LV é suportado pela NSF (IOS 1002837)

1. Tornando o Protoplastos <ol><li> Adicionar 9 ml de 8% manitol a uma placa de Petri. </li><li> Usando uma espátula, coloque sete dias de idade musgo 2-3 PPNH4 placas de 5 - na placa de Petri contendo manitol. </li><li> Adicionar 3 mL de Driselase 2% (Sigma D9515-25G) para a placa de Petri. </li><li> Incubar a placa de Petri, à temperatura ambiente com agitação suave durante 1 hora. </li><li> Filtrar a suspensão de protoplastos através de uma malha M 100 (BD Falcon 352.350). </li><li> Gire a suspensão filtrada em 250 g por 5 minutos. Remover o sobrenadante. </li><li> Re-suspender a protoplastos muito suavemente, com 500 mL de manitol 8%. </li><li> Adicionar 9,5 mL de manitol 8% no tubo de cultura. Certifique-se de protoplastos são totalmente suspensos. </li><li> Repita a filtragem e re-suspensão etapas (1.6, 1.7 e 1.8) mais duas vezes. </li><li> Tomar 10 mL da solução de protoplastos e contar os protoplastos em um hemocitômetro. </li><li> Multiplicar o número de protoplastos em uma área de 16 quadrados por 10 mil para obter o número de protoplastos por mL (Figura 1). </li></ol> 2. Transformação <ol><li> Gire a suspensão de protoplastos em 250 g por 5 minutos. Remover o sobrenadante. </li><li> Re-suspender de protoplastos em solução de MMG na concentração de 1,6 milhões de protoplastos / mL. </li><li> Incubar a suspensão de protoplastos em temperatura ambiente por 20 minutos. </li><li> Adicionar 600 mL da suspensão de protoplastos em um tubo de cultura contendo 60 g de DNA. Agitar o tubo cuidadosamente. </li><li> Adicionar 700 mL de PEG / Ca solução na mistura de protoplastos / DNA. Redemoinho suavemente o tubo até que a mistura toda é homogênea. </li><li> Incubar a mistura à temperatura ambiente por 30 minutos. </li><li> Durante este período de espera, cubra PRM-B placas com papel celofane (80 círculos mm de diâmetro, AA Packaging Limited, UK); permitir celofane para hidratar na superfície da placa durante pelo menos 5 min. </li><li> Com uma espátula, retire as bolhas de ar presas entre o celofane eo prato. </li><li> Diluir a mistura com 3 mL de solução W5. </li><li> Girar a mistura de 250 g por 5 minutos. Remover o sobrenadante. </li><li> Re-suspender de protoplastos em 2 mL da PRM derreteu-T (antes de adicioná-lo ao protoplastos esta solução pode ser mantido líquido a 42 ° C). Placa de 1 mL de re-suspensão de protoplastos por PRM-B placa coberta por papel celofane. Enrole as placas com micropore (3M, Cuidados de Saúde). Mantenha as placas a 25 ° C em câmara de crescimento. </li><li> Alternativamente, protoplastos pode ser re-suspenso em 1 mL de meio de revestimento líquido e 0,5 mL semeadas em uma placa de PRM-B coberto com papel celofane. Isto permite acelerar a regeneração, mas o número de plantas em regeneração é menor. </li><li> Câmara de crescimento é definida para uma hr 16. luz e 8 horas. ciclo escuro. </li><li> Mover o celofane para a uma placa nova seleção quatro dias após a transformação. </li></ol> 3. Resultados representativos: Um exemplo de transformação é mostrado na Figura 2. O DNA utilizado neste exemplo foi um superenrolado 7,8 kb do plasmídeo carregando uma fita resistente à higromicina. A quantidade de protoplastos banhado foi reduzida para 50% para a fotografia. As fotos foram tiradas duas semanas depois que as plantas foram transferidas para as placas de seleção. A eficiência de transformação não é afetada pela concentração de DNA até 60 mg. Maiores concentrações de DNA são prejudiciais para a eficiência de transformação. Os dados mostraram que este método proporciona resultados consistentes em uma ampla faixa de quantidades de DNA. Este método também pode gerar transformantes estáveis ​​de forma eficaz, como mostrado na Tabela 1, podemos obter cerca de 4 transformantes estáveis ​​por micrograma de DNA linearizado, que é muito maior do que o que foi relatado anteriormente 14. Também testamos o efeito do choque térmico sobre a eficiência de transformação, transformando um plasmídeo superenrolado carregando um gene de uma proteína fluorescente como um marcador. O choque térmico foi realizada 10 minutos após incubação à temperatura ambiente (dentro de Passo 2.6), incubando a mistura em banho-maria a 45 ° C por 3 minutos. A mistura foi incubada em banho-maria à temperatura ambiente imediatamente após o choque térmico durante 17 min. Os resultados foram registrados 22 horas após a transformação. Os rácios de transformantes obtidos com e sem choque térmico foram muito semelhantes (~ 25%), mas observamos um efeito adverso de choque térmico sobre a viabilidade da protoplastos (dados não mostrados). <img src="/files/ftp_upload/2560/2560fig1.jpg" alt="Figura 1" /> Figura 1. Ver de protoplastos em hemocitômetro. A seção necessária para a contagem de protoplastos é indicado por um quadrado vermelho. Observe a 16 quadrados necessários para a contagem de protoplastos (ver 1.11), a imagem mostra uma contagem de 24 de protoplastos (240.000 células / mL). <img src="/files/ftp_upload/2560/2560fig2.jpg" alt="Figura 2" /> Figura 2. Fotos de 18 dias de idade transformantes transitória. Protoplastos foram transformado com a quantidade indicada de plasmídeo superenrolado. A: 15μg; B: 30μg; C: 60μg; D: 120μg. <table border="1"><tbody><tr><td> Quantidade de DNA (mg) </td><td> Eficiência (tranformants / DNA mg) </td></tr><tr><td> 15, superenrolado plasmídeo </td><td> 120 ± 24 </td></tr><tr><td> 30, superenrolado plasmídeo </td><td> 145 ± 54 </td></tr><tr><td> 60, superenrolado plasmídeo </td><td> 121 ± 60 </td></tr><tr><td> 120, superenrolado plasmídeo </td><td> 71 ± 38 </td></tr><tr><td> 60, linearizado plasmídeo </td><td> 4 ± 1 </td></tr></tbody></table> Tabela de eficiência de transformação 1. DNA em quantidades diferentes.

<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>

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O método aqui apresentado permite uma transformação DNA simples e consistente em protoplastos Physcomitrella patens. Este método foi originalmente desenvolvido para Arabidopsis 1, mas aqui nós demonstramos que ele executa bem com protoplastos de Physcomitrella patens, uma planta simples e diferente. Portanto, esse método pode ser aplicado a outras espécies vegetais com pequenas modificações. Possíveis modificações para a otimização incluem concentração de protoplastos em MMG, tempo de incubação em solução de MMG e PEG / Ca. Nós escolhemos usar 60 mg de DNA em nossos experimentos de rotina porque o número de transformantes aumenta linearmente com a concentração de DNA até este ponto (Tabela 1). Poderíamos obter aproximadamente 7.000 transformantes transitória ou 200 transformantes estáveis ​​em um único transformação DNA 60 mg, que permite a triagem e análise de uma grande população de plantas. Os protoplastos transformados podem ser distribuídos com o meio de revestimento líquido ou PRM-T. Embora a eficiência de regeneração é menor quando o meio de revestimento líquido é usado, escolhemos este método quando os protoplastos foram transformadas com plasmídeos superenrolado. Como mostrado na Tabela 1, as transformações feitas com DNA plasmidial superenrolado transformantes gerar muitos mais e, assim, ainda podemos obter um grande número de plantas com meio de revestimento líquido. Além disso, espalhando protoplastos com o meio líquido de revestimento permite acelerar a regeneração, o que é importante para experimentos envolvendo fenótipos transitórios, tais como RNAi derrubar experimentos 9,15. Além disso, a ausência de agar top permite o isolamento planta fácil para a imunocoloração e RT-PCR 9,15.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Ingrediente </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 de tartarato diamónico 54 M FeSO 4 .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 Adicionar agar 0,7% para meio sólido. </td></tr><tr><td> PRM-B </td><td> PPNH 4 com manitol 6% e ágar 0,8%. Adicionar 10 mL 1M CaCl 2 a 1 L antes de coloca placas. </td></tr><tr><td> PRM-T </td><td> PPNH 4 com manitol 6% e ágar 0,6%. Adicionar 0,5 mL 1M CaCl 2 a 50 mL antes do uso. </td></tr><tr><td> Médio Plating líquido </td><td> PPNH 4 com manitol 8,5%, sem agar. Adicionar 0,5 mL 1M CaCl 2 a 50 mL antes do uso. </td></tr><tr><td> Driselase 2% </td><td> Adicionar 4 g de Driselase (Sigma D9515-25G) para 200 mL de manitol 8%. Mexa delicadamente por 30 min. à temperatura ambiente, incubar 30 min. a 4 ° C, e mexa delicadamente por 5 min. à temperatura ambiente. Spin-2500 g por 10 min, descarte da pelota. Filtro com 0,22 mM, e armazenar congelados como alíquotas de 10 mL. Descongelar em banho-maria à temperatura ambiente antes de usar. </td></tr><tr><td> MMG </td><td> 0,4 M manitol 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 manitol 1 mL CaCl 2 1M </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; Um método simples e eficiente para transformar<em&gt; Physcomitrella pantens</em&gt; Protoplastos é descrito. Este método é adaptado de protocolos para<em&gt; Physocmitrella</em&gt; Protonemal protoplastos e<em&gt; Arabidopsis</em&gt; Transformação de protoplastos do mesófilo<sup&gt; 1</sup&gt;. Devido à sua capacidade de se submeter a mitose eficiente recombinação homóloga<em&gt;, Physcomitrella patens</em&gt; Surgiu como um sistema modelo importante nos últimos anos<sup&gt; 2</sup&gt;. Esta capacidade permite que altas freqüências de gene targeting<sup&gt; 09/03</sup&gt;, Que não é visto em plantas modelo, tais como<em&gt; Arabidopsis</em&gt;. Para tirar o máximo partido deste sistema, precisamos de um método eficaz e fácil de entregar DNA em células de musgo. As formas mais comuns de transformar este musgo são bombardeio de partículas<sup&gt; 10</sup&gt; E DNA mediada por PEG captação<sup&gt; 11</sup&gt;. Embora bombardeio de partículas pode produzir uma alta eficiência de transformação<sup&gt; 12</sup&gt;, Armas gene não são prontamente disponíveis para muitos laboratórios e que o protocolo é de difícil padronização. Por outro lado, PEG transformação mediada não requer equipamentos especializados, e pode ser realizada em qualquer laboratório com um capuz estéril. Aqui, vamos mostrar um método simples e altamente eficiente para a transformação de protoplastos musgo. Este método pode gerar mais de 120 transformantes por micrograma transitória de DNA, que é uma melhoria a partir do protocolo mais eficiente relatado anteriormente<sup&gt; 13</sup&gt;. Devido à sua simplicidade, eficiência e reprodutibilidade, este método pode ser aplicado a projetos que exigem grande número de transformantes, bem como para a transformação de rotina.</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

Um método simples e eficiente para transformar<em&gt; Physcomitrella pantens</em&gt; Protoplastos é descrito. Este método é adaptado de protocolos para<em&gt; Physocmitrella</em&gt; Protonemal protoplastos e<em&gt; Arabidopsis</em&gt; Transformação de protoplastos do mesófilo<sup&gt; 1</sup&gt;.

Polietileno Glicol eficiente Transformação Mediada (PEG) do 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 visualizações.  Worcester Polytechnic Institute- WPI. Um método simples e eficiente para transformar Physcomitrella pantens Protoplastos é descrito. Este método é adaptado de protocolos para Physocmitrella Protonemal protoplastos e Arabidopsis Transformação de protoplastos do mesófilo 1.

Vídeo: 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.

Ambientalmente induzido mudanças hereditárias em Linho

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

Biologia

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.

Criopreservação de embriões de camundongos por Vitrificação Etileno Glicol-Based

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.

Transformação genômica do 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-Transformação de embrião imaturo mediado de linhas de milho recalcitrante usando genes morfogênicos

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.

Preparando e injetando embriões de mosquitos Culex para gerar mutações nulas usando 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-Transformação Genética Mediada, Produção Transgênica e Sua Aplicação para o Estudo do Desenvolvimento Reprodutivo Masculino no Arroz

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.

Transformação germline hiperativa da Transposase mediada pela Transposase no Verme do Exército de Outono, 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%.

Transformação Genética Mediada por Agrobacterium tumefaciens de Plátano de Folha Estreita

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 ...

Método de transformação de raiz peluda em uma etapa para o avanço da biologia relacionada à raiz

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 em destaque: Simplificando a produção de plantas compostas por meio da transformação de raízes pilosas mediada por Agrobacterium rhizogenes 

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

Um Método Eficiente e Reprodutível para Produção de Compósitos por Transformação de Raízes Pelosas à Base de Agrobacterium rhizogenes

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