage alten transiente Transformanten. Protoplasten wurden transgebildet mit der angegebenen Menge supercoiled Plasmid. A: 15μg; B: 30 &amp;mgr; g; C: 60μg; D: 120μg. <table border="1"><tbody><tr><td> DNA-Menge (pg) </td><td> Efficiency (Transformanten / ug DNA) </td></tr><tr><td> 15, supercoiled Plasmid </td><td> 120 ± 24 </td></tr><tr><td> 30, supercoiled Plasmid </td><td> 145 ± 54 </td></tr><tr><td> 60, supercoiled Plasmid </td><td> 121 ± 60 </td></tr><tr><td> 120, supercoiled Plasmid </td><td> 71 ± 38 </td></tr><tr><td> 60, linearisierte Plasmid </td><td> 4 ± 1 </td></tr></tbody></table> Tabelle 1. Transformation Effizienz auf verschiedenen DNA-Mengen.

<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>

Die hier vorgestellte Methode ermöglicht eine einfache und konsistente DNA-Transformation in Physcomitrella patens Protoplasten. Diese Methode wurde ursprünglich für Arabidopsis 1 entwickelt, aber hier haben wir gezeigt, dass es gut funktioniert mit Protoplasten von Physcomitrella patens, eine einfachere und verschiedene Pflanzenarten. Daher kann diese Methode auf andere Pflanzenarten mit geringfügigen Änderungen angewendet werden. Mögliche Modifikationen zur Optimierung gehören Protoplastenkonzentration in der MMG, Inkubationszeit in MMG und PEG / Ca-Lösung. Wir entscheiden uns für 60 ug DNA in unseren Routineversuche zu verwenden, da die Zahl der Transformanten steigt linear mit der DNA-Konzentration bis zu diesem Zeitpunkt (Tabelle 1). Wir konnten etwa 7000 transiente Transformanten oder 200 stabile Transformanten auf einer einzigen 60 ug DNA-Transformation, die zur weiteren Überprüfung und Analyse einer großen Population von Pflanzen ermöglicht. Die transformierten Protoplasten können entweder verteilt mit Flüssigkeit plating Medium oder PRM-T werden. Obwohl die Regeneration Wirkungsgrad ist niedriger, wenn flüssige Beschichtung Medium verwendet wird, wählen wir diese Methode, wenn die Protoplasten mit supercoiled Plasmiden transformiert wurden. Da zeigten die in Tabelle 1, erzeugen Transformationen mit supercoiled Plasmid-DNA erfolgt viel mehr Transformanten und somit können wir immer noch erhalten eine große Anzahl von Anlagen mit flüssigen Beschichtung Medium. Darüber hinaus verbreiten Protoplasten mit flüssigen Beschichtung Medium ermöglicht eine schnellere Regeneration, was wichtig ist für Experimente, die transiente Phänotypen, wie RNAi knock down Experimenten 9,15. Neben dem Fehlen von Top-Agar ermöglicht eine einfache Anlage Trennung für Immunfärbung und RT-PCR 9,15.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Zutat </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 mM Diammonium Tartrat 54 um FeSO 4 .7 H 2 O 9,93 uM H 3 BO 3 1,97 pM MnCl 2 .4 H 2 O 0,23 um CoCl 2 .6 H 2 O 0,19 pM ZnSO 4. 7H 2 O 0,22 &amp;mgr; M CuSO 4. 5H 2 O 0,10 um Na 2 MoO 4 .2 H 2 O 0,168 uM KI Add 0,7% Agar für festes Medium. </td></tr><tr><td> PRM-B </td><td> PPNH 4 mit 6% Mannitol und 0,8% Agar. Fügen Sie 10 ml 1M CaCl 2 bis 1 L vor dem Gießen-Platten. </td></tr><tr><td> PRM-T </td><td> PPNH 4 mit 6% Mannitol und 0,6% Agar. Fügen Sie 0,5 ml 1M CaCl 2 bis 50 mL vor dem Gebrauch. </td></tr><tr><td> Flüssige Mittel Plating </td><td> PPNH 4 mit 8,5% Mannit ohne Agar. Fügen Sie 0,5 ml 1M CaCl 2 bis 50 mL vor dem Gebrauch. </td></tr><tr><td> 2% Driselase </td><td> 4 g Driselase (Sigma D9515-25G) auf 200 ml 8% Mannitol. Leicht umrühren für 30 min. bei Raumtemperatur inkubieren 30 min. bei 4 ° C, und sanft rühren 5 min. bei Raumtemperatur. Spin 2.500 g für 10 min, verwerfen Pellet. Filter mit 0,22 um, und speichern Sie bis zu 10 mL Aliquots eingefroren. Tauwetter in einer Raumtemperatur Wasserbad vor dem Gebrauch. </td></tr><tr><td> MMG </td><td> 0,4 M Mannitol 15 mM MgCl 2 4 mM MES (pH 5,7) </td></tr><tr><td> PEG / Ca </td><td> 4 g PEG4000 3 mL H 2 O 2,5 ml 0,8 M Mannit 1 mL 1 M CaCl 2 </td></tr><tr><td> W5 </td><td> 154 mM NaCl 125 mM CaCl 2 5 mM KCl 2 mM MES (pH 5,7) </td></tr></tbody></table>

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

<p class="jove_content"&gt; Eine einfache und effiziente Methode zur Umwandlung<em&gt; Physcomitrella pantens</em&gt; Protoplasten beschrieben. Diese Methode wird von Protokollen für angepasste<em&gt; Physocmitrella</em&gt; Protonema Protoplasten und<em&gt; Arabidopsis</em&gt; Mesophyll Protoplasten-Transformation<sup&gt; 1</sup&gt;. Aufgrund seiner Fähigkeit, effiziente mitotischen unterziehen homologe Rekombination<em&gt;, Physcomitrella patens</em&gt; Hat als ein wichtiges Modellsystem in den letzten Jahren entstanden<sup&gt; 2</sup&gt;. Diese Fähigkeit erlaubt hohe Frequenzen von gene targeting<sup&gt; 3-9</sup&gt;, Die nicht in andere Modell Pflanzen wie man sieht<em&gt; Arabidopsis</em&gt;. Um den vollen Nutzen dieses Systems, brauchen wir eine effektive und einfache Methode, um DNA in Moos-Zellen liefern. Die gängigsten Methoden, um dieses Moos zu verwandeln sind Partikelbeschuss<sup&gt; 10</sup&gt; Und PEG-vermittelter DNA-Aufnahme<sup&gt; 11</sup&gt;. Obwohl Partikelbeschuss kann eine hohe Transformationseffizienz<sup&gt; 12</sup&gt; Sind Genkanonen nicht ohne weiteres verfügbar, viele Labors und das Protokoll ist schwer zu standardisieren. Auf der anderen Seite, ist PEG-vermittelte Transformation keine spezielle Ausrüstung, und kann in jedem Labor mit einem sterilen Abzug durchgeführt werden. Hier zeigen wir eine einfache und sehr effiziente Methode zur Umwandlung von Moos-Protoplasten. Diese Methode kann mehr als 120 transiente Transformanten pro Mikrogramm DNA, die eine Verbesserung der effizientesten Protokoll bereits berichtet wird<sup&gt; 13</sup&gt;. Aufgrund seiner Einfachheit, Effizienz und Reproduzierbarkeit, kann diese Methode, um Projekte, die große Zahl der Transformanten als auch für Routine-Transformation angewendet werden.</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

Eine einfache und effiziente Methode zur Umwandlung<em&gt; Physcomitrella pantens</em&gt; Protoplasten beschrieben. Diese Methode wird von Protokollen für angepasste<em&gt; Physocmitrella</em&gt; Protonema Protoplasten und<em&gt; Arabidopsis</em&gt; Mesophyll Protoplasten-Transformation<sup&gt; 1</sup&gt;.

Effiziente Polyethylenglykol (PEG) vermittelten Transformation von der Moss 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

40.3K Aufrufe.  Worcester Polytechnic Institute- WPI. Eine einfache und effiziente Methode zur Umwandlung Physcomitrella pantens Protoplasten beschrieben. Diese Methode wird von Protokollen für angepasste Physocmitrella Protonema Protoplasten und Arabidopsis Mesophyll Protoplasten-Transformation 1.

Serie: 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.

Umweltfreundliche Induced vererbbare Veränderungen in Flax

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

Forschung

Biologie

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.

Kryokonservierung von Maus Embryonen durch Ethylen-Glykol-Based Vitrification

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.

Genomic Transformation des 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 ...

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- Vermitteltune unreife Embryo-Transformation von widerspenstigen Mais Inzuchtlinien mit morphogenen Genen

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.

Vorbereitung und Injektion von Embryonen von Culex-Moskitos zur Erzeugung von Nullmutationen mit 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.

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.

Hyperaktives SchweinchenBac Transposase-vermittelte Keimbahntransformation im Herbst-Heerwurm, Spodoptera frugiperda

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

Agrobacterium tumefaciens-Mediated Genetic Transformation of Narrowleaf Plantain

Species in the genus Plantago have several unique traits that have led to them being adapted as model plants in various fields of study. However, the lack of a genetic manipulation system prevents in-depth investigation of gene function, limiting the versatility of this genus as a model. Here, a transformation protocol is presented for Plantago lanceolata, the most commonly studied Plantago species. Using Agrobacterium tumefaciens-mediated transformation, 3 week-old roots of aseptically grown P. lanceolata plants were infected with bacteria, incubated for 2-3 days, and then transferred to a shoot induction medium with appropriate antibiotic selection. Shoots typically emerged from the medium after 1 month, and roots developed 1-4 weeks after the shoots were transferred to the root induction medium. The plants were then acclimated to a soil environment and tested for the presence of a transgene using the &#946;-glucuronidase (GUS) reporter assay. The transformation efficiency of the current method is ~20%, with two transgenic plants emerging per 10 root tissues transformed. Establishing a transformation protocol for narrowleaf plantain will facilitate the adoption of this plant as a new model species in various areas.

Agrobacterium tumefaciens-Mediated Genetic Transformation of Narrowleaf Plantain

Because of its versatile application as a model species in various fields of study, there is a need for a genetic transformation toolkit in narrowleaf plantain (Plantago lanceolata). Here, using Agrobacterium tumefaciens-mediated transformation, a protocol is presented that results in stable transgenic lines with a transformation efficiency of 20%.

Agrobacterium tumefaciens-vermittelte genetische Transformation des Spitzwegerichs

Species in the genus Plantago have several unique traits that have led to them being adapted as model plants in various fields of study. However, the lack ...

Einstufige Methode zur Transformation der haarigen Wurzel zur Weiterentwicklung der wurzelbezogenen Biologie

An Efficient and Reproducible Method for Producing Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation

Producing composite plants with transgenic roots and nontransgenic stems and buds using Agrobacterium rhizogenes-mediated hairy root transformation is a powerful tool to study root-related biology. Hairy root transformation is established in a wide range of dicotyledons and in several monocotyledon species and is almost independent of the genotype. The traditional method of hypocotyl injection with A. rhizogenes to obtain composite plants is inefficient, time-consuming, laborious, and frequently causes the death of tender and tiny hypocotyl plants. A highly efficient, one-step hairy root transformation mediated by A. rhizogenes was established previously, which eliminates the need for transplanting after producing hairy roots. In this study, a partial hypocotyl and primary root were removed, the hypocotyl incision site was coated with A. rhizogenes, and&#160;then hypocotyls were planted in sterile vermiculite. After 12 days of cultivation, the hypocotyl incision expanded and new hairy roots were induced. This article provides the detailed protocol of a one-step transformation method mediated by A. rhizogenes, with its effectiveness demonstrated by producing composite plants of wild soybean, Solanum americanum, and pumpkin.

Autor im Rampenlicht: Rationalisierung der Produktion von Mischpflanzen durch Agrobacterium Rhizogene-vermittelte Haarwurzeltransformation 

Here, we provide the detailed protocol of a one-step transformation method mediated by Agrobacterium tumefaciens to produce composite plants.

Eine effiziente und reproduzierbare Methode zur Herstellung von Kompositpflanzen durch Agrobacterium rhizogenes-basierte Haarwurzeltransformation

Producing composite plants with transgenic roots and nontransgenic stems and buds using Agrobacterium rhizogenes-mediated hairy root transformation is a ...