BT est soutenu par la NSF (IOS 1002837)

1. Rendre les protoplastes <ol><li> Ajouter 9 ml de 8% de mannitol à une boîte de Pétri. </li><li> Avec une spatule, mis 7 jours vieille mousse 2-3 PPNH4 plaques de 5 - dans la boîte de Pétri contenant du mannitol. </li><li> Ajouter 3 ml de Driselase 2% (Sigma D9515-25G) pour la boîte de Pétri. </li><li> Incuber les boîtes de Pétri à température ambiante avec agitation douce pendant 1 heure. </li><li> Filtrer la suspension de protoplastes à travers une maille 100 um (BD Falcon 352 350). </li><li> Spin filtré la suspension à 250 g pendant 5 minutes. Enlever le surnageant. </li><li> Re-suspendre les protoplastes très doucement avec 500 uL de mannitol 8%. </li><li> Ajouter 9,5 ml de mannitol 8% dans le tube de culture. Assurez-vous que les protoplastes sont entièrement suspendus. </li><li> Répétez la filtration et la remise en suspension des mesures (1.6, 1.7 et 1.8) deux fois plus. </li><li> Prenez 10 ul de la solution de protoplastes et de compter les protoplastes dans un hématimètre. </li><li> Multiplier le nombre de protoplastes dans une zone de 16 carrés par 10 000 pour obtenir le nombre de protoplastes par ml (voir figure 1). </li></ol> 2. Transformation <ol><li> Faites tourner la suspension de protoplastes à 250 g pendant 5 minutes. Enlever le surnageant. </li><li> Re-suspendre protoplastes dans une solution de MMG à la concentration de 1,6 million de protoplastes / mL. </li><li> Incuber la suspension de protoplastes à température ambiante pendant 20 minutes. </li><li> Ajouter 600 uL de suspension de protoplastes dans un tube de culture contenant 60 ug d&#39;ADN. Agiter le tube doucement. </li><li> Ajouter 700 ul de PEG / Ca solution dans le mélange de protoplastes / ADN. Agiter le tube doucement jusqu&#39;à ce mélange est homogène. </li><li> Incuber le mélange à la température ambiante pendant 30 minutes. </li><li> Pendant cette période d&#39;attente, la couverture PRM-B assiettes avec du cellophane (80 mm de diamètre des cercles, AA Packaging Limited, UK); permettent d&#39;hydrater la cellophane sur la surface de la plaque pendant au moins 5 min. </li><li> Avec une spatule, retirer les bulles d&#39;air piégées entre la cellophane et de la plaque. </li><li> Diluer le mélange avec 3 ml de solution de W5. </li><li> Spin le mélange à 250 g pendant 5 minutes. Enlever le surnageant. </li><li> Re-suspendre protoplastes dans fondu 2 mL de PRM-T (avant de l&#39;ajouter aux protoplastes cette solution peut être maintenu liquide à 42 ° C). Planche 1 mL de remettre en suspension protoplastes par PRM-B plaque recouverte par du cellophane. Envelopper les plaques avec Micropore (3M, les soins de santé). Garder les assiettes à 25 ° C dans une chambre de croissance. </li><li> Alternativement, des protoplastes peuvent être remis en suspension dans 1 ml de milieu liquide et placage plaqué de 0,5 mL sur une plaque PRM-B recouverte de cellophane. Ceci permet une régénération plus rapide, mais le nombre de régénération des plantes est plus faible. </li><li> Chambre de croissance est fixé pour un h 16. lumière et 8 heures. cycle de sombre. </li><li> Déplacer le cellophane sur une assiette nouvelle sélection 4 jours après la transformation. </li></ol> 3. Les résultats représentatifs: Un exemple de transformation est montré dans la figure 2. L&#39;ADN utilisé dans cet exemple était de 7,8 kb superenroulé plasmide portant une cassette résistante hygromycine. Le montant de protoplastes plaqués a été réduit à 50% pour la photographie. Les photos ont été prise deux semaines après que les plantes ont été déplacés vers les plaques de sélection. L&#39;efficacité de transformation n&#39;est pas affectée par la concentration d&#39;ADN jusqu&#39;à 60 pg. Des concentrations plus élevées de l&#39;ADN sont préjudiciables à l&#39;efficacité de transformation. Les données ont montré que cette méthode offre des résultats cohérents sur une large gamme de quantités d&#39;ADN. Cette méthode peut également générer des transformants stables de manière efficace comme le montre le tableau 1, nous pouvons obtenir environ 4 transformants stables par microgramme d&#39;ADN linéarisé, qui est beaucoup plus élevé que ce qui a été indiqué précédemment 14. Nous avons également testé l&#39;effet du choc thermique sur l&#39;efficacité de transformation en transformant un plasmide superenroulé transportant un gène d&#39;une protéine fluorescente comme un marqueur. Le choc thermique a été effectué 10 minutes après l&#39;incubation température ambiante (dans l&#39;étape 2.6), en incubant le mélange dans un bain d&#39;eau à 45 ° C pendant 3 minutes. Le mélange a été incubé dans un bain d&#39;eau à température ambiante immédiatement après choc thermique pendant 17 min. Les résultats ont été enregistrés 22 heures après la transformation. Les ratios des transformants obtenus avec et sans choc thermique étaient très similaires (~ 25%), mais nous avons observé un effet négatif du choc thermique sur la viabilité des protoplastes (données non présentées). <img src="/files/ftp_upload/2560/2560fig1.jpg" alt="Figure 1" /> Figure 1. Vue de protoplastes dans hémocytomètre. La section nécessaire pour le comptage des protoplastes est indiquée par un carré rouge. Notez les 16 carrés nécessaires pour le comptage des protoplastes (voir 1.11), l&#39;image montre un décompte de 24 protoplastes (240 000 cellules / mL). <img src="/files/ftp_upload/2560/2560fig2.jpg" alt="Figure 2" /> Figure 2. Photos de 18 jours anciens transformants transitoires. Protoplastes ont été transformé avec le montant indiqué de plasmide superenroulé. A: 15μg; B: 30μg; C: 60μg; D: 120μg. <table border="1"><tbody><tr><td> Montant de l&#39;ADN (pg) </td><td> Efficacité (tranformants / ug d&#39;ADN) </td></tr><tr><td> 15, plasmide superenroulé </td><td> 120 ± 24 </td></tr><tr><td> 30, plasmide superenroulé </td><td> 145 ± 54 </td></tr><tr><td> 60, plasmide superenroulé </td><td> 121 ± 60 </td></tr><tr><td> 120, plasmide superenroulé </td><td> 71 ± 38 </td></tr><tr><td> 60, plasmide linéarisé </td><td> 4 ± 1 </td></tr></tbody></table> Efficacité de transformation tableau 1. ADN à des montants différents.

<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>

La méthode présentée ici permet une transformation de l&#39;ADN simple et cohérente dans des protoplastes Physcomitrella patens. Cette méthode a été initialement développé pour une Arabidopsis, mais ici nous avons démontré qu&#39;il se comporte bien avec des protoplastes de Physcomitrella patens, une plante simple et différent. Par conséquent, cette méthode peut être appliquée à d&#39;autres espèces végétales avec des modifications mineures. Modifications possibles pour l&#39;optimisation incluent la concentration de protoplastes MMG, le temps d&#39;incubation dans une solution de MMG et PEG / Ca. Nous avons choisi d&#39;utiliser 60 ug d&#39;ADN dans nos expériences de routine parce que le nombre de transformants augmente linéairement avec la concentration d&#39;ADN jusqu&#39;à ce point (tableau 1). Nous pourrions obtenir environ 7000 transformants transitoires ou 200 transformants stables sur une seule transformation de 60 ug d&#39;ADN, qui permet de dépistage et d&#39;analyse d&#39;une grande population de plantes. Les protoplastes transformés peuvent être réparties en milieu d&#39;étalement de liquide ou PRM-T. Bien que l&#39;efficacité de la régénération est plus faible lorsque milieu d&#39;étalement liquide est utilisé, nous avons choisi cette méthode lorsque les protoplastes ont été transformées avec les plasmides superenroulé. Comme montré dans le tableau 1, les transformations fait avec l&#39;ADN plasmidique superenroulé générer des transformants beaucoup plus et donc, nous pouvons encore obtenir un grand nombre de plantes avec des moyennes plaquage liquide. Par ailleurs, la propagation protoplastes avec milieu d&#39;étalement liquide permet une régénération plus rapide, ce qui est important pour les expériences impliquant des phénotypes transitoires, telles que RNAi renverser expériences 9,15. En plus de l&#39;absence de top agar permet d&#39;isoler des plantes faciles pour immunomarquage et RT-PCR 9,15.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Ingrédient </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 tartrate de diammonium mM 54 uM FeSO 4 .7 H 2 O 9,93 uM 3 H 3 BO 1,97 uM MnCl2 0,4 H 2 O 0,23 uM 2 .6 H 2 O CoCl 0,19 uM ZnSO 4. 7H 2 O 0,22 uM CuSO 4. 5H 2 O 0,10 uM Na 2 MoO 4 .2 H 2 O 0.168 KI uM Ajouter agar 0,7% pour un milieu solide. </td></tr><tr><td> PRM-B </td><td> PPNH 4 avec le mannitol 6% et 0,8% d&#39;agar. Ajouter 10 ml de CaCl 2 1M à 1 L avant de couler les plaques. </td></tr><tr><td> PRM-T </td><td> PPNH 4 avec le mannitol 6% et 0,6% d&#39;agar. Ajouter 0,5 ml de CaCl 2 1M à 50 mL avant l&#39;utilisation. </td></tr><tr><td> Milieu d&#39;étalement liquide </td><td> PPNH 4 avec le mannitol 8,5% sans agar. Ajouter 0,5 ml de CaCl 2 1M à 50 mL avant l&#39;utilisation. </td></tr><tr><td> Driselase 2% </td><td> Ajouter 4 g de Driselase (Sigma D9515-25G) à 200 ml de mannitol à 8%. Remuer doucement pendant 30 min. à température ambiante, incuber 30 min. à 4 ° C, et remuer doucement pendant 5 min. à température ambiante. Spin 2500 g pendant 10 min, jeter à granulés. Filtre avec 0,22 um, et de stocker gelés aliquotes de 10 ml. Dégel dans un bain d&#39;eau à température ambiante avant utilisation. </td></tr><tr><td> MMG </td><td> 0,4 M mannitol 15 mM MgCl 2 4 mM de MES (pH 5,7) </td></tr><tr><td> PEG / Ca </td><td> 4 g PEG4000 3 ml H 2 O 2,5 ml à 0,8 M mannitol 1 ml de CaCl 2 1M </td></tr><tr><td> W5 </td><td> 154 mM de NaCl 125 mM de CaCl2 5 mM de KCl 2 mM de MES (pH 5,7) </td></tr></tbody></table>

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

<p class="jove_content"&gt; Une méthode simple et efficace pour transformer<em&gt; Pantens Physcomitrella</em&gt; Protoplastes est décrite. Cette méthode est adaptée à partir des protocoles de<em&gt; Physocmitrella</em&gt; Protonemal protoplastes et<em&gt; Arabidopsis</em&gt; Transformation de protoplastes de mésophylle<sup&gt; 1</sup&gt;. Grâce à sa capacité à subir mitotique efficaces recombinaison homologue<em&gt;, Physcomitrella patens</em&gt; A émergé comme un système modèle important ces dernières années<sup&gt; 2</sup&gt;. Cette capacité permet à des fréquences élevées de ciblage génique<sup&gt; 3-9</sup&gt;, Qui ne se voit pas dans les plantes telles que le modèle d&#39;autres<em&gt; Arabidopsis</em&gt;. Pour profiter pleinement de ce système, nous avons besoin d&#39;une méthode efficace et facile à transporter l&#39;ADN dans les cellules de la mousse. Les façons les plus courantes pour transformer cette mousse sont bombardement de particules<sup&gt; 10</sup&gt; Et le PEG-médiée ADN absorption<sup&gt; 11</sup&gt;. Bien que le bombardement de particules peut produire un rendement de transformation de haute<sup&gt; 12</sup&gt;, Canons à gènes ne sont pas facilement disponibles pour de nombreux laboratoires et le protocole est difficile à standardiser. D&#39;autre part, le PEG transformation médiée ne nécessite pas d&#39;équipements spécialisés, et peut être réalisée dans tout laboratoire avec une hotte stérile. Ici, nous montrons une méthode simple et très efficace pour la transformation de protoplastes de mousse. Cette méthode peut générer plus de 120 transformants transitoires par microgramme d&#39;ADN, qui est une amélioration par rapport au protocole le plus efficace indiqué précédemment<sup&gt; 13</sup&gt;. En raison de sa simplicité, l&#39;efficacité et la reproductibilité, cette méthode peut être appliquée à des projets nécessitant grand nombre de transformants ainsi que pour la transformation de la 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

Une méthode simple et efficace pour transformer<em&gt; Pantens Physcomitrella</em&gt; Protoplastes est décrite. Cette méthode est adaptée à partir des protocoles de<em&gt; Physocmitrella</em&gt; Protonemal protoplastes et<em&gt; Arabidopsis</em&gt; Transformation de protoplastes de mésophylle<sup&gt; 1</sup&gt;.

Efficace polyéthylène glycol transformation médiée (PEG) de la mousse 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

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Biology

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

40.3K vues.  Worcester Polytechnic Institute- WPI. Une méthode simple et efficace pour transformer Pantens Physcomitrella Protoplastes est décrite. Cette méthode est adaptée à partir des protocoles de Physocmitrella Protonemal protoplastes et Arabidopsis Transformation de protoplastes de mésophylle 1.

Vidéo: 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.

Induites par l&#39;environnement changements héréditaires dans lin

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

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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.

Cryoconservation d&#39;embryons de souris par base d&#39;éthylène glycol 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.

Transformation génomique de la 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-Transformation d’embryon immature médié des lignées consanguines de maïs récalcitrants utilisant des gènes morphogènes

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.

Préparation et injection d’embryons de moustiques Culex pour générer des mutations nulles à l’aide de 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-Transformation génétique médiée, production transgénique et application pour l’étude du développement de la reproduction masculine dans le riz

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.

Transformation germinale hyperactive médiée par la transposase chez la chenille légionnaire d’automne, 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%.

Transformation génétique médiée par Agrobacterium tumefaciens du plantain à feuilles étroites

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 ...

Méthode de transformation de racines poilues en une étape pour faire progresser la biologie liée aux racines

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.

Pleins feux sur l’auteur : Rationalisation de la production de plantes composites grâce à la transformation des racines poilues médiée par Agrobacterium rhizogenes 

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

Procédé efficace et reproductible de production de plantes composites par transformation racinaire velue basée sur Agrobacterium rhizogenes

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