LV è supportato dalla NSF (IOS 1002837)

1. Rendere il protoplasti <ol><li> Aggiungere 9 ml di mannitolo 8% a una piastra di Petri. </li><li> Con una spatola, mettere 7 giorni muschio vecchio 2-3 PPNH4 lastre di 5 - nella capsula di Petri contenente mannitolo. </li><li> Aggiungere 3 ml del 2% Driselase (Sigma D9515-25G) per la piastra di Petri. </li><li> Incubare la piastra Petri a temperatura ambiente agitando delicatamente per 1 ora. </li><li> Filtrare la sospensione di protoplasti attraverso una maglia di 100 micron (BD Falcon 352350). </li><li> Spin la sospensione filtrata a 250 g per 5 minuti. Rimuovere il surnatante. </li><li> Risospendere il protoplasti molto delicatamente con 500 ml di mannitolo 8%. </li><li> Aggiungere 9,5 ml di mannitolo 8% nel tubo di cultura. Assicurarsi protoplasti sono completamente sospesi. </li><li> Ripetere la filtrazione e la risospensione passi (1,6, 1,7 e 1,8) altre due volte. </li><li> Prendere 10 microlitri della soluzione del protoplasti e contare i protoplasti in un emocitometro. </li><li> Moltiplicare il numero di protoplasti in una zona con 16 quadrati da 10.000 a ottenere il numero di protoplasti per ml (vedi figura 1). </li></ol> 2. Trasformazione <ol><li> Spin la sospensione di protoplasti a 250 g per 5 minuti. Rimuovere il surnatante. </li><li> Risospendere protoplasti MMG in soluzione alla concentrazione di 1,6 milioni di protoplasti / mL. </li><li> Incubare la sospensione di protoplasti a temperatura ambiente per 20 minuti. </li><li> Aggiungere 600 ml di sospensione di protoplasti in un tubo cultura del DNA contenente 60 mcg. Agitare delicatamente il tubo. </li><li> Aggiungere 700 ml di PEG / Ca soluzione nel protoplasti / DNA miscela. Agitare delicatamente fino a quando il tubo miscela è omogenea. </li><li> Incubare la miscela a temperatura ambiente per 30 minuti. </li><li> Durante questo periodo di attesa, copertura PRM-B piatti con cellophane (80 mm di diametro cerchi, AA Packaging Limited, UK); permettono cellophane per idratare sulla superficie della piastra per almeno 5 minuti. </li><li> Con una spatola, rimuovere eventuali bolle d&#39;aria intrappolate tra il cellophane e la piastra. </li><li> Diluire il composto con 3 ml di soluzione W5. </li><li> Spin la miscela a 250 g per 5 minuti. Rimuovere il surnatante. </li><li> Risospendere protoplasti nel fuso 2 ml di PRM-T (prima di aggiungerlo al protoplasti questa soluzione può essere mantenuta liquida a 42 ° C). Tavola 1 ml di risospendere protoplasti per PRM-B piatto coperto da cellophane. Avvolgere le piastre con Micropore (3M, Health Care). Tenere le piastre a 25 ° C in una camera di crescita. </li><li> In alternativa, protoplasti può essere risospeso in 1 ml di terreno liquido e placcato placcatura 0,5 ml su un PRM-B piatto coperto da cellophane. Questo permette una più veloce rigenerazione, ma il numero di impianti di rigenerazione è più basso. </li><li> Camera di crescita è impostato per un ore 16. luce e 8 ore. ciclo scuro. </li><li> Spostare il cellophane su di una piastra nuova selezione 4 giorni dopo la trasformazione. </li></ol> 3. Rappresentante dei risultati: Un esempio di trasformazione è mostrato nella Figura 2. Il DNA utilizzato in questo esempio è stato un superavvolto 7,8 kb plasmide porta una cassetta resistente Hygromycin. La quantità di protoplasti placcato è stata ridotta al 50% per la fotografia. Le foto sono state scattate due settimane dopo che le piante sono state spostate le piastre di selezione. L&#39;efficienza di trasformazione non è influenzata dalla concentrazione di DNA fino a 60 mcg. Concentrazioni più elevate di DNA sono dannose per l&#39;efficienza della trasformazione. I dati hanno mostrato che questo metodo fornisce risultati coerenti in un ampio intervallo di quantità di DNA. Questo metodo può anche generare trasformanti stabili in modo efficace, come illustrato nella tabella 1, possiamo ottenere circa 4 trasformanti stabili per microgrammo di DNA linearizzato, che è molto più alta di quanto è stato riportato in precedenza 14. Abbiamo anche testato l&#39;effetto dello shock termico sulla efficienza della trasformazione, trasformando un plasmide superavvolto portatrice di un gene per una proteina fluorescente come marcatore. Lo shock termico è stata effettuata 10 minuti dopo l&#39;incubazione a temperatura ambiente (entro Passo 2.6) incubando la miscela in un bagno d&#39;acqua a 45 ° C per 3 minuti. La miscela è stata incubata in un bagno di acqua a temperatura ambiente subito dopo la scossa di calore per 17 min. I risultati sono stati registrati 22 ore dopo la trasformazione. I rapporti di trasformanti ottenuti con e senza shock termico erano molto simili (~ 25%), ma abbiamo osservato un effetto negativo di shock termico sulla redditività dei protoplasti (dati non riportati). <img src="/files/ftp_upload/2560/2560fig1.jpg" alt="Figura 1" /> Figura 1. Vista di protoplasti in emocitometro. La sezione necessaria per il conteggio di protoplasti è indicata da un quadrato rosso. Si noti il ​​16 quadrati necessari per il conteggio protoplasti (vedi 1.11), l&#39;immagine mostra un conteggio di 24 protoplasti (240.000 cellule / ml). <img src="/files/ftp_upload/2560/2560fig2.jpg" alt="Figura 2" /> Figura 2. Foto di 18 giorni trasformanti vecchio transitori. Protoplasti sono stati transformato con la quantità indicata di plasmide superavvolto. A: 15μg; B: 30 mcg; C: 60μg; D: 120μg. <table border="1"><tbody><tr><td> Quantità di DNA (mg) </td><td> Efficienza (tranformants / DNA mcg) </td></tr><tr><td> 15, superavvolto plasmide </td><td> 120 ± 24 </td></tr><tr><td> 30, superavvolto plasmide </td><td> 145 ± 54 </td></tr><tr><td> 60, superavvolto plasmide </td><td> 121 ± 60 </td></tr><tr><td> 120, superavvolto plasmide </td><td> 71 ± 38 </td></tr><tr><td> 60, plasmide linearizzato </td><td> 4 ± 1 </td></tr></tbody></table> Tabella 1. Efficienza di trasformazione del DNA in quantità diverse.

<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>

Nessun conflitto di interessi dichiarati.

Il metodo presentato qui permette una trasformazione del DNA lineare e coerente in Physcomitrella protoplasti patene. Questo metodo è stato originariamente sviluppato per Arabidopsis 1, ma qui abbiamo dimostrato che si comporta bene con protoplasti di Physcomitrella patene, un impianto più semplice e diverso. Pertanto, questo metodo può essere applicato ad altre specie di piante, con piccole modifiche. Possibili modifiche per l&#39;ottimizzazione comprendono concentrazione di protoplasti in MMG, tempo di incubazione in soluzione MMG e PEG / Ca. Abbiamo scelto di utilizzare il DNA 60 mg nei nostri esperimenti di routine perché il numero di trasformanti cresce linearmente con la concentrazione di DNA fino a questo punto (Tabella 1). Potremmo avere circa 7000 trasformanti transitori o 200 trasformanti stabili su un singolo 60 mcg trasformazione del DNA, che permette per lo screening e analisi di una vasta popolazione di piante. Il protoplasti trasformato può essere diffuso con il mezzo liquido o placcatura PRM-T. Sebbene l&#39;efficienza di rigenerazione è più bassa quando medie placcatura liquida è utilizzato, abbiamo scelto questo metodo quando il protoplasti sono stati trasformati con plasmidi superavvolto. Come mostrato nella tabella 1, le trasformazioni fatto con superavvolto DNA plasmidico generare trasformanti molti altri e, quindi, possiamo ancora ottenere un gran numero di impianti di media placcatura liquido. Inoltre, la diffusione protoplasti con placcatura medio liquido permette una più veloce rigenerazione, che è importante per gli esperimenti che coinvolgono fenotipi transitoria, come RNAi abbattere esperimenti 9,15. Inoltre l&#39;assenza di agar in alto permette di isolamento pianta facile per immunoistochimica e RT-PCR 9,15.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Ingrediente </td></tr><tr><td> PPNH 4 </td><td> 1,84 mM KH 2 PO 4 3,4 mm di Ca (NO 3) 2 1 mM MgSO 4 2,72 mM di tartrato di diammonio 54 mM FeSO4 0,7 H 2 O 9,93 mM H 3 BO 3 1,97 mM MnCl 2 .4 H 2 O 0,23 mM CoCl 2 .6 H 2 O 0,19 mM ZnSO 4. 7H 2 O 0,22 mM CuSO 4. 5H 2 O 0,10 mM Na 2 MoO 4 .2 H 2 O 0,168 mM KI Aggiungere 0,7% agar per terreno solido. </td></tr><tr><td> PRM-B </td><td> PPNH 4 con il 6% mannitolo e 0,8% agar. Aggiungere 10 ml 1M CaCl 2 a 1 L prima di versare piatti. </td></tr><tr><td> PRM-T </td><td> PPNH 4 con il 6% mannitolo e 0,6% agar. Aggiungere 0,5 ml 1M CaCl 2 a 50 ml prima dell&#39;uso. </td></tr><tr><td> Placcatura Media liquido </td><td> PPNH 4 con 8,5% di mannitolo senza agar. Aggiungere 0,5 ml 1M CaCl 2 a 50 ml prima dell&#39;uso. </td></tr><tr><td> 2% Driselase </td><td> Aggiungere 4 g di Driselase (Sigma D9515-25G) a 200 ml di mannitolo 8%. Mescolare delicatamente per 30 min. a temperatura ambiente, incubare 30 min. a 4 ° C, e mescolare delicatamente per 5 minuti. a temperatura ambiente. Spin 2.500 g per 10 minuti, scartare pellet. Filtro con 0,22 micron, e conservare congelato aliquote da 10 ml. Scongelare in un bagno d&#39;acqua a temperatura ambiente prima dell&#39;uso. </td></tr><tr><td> MMG </td><td> 0,4 M di mannitolo 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 mannitolo 1 ml 1M 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; Un metodo semplice ed efficace per trasformare<em&gt; Physcomitrella pantens</em&gt; Protoplasti è descritto. Questo metodo è adattato da protocolli per<em&gt; Physocmitrella</em&gt; Protonemal protoplasti e<em&gt; Arabidopsis</em&gt; Mesofillo trasformazione di protoplasti<sup&gt; 1</sup&gt;. Grazie alla sua capacità di subire mitosi efficiente ricombinazione omologa<em&gt;, Physcomitrella patene</em&gt; È emerso come un sistema modello importante negli ultimi anni<sup&gt; 2</sup&gt;. Questa capacità consente di alte frequenze del gene targeting<sup&gt; 3-9</sup&gt;, Che non si vede nelle piante altri modelli come ad esempio<em&gt; Arabidopsis</em&gt;. Per trarre il massimo vantaggio di questo sistema, abbiamo bisogno di un metodo efficace e semplice per fornire il DNA nelle cellule muschio. I modi più comuni di trasformare questo muschio sono bombardamento di particelle<sup&gt; 10</sup&gt; E PEG-mediata del DNA uptake<sup&gt; 11</sup&gt;. Anche se bombardamento di particelle in grado di produrre una elevata efficienza di trasformazione<sup&gt; 12</sup&gt;, Pistole gene non sono prontamente disponibili per molti laboratori e il protocollo è difficile da standardizzare. D&#39;altra parte, la trasformazione PEG mediata non richiede attrezzature specializzate, e può essere eseguita in qualsiasi laboratorio, con un cappuccio sterile. Qui vi mostriamo un metodo semplice ed altamente efficiente per la trasformazione di protoplasti muschio. Questo metodo può generare più di 120 trasformanti transitoria per microgrammo di DNA, che è un miglioramento rispetto al protocollo più efficiente precedentemente riportato<sup&gt; 13</sup&gt;. Grazie alla sua semplicità, efficienza e riproducibilità, questo metodo può essere applicato a progetti che richiedono grande numero di trasformanti così come per la trasformazione di routine.</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

Un metodo semplice ed efficace per trasformare<em&gt; Physcomitrella pantens</em&gt; Protoplasti è descritto. Questo metodo è adattato da protocolli per<em&gt; Physocmitrella</em&gt; Protonemal protoplasti e<em&gt; Arabidopsis</em&gt; Mesofillo trasformazione di protoplasti<sup&gt; 1</sup&gt;.

Efficiente polietilene glicole (PEG) Trasformazione mediata della Moss Physcomitrella patene</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

40.4K Views.  Worcester Polytechnic Institute- WPI. Un metodo semplice ed efficace per trasformare Physcomitrella pantens Protoplasti è descritto. Questo metodo è adattato da protocolli per Physocmitrella Protonemal protoplasti e Arabidopsis Mesofillo trasformazione di protoplasti 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.

Indotta da fattori ambientali modifiche ereditabili nella Lino

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

Ricerca

Biologia

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.

Trasformazione genomica del 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 efficiente di impianti di alto livello espressione transiente di proteine ​​ricombinanti

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

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- Trasformazione dell'embrione immaturo mediato delle linee inbrete di mais recalcitranti utilizzando geni morfogenici

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.

Preparazione e iniezione di embrioni di zanzare Culex per generare mutazioni nulle utilizzando 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.

Agrobacterium-Mediato Trasformazione Genetica, Produzione Transgenica, e la sua applicazione per lo studio dello sviluppo riproduttivo maschile nel riso

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.

Piggy iperattivoTrasponsi-mediata dalla trasposizione della linea germinale nel verme dell'esercito autunnale, 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-Trasformazione genetica mediata della piantaggine a foglia stretta

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 ...

Metodo di trasformazione della radice pelosa in un solo passaggio per l'avanzamento della biologia correlata alle radici

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.

Autore in primo piano: Razionalizzazione della produzione di piante composite attraverso la trasformazione delle radici pelose mediata da Agrobacterium rhizogenes 

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

Un metodo efficiente e riproducibile per la produzione di piante composite mediante trasformazione delle radici pelose a base di Agrobacterium rhizogenes

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