Esta pesquisa é suportada pelo holandês tecnologia Foundation STW (12385), que faz parte da organização de países baixos para a pesquisa científica (NWO), e que é parcialmente financiado pelo Ministério dos assuntos económicos (OTP Grant 12385-MS).  Agradecemos a Carol M. Hamilton (Universidade de Cornell, Estados Unidos) para a prestação de pCH20, a espinha dorsal dos vetores BIBAC-GW.

<ol>
	<li>Jorgensen, R. A., Cluster, P. D., English, J., Que, Q., Napoli, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chalcone+synthase+cosuppression+phenotypes+in+petunia+flowers:+comparison+of+sense+vs.+antisense+constructs+and+single-copy+vs.+complex+T-DNA+sequences.">Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and single-copy vs. complex T-DNA sequences.</a> Plant Mol Biol. 31 (5), 957-973 (1996).</li><li>Stam, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-transcriptional+silencing+of+chalcone+synthase+in+Petunia+by+inverted+transgene+repeats.">Post-transcriptional silencing of chalcone synthase in Petunia by inverted transgene repeats.</a> Plant J. 12, 63-82 (1997).</li><li>Stam, M., Viterbo, A., Mol, J. N., Kooter, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Position-dependent+methylation+and+transcriptional+silencing+of+transgenes+in+inverted+T-DNA+repeats:+implications+for+posttranscriptional+silencing+of+homologous+host+genes+in+plants.">Position-dependent methylation and transcriptional silencing of transgenes in inverted T-DNA repeats: implications for posttranscriptional silencing of homologous host genes in plants.</a> Mol Cell Biol. 18 (11), 6165-6177 (1998).</li><li>Jin, Y., Guo, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgene-induced+gene+silencing+in+plants.">Transgene-induced gene silencing in plants.</a> Methods Mol Biol. 1287, 105-117 (2015).</li><li>Oltmanns, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+Backbone-Free,+Low+Transgene+Copy+Plants+by+Launching+T-DNA+from+the+Agrobacterium+Chromosome+1[W][OA].">Generation of Backbone-Free, Low Transgene Copy Plants by Launching T-DNA from the Agrobacterium Chromosome 1[W][OA].</a> Plant Physiol. , (2010).</li><li>Ye, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+production+of+single+copy+backbone-free+transgenic+plants+in+multiple+crop+species+using+binary+vectors+with+a+pRi+replication+origin+in+Agrobacterium+tumefaciens.">Enhanced production of single copy backbone-free transgenic plants in multiple crop species using binary vectors with a pRi replication origin in Agrobacterium tumefaciens.</a> Transgenic Res. , (2011).</li><li>Hamilton, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+binary-BAC+system+for+plant+transformation+with+high-molecular-weight+DNA.">A binary-BAC system for plant transformation with high-molecular-weight DNA.</a> Gene. 200 (1-2), 107-116 (1997).</li><li>Frary, A., Hamilton, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficiency+and+stability+of+high+molecular+weight+DNA+transformation:+an+analysis+in+tomato.">Efficiency and stability of high molecular weight DNA transformation: an analysis in tomato.</a> Transgenic Res. 10 (2), 121-132 (2001).</li><li>Vega, J. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agrobacterium-mediated+transformation+of+maize+(Zea+mays)+with+Cre-lox+site+specific+recombination+cassettes+in+BIBAC+vectors.">Agrobacterium-mediated transformation of maize (Zea mays) with Cre-lox site specific recombination cassettes in BIBAC vectors.</a> Plant Mol Biol. 66 (6), 587-598 (2008).</li><li>Anggoro, D. T., Tark-Dame, M., Walmsley, A., Oka, R., de Sain, M., Stam, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BIBAC-GW-based+vectors+for+generating+reporter+lines+for+site-specific+genome+editing+in+planta.">BIBAC-GW-based vectors for generating reporter lines for site-specific genome editing in planta.</a> Plasmid. 89, 27-36 (2017).</li><li>Hamilton, C. M., Frary, A., Lewis, C., Tanksley, S. 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+transfer+of+intact+high+molecular+weight+DNA+into+plant+chromosomes.">Stable transfer of intact high molecular weight DNA into plant chromosomes.</a> Proc Natl Acad Sci U S A. 93 (18), 9975-9979 (1996).</li><li>Belele, C. L., Sidorenko, L., Stam, M., Bader, R., Arteaga-Vazquez, M. A., Chandler, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+tandem+repeats+are+sufficient+for+paramutation-induced+trans-generational+silencing.">Specific tandem repeats are sufficient for paramutation-induced trans-generational silencing.</a> PLoS Genet. 9 (10), e1003773 (2013).</li><li>Shizuya, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+stable+maintenance+of+300-kilobase-pair+fragments+of+human+DNA+in+Escherichia+coli+using+an+F-factor-based+vector.">Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector.</a> Proc Natl Acad Sci U S A. 89 (18), 8794-8797 (1992).</li><li>Shi, X., Zeng, H., Xue, Y., Luo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pair+of+new+BAC+and+BIBAC+vectors+that+facilitate+BAC/BIBAC+library+construction+and+intact+large+genomic+DNA+insert+exchange.">A pair of new BAC and BIBAC vectors that facilitate BAC/BIBAC library construction and intact large genomic DNA insert exchange.</a> Plant Methods. 7, 33 (2011).</li><li>Woodman, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+PCR+of+Intact+Bacteria+(Colony+PCR).">Direct PCR of Intact Bacteria (Colony PCR).</a> Curr Protoc Microbiol. , A.3D.1-A.3D.7 (2016).</li><li>Ausubel, F. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mol+Biol.">Mol Biol.</a> Current Protocols in Molecular Biology. 1, (2003).</li><li>Edwards, K., Johnstone, C., Thompson, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+rapid+method+for+the+preparation+of+plant+genomic+DNA+for+PCR+analysis.">A simple and rapid method for the preparation of plant genomic DNA for PCR analysis.</a> Nucleic Acids Res. 19 (6), 1349 (1991).</li><li>Clarke, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cetyltrimethyl+ammonium+bromide+(CTAB)+DNA+miniprep+for+plant+DNA+isolation.">Cetyltrimethyl ammonium bromide (CTAB) DNA miniprep for plant DNA isolation.</a> Cold Spring Harb Protoc. (3), (2009).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+Gel+Electrophoresis+for+the+Separation+of+DNA+Fragments.">Agarose Gel Electrophoresis for the Separation of DNA Fragments.</a> J Vis Exp. (62), e3923 (2012).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning. , (2012).</li><li>Sosa, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatography+with+Sephadex+Gels.">Chromatography with Sephadex Gels.</a> Anal Chem. 52 (6), 910-912 (1980).</li><li>Depicker, A., Stachel, S., Dhaese, P., Zambryski, P., Goodman, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nopaline+synthase:+transcript+mapping+and+DNA+sequence.">Nopaline synthase: transcript mapping and DNA sequence.</a> J Mol Appl Genet. 1 (6), 561-573 (1982).</li><li>Feng, J., Vick, B. A., Lee, M. K., Zhang, H. B., Jan, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+BAC+and+BIBAC+libraries+from+sunflower+and+identification+of+linkage+group-specific+clones+by+overgo+hybridization.">Construction of BAC and BIBAC libraries from sunflower and identification of linkage group-specific clones by overgo hybridization.</a> Theor Appl Genet. 113 (1), 23-32 (2006).</li><li>Lee, M. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Construction+of+a+plant-transformation-competent+BIBAC+library+and+genome+sequence+analysis+of+polyploid+Upland+cotton+(Gossypium+hirsutum+L).">Construction of a plant-transformation-competent BIBAC library and genome sequence analysis of polyploid Upland cotton (Gossypium hirsutum L).</a> BMC Genomics. 14, 208 (2013).</li><li>Wang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large+insert+Thellungiella+halophila+BIBAC+library+for+genomics+and+identification+of+stress+tolerance+genes.">A large insert Thellungiella halophila BIBAC library for genomics and identification of stress tolerance genes.</a> Plant Mol Biol. 72 (1-2), 91-99 (2010).</li><li>Wu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+BAC-+and+BIBAC-based+physical+map+of+the+soybean+genome.">A BAC- and BIBAC-based physical map of the soybean genome.</a> Genome Res. 14 (2), 319-326 (2004).</li><li>Xu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+physical+mapping+from+large-insert+clones+by+fingerprint+analysis+with+capillary+electrophoresis:+a+robust+physical+map+of+Penicillium+chrysogenum.">Genome physical mapping from large-insert clones by fingerprint analysis with capillary electrophoresis: a robust physical map of Penicillium chrysogenum.</a> Nucleic Acids Res. 33 (5), e50 (2005).</li><li>Zhang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+physical+mapping+of+polyploids:+a+BIBAC+physical+map+of+cultivated+tetraploid+cotton,+Gossypium+hirsutum+L.">Genome physical mapping of polyploids: a BIBAC physical map of cultivated tetraploid cotton, Gossypium hirsutum L.</a> PLoS One. 7 (3), e33644 (2012).</li><li>Frary, A., Hamilton, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficiency+and+stability+of+high+molecular+weight+DNA+transformation:+An+analysis+in+tomato.">Efficiency and stability of high molecular weight DNA transformation: An analysis in tomato.</a> Transgenic Res. , (2001).</li><li>Stam, M., Viterbo, A., Mol, J. N. M., Kooter, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Position-Dependent+Methylation+and+Transcriptional+Silencing+of+Transgenes+in+Inverted+T-DNA+Repeats:+Implications+for+Posttranscriptional+Silencing+of+Homologous+Host+Genes+in+Plants.">Position-Dependent Methylation and Transcriptional Silencing of Transgenes in Inverted T-DNA Repeats: Implications for Posttranscriptional Silencing of Homologous Host Genes in Plants.</a> Cell Biol. 18 (11), 6165-6177 (1998).</li><li>G&#322;owacka, K., Kromdijk, J., Leonelli, L., Niyogi, K. K., Clemente, T. E., Long, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+evaluation+of+new+and+established+methods+to+determine+T-DNA+copy+number+and+homozygosity+in+transgenic+plants.">An evaluation of new and established methods to determine T-DNA copy number and homozygosity in transgenic plants.</a> Plant Cell Environ. 39 (4), 908-917 (2016).</li><li>Stefano, B., Patrizia, B., Matteo, C., Massimo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inverse+PCR+and+Quantitative+PCR+as+Alternative+Methods+to+Southern+Blotting+Analysis+to+Assess+Transgene+Copy+Number+and+Characterize+the+Integration+Site+in+Transgenic+Woody+Plants.">Inverse PCR and Quantitative PCR as Alternative Methods to Southern Blotting Analysis to Assess Transgene Copy Number and Characterize the Integration Site in Transgenic Woody Plants.</a> Biochem Genet. 54 (3), 291-305 (2016).</li></ol>

Os autores declaram não concorrentes interesses financeiros ou outros conflitos de interesse.

Crítico para a geração de transgênicos com integrações único, intactos de um transgene é a escolha do vetor binário usado. Vetores de família BIBAC têm sido utilizados para entregar muitos planta espécie23,24,25,26,,27,28-sequências de interesses. Vetores BIBAC, incluindo BIBAC-GW, rendem cópia única integraç?...

Ao gerar plantas transgênicas, geralmente, o objetivo é ter o transgene integrado estàvel expressa. Isto pode ser conseguido por integrações intacta cópia única de um transgene. Múltiplas integrações podem levar ao aumento da expressão de um transgene, mas também para o silenciamento de genes. Silenciamento de transgenes é mais provável se sequências inseridas são dispostas em tandem ou repetições invertidas1,2,3,4. Vetores binários são usadas como naves em Agrobacterium-mediada experimentos de transformação para entregar as sequências de interesse em genomas de planta. O número de integrações no genoma planta é dependente do número de cópia do vetor binário em Agrobacterium tumefaciens5,6. Muitos vetores binários comumente usados são vetores de alta cópia e, portanto, produzir um número de cópia do transgene média alta: cópias de 3.3 para 4,9 em Arabidopsis5.
O número de integrações de T-DNA pode ser diminuído usando vetores binários que têm um número baixo-cópia em a. tumefaciens, tais como BIBAC7, ou através do lançamento de um T-DNA da . tumefaciens cromossoma5. O número médio de integrações do transgene em tais casos é inferior a 25,8,9,10. Devido a ser cópia única na . tumefaciens e também em Escherichia coli, BIBAC-derivados podem manter e entregar as construções tão grandes quanto 150 kb,11.
Compatível com o GW BIBAC vetores10,12 permitir fácil introdução de genes de interesse para o vetor usando Gateway clonagem. O uso da tecnologia de Gateway simplifica o procedimento de clonagem, mas também supera os problemas comuns associados com13,grandes vetores de baixo-cópia-número14, como um baixo rendimento de DNA e uma seleção limitada de restrição exclusiva sites disponíveis para clonagem de7,11. Os derivados de pBIBAC-GW estão disponíveis com qualquer resistência ao glufosinato-amônio (pBIBAC-BAR-GW) ou DsRed fluorescência em revestimentos de semente (pBIBAC-RFP-GW) para seleção em plantas (Figura 1)10,12. Para os dois vetores, um gene de resistência canamicina é usado como marcador de seleção em bactérias.
Combinam os vetores de pBIBAC-GW: design (1) fácil e manipulação genética em e. colie integrações de cópia única (2) intacto em planta com alta eficiência. O rendimento de vetores de pBIBAC-GW em integrações de média 1,7 em Arabidopsis com aproximadamente metade das plantas transgênicas carregando um único integrado T-DNA10.
Expressao de transgenes é um requisito para a maioria dos transgênicos gerados. Expressão do transgene estável pode ser conseguida integrações intactas, cópia única. Trabalhar com plantas transgênicas carregando integrações intactas, cópia única é, no entanto, ainda mais importante, se por exemplo, o objetivo é estudar a eficiência de processos baseados em cromatina, tais como recombinação, mutagênese ou reparação e a dependência destes processos no genoma e a estrutura da cromatina no local da inserção. Para nosso interesse, para estudar a dependência de mutagênese do oligonucleotide dirigido (ODM) no contexto genômico do local, um conjunto de linhas de repórter com integrações intactos, cópia única de um gene repórter de mutagénese foi gerado (Figura 2)10. Usando este conjunto de linhas, foi demonstrado que a eficiência ODM varia entre loci transgénicos integrado em diferentes localizações genômicas, apesar dos níveis de expressão do transgene ser bastante semelhante.

<table><tbody><tr><td>Kanamycin sulphate monohydrate</td><td>Duchefa</td><td>K0126</td><td></td></tr><tr><td>Gentamycin sulphate</td><td>Duchefa</td><td>G0124</td><td></td></tr><tr><td>Rifampicin</td><td>Duchefa</td><td>R0146</td><td></td></tr><tr><td>Tetracycline hydrochloride</td><td>Sigma</td><td>T-3383</td><td></td></tr><tr><td>DB3.1 competent cells</td><td>Thermo Scientific - Invitrogen</td><td>11782-018</td><td>One Shot &lt;em&gt;ccdB&lt;/em&gt; Survival 2 T1R Competent Cells (A10460) by Invitrogen or any other ccdB resistant &lt;em&gt;E. coli&lt;/em&gt; strain can be used instead&amp;nbsp;&amp;nbsp;</td></tr><tr><td>DH10B competent cells</td><td>Thermo Scientific - Invitrogen</td><td>18290-015</td><td></td></tr><tr><td>Gateway LR clonase enzyme mix&amp;nbsp;</td><td>Thermo Scientific - Invitrogen</td><td>11791-019</td><td></td></tr><tr><td>tri-Sodium citrate dihydrate</td><td>Merck</td><td>106432</td><td></td></tr><tr><td>Trizma base</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>EDTA disodium dihydrate</td><td>Duchefa</td><td>E0511</td><td></td></tr><tr><td>Proteinase K</td><td>Thermo Scientific&amp;nbsp;</td><td>EO0491</td><td></td></tr><tr><td>Bacto tryptone</td><td>BD</td><td>211705</td><td></td></tr><tr><td>Yeast extract</td><td>BD</td><td>212750</td><td></td></tr><tr><td>Sodium chloride</td><td>Honeywell Fluka</td><td>13423</td><td></td></tr><tr><td>Potassium chloride</td><td>Merck</td><td>104936</td><td></td></tr><tr><td>D(+)-Glucose monohydrate</td><td>Merck</td><td>108346</td><td></td></tr><tr><td>Electroporation Cuvettes, 0.1 cm gap</td><td>Biorad</td><td>1652089</td><td></td></tr><tr><td>Electroporator Gene Pulser</td><td>BioRad</td><td></td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Calbiochem</td><td>442613</td><td></td></tr><tr><td>D(+)-Maltose monohydrate 90%</td><td>Acros Organics</td><td>32991</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>84100</td><td></td></tr><tr><td>Silwet L-77</td><td>Fisher Scientific</td><td>NC0138454</td><td></td></tr><tr><td>Murashige Skoog medium</td><td>Duchefa</td><td>M0221</td><td></td></tr><tr><td>Agar</td><td>BD</td><td>214010</td><td></td></tr><tr><td>Glufosinate-ammonium (Basta)</td><td>Bayer</td><td>79391781</td><td></td></tr><tr><td>Restriction enzymes</td><td>NEB</td><td></td><td></td></tr><tr><td>Ethidium Bromide</td><td>Bio-Rad</td><td>1610433</td><td></td></tr><tr><td>Electrophoresis system</td><td>Bio-Rad</td><td></td><td></td></tr><tr><td>Sodium hydroxide</td><td>Merck</td><td>106498</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Merck</td><td>100316</td><td></td></tr><tr><td>Blotting nylon membrane Hybond N+</td><td>Sigma Aldrich</td><td>15358</td><td>or GE Healthcare Life Sciences (RPN203B)</td></tr><tr><td>Whatman 3MM Chr blotting paper</td><td>GE Healthcare Life Sciences</td><td>3030-931</td><td></td></tr><tr><td>dNTP</td><td>Thermo Fisher</td><td>R0181</td><td></td></tr><tr><td>Acetylated BSA</td><td>Sigma-Aldrich</td><td>B2518</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H4034</td><td></td></tr><tr><td>2-Mercaptoethanol</td><td>Merck</td><td>805740</td><td></td></tr><tr><td>Sephadex G-50 Coarse</td><td>GE Healthcare Life Sciences</td><td>17004401</td><td>or Sephadex G-50 Medium (17004301)</td></tr><tr><td>Dextran sulfate sodium salt</td><td>Sigma-Aldrich</td><td>D8906</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate&amp;nbsp;</td><td>US Biological</td><td>S5010</td><td></td></tr><tr><td>Salmon Sperm DNA</td><td>Sigma-Aldrich</td><td>D7656</td><td></td></tr><tr><td>Sodium dihydrogen phosphate monohydrate</td><td>Merck</td><td>106346</td><td></td></tr><tr><td>Storage Phosphor screen and casette</td><td>GE Healthcare Life Sciences</td><td>28-9564-74</td><td></td></tr><tr><td>Phosphor imager</td><td>GE Healthcare Life Sciences</td><td>Typhoon FLA 7000</td><td></td></tr><tr><td>UV Crosslinker</td><td>Stratagene</td><td>Stratalinker 1800</td><td></td></tr><tr><td>cling film (Saran wrap)</td><td>Omnilabo</td><td>1090681</td><td></td></tr><tr><td>Agarose</td><td>Thermo Scientific - Invitrogen</td><td>16500</td><td></td></tr><tr><td>Boric acid</td><td>Merck</td><td>100165</td><td></td></tr><tr><td>DNA marker &amp;lsquo;Blauw&amp;rsquo;; DNA ladder.</td><td>MRC Holland</td><td>MCT8070</td><td></td></tr><tr><td>DNA marker &amp;lsquo;Rood&amp;rsquo;; DNA ladder</td><td>MRC Holland</td><td>MCT8080</td><td></td></tr><tr><td>Hexanucleotide Mix</td><td>Roche</td><td>11277081001</td><td></td></tr><tr><td>Large-Construct Kit</td><td>Qiagen</td><td>12462</td><td></td></tr><tr><td>Heat-sealable polyethylene tubing, clear</td><td>various providers</td><td></td><td>the width of the tubing should be wider than that of blotting membrane</td></tr><tr><td>Heat sealer</td><td></td><td></td><td></td></tr><tr><td>Membrane filter disk</td><td>Merck</td><td>VSWP02500</td><td></td></tr><tr><td>Magnesium chloride</td><td>Merck</td><td>105833</td><td></td></tr><tr><td>Hybridization mesh</td><td>GE Healthcare Life Sciences</td><td>RPN2519</td><td></td></tr></tbody></table>

generating transgenic plants with single-copy insertions using bibac-gw binary vector

Ao gerar plantas transgênicas, em geral, o objetivo é ter expressão estável de um transgene. Isto requer única intacta de uma integração do transgene, como cópia multi integrações são frequentemente sujeitas a silenciamento de genes. O vetor binário compatível com o Gateway com base em cromossomos artificiais bacterianos (pBIBAC-GW), como outros derivados de pBIBAC, permite a inserção dos transgenes cópia única com alta eficiência. Como uma melhoria para o pBIBAC original, uma gaveta de Gateway foi clonada em pBIBAC-GW, para que as sequências de interesse podem agora ser facilmente incorporadas a transferência de vector DNA (T-DNA) clonando Gateway. Comumente, a transformação com pBIBAC-GW resulta em uma eficiência de 0,2-0,5%, segundo o qual metade dos transgênicos a transportar uma integração de cópia única intacta do T-DNA. Os vetores de pBIBAC-GW estão disponíveis com resistência ao glufosinato-amônio ou DsRed fluorescência em revestimentos de semente para seleção em plantas e com resistência à canamicina como uma seleção em bactérias. Aqui, uma série de protocolos é apresentada que guia o leitor através do processo de geração de plantas transgênicas usando pBIBAC-GW: a partir de recombinar as sequências de interesse em vetor de escolha, para plantar a transformação com pBIBAC-GW Agrobacterium, seleção dos transgênicos e teste as plantas para integridade e cópia número de inserções usando a DNA mancha. Atenção é dada a concepção de uma estratégia de mancha de DNA para reconhecer integrações de single e multi cópias em único e múltiplos loci.

Usando o sistema de BIBAC-GW, construções de repórter para estudar ODM em plantas foram gerados10. Construções foram projetadas no Gateway de entrada vetor pENTR-gm12 e inseridas no pBIBAC-BAR-GW (Figura 1), usando a reação de recombinação Gateway LR.
Arabidopsis foram transformados com pDM19, um BIBAC-BAR-GW plasmídeo com um repórter de mTu...

Watch this Scientific Journal Video about Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector at JoVE.com

Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

Usando um vetor binário pBIBAC-GW faz gerar plantas transgénicas com inserções de cópia única intactas, um processo fácil. Aqui, uma série de protocolos é apresentada que guia o leitor através do processo de geração de plantas transgênicas de Arabidopsis e teste as plantas para integridade e copiar o número de inserções.

Geração de plantas transgênicas com inserções de cópia única usando vetor binário BIBAC-GW

When generating transgenic plants, generally the objective is to have stable expression of a transgene. This requires a single, intact integration of the ...

generating-transgenic-plants-with-single-copy-insertions-using-bibac

Research

JoVE Journal

Genetics

12.4K visualizações.  University of Amsterdam. Usando um vetor binário pBIBAC-GW faz gerar plantas transgénicas com inserções de cópia única intactas, um processo fácil. Aqui, uma série de protocolos é apresentada que guia o leitor através do processo de geração de plantas transgênicas de Arabidopsis e teste as plantas para integridade e copiar o número de inserções.

Vídeo: Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector

Transient Gene Expression in Tobacco using Gibson Assembly and the Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately using complex regulatory strategies including differential promoters and/or signal sequences. Metabolic engineers and synthetic biologists interested in targeting enzymes to a particular organelle are faced with a challenge: For a protein that is to be localized to more than one organelle, the engineer must clone the same gene multiple times. This work presents a solution to this strategy: harnessing alternative splicing of mRNA. This technology takes advantage of established chloroplast and peroxisome targeting sequences and combines them into a single mRNA that is alternatively spliced. Some splice variants are sent to the chloroplast, some to the peroxisome, and some to the cytosol. Here the system is designed for multiple-organelle targeting with alternative splicing. In this work, GFP was expected to be expressed in the chloroplast, cytosol, and peroxisome by a series of rationally designed 5&#8217; mRNA tags. These tags have the potential to reduce the amount of cloning required when heterologous genes need to be expressed in multiple subcellular organelles. The constructs were designed in previous work11, and were cloned using Gibson assembly, a ligation independent cloning method that does not require restriction enzymes. The resultant plasmids were introduced into Nicotiana benthamiana epidermal leaf cells with a modified Gene Gun protocol. Finally, transformed leaves were observed with confocal microscopy.

This work describes a novel method for selectively targeting subcellular organelles in plants, assayed using the BioRad Gene Gun.

Expressão Gênica transiente em tabaco usando Assembleia Gibson eo Gene Gun

In order to target a single protein to multiple subcellular organelles, plants typically duplicate the relevant genes, and express each gene separately ...

Ambiente

Modifying Baculovirus Expression Vectors to Produce Secreted Plant Proteins in Insect Cells

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The baculovirus-mediated insect cell expression system is one of the systems used to express recombinant eukaryotic secretory proteins with some post-translational modifications. The secretory proteins need to be routed through the secretory pathways for protein glycosylation, disulfide bonds formation, and other post-translational modifications. To improve the existing insect cell expression of secretory plant proteins, a baculovirus expression vector is modified by the addition of either a GP67 or a hemolin signal peptide sequence between the promoter and multiple-cloning sites. This newly designed modified vector system successfully produced a high yield of soluble recombinant secreted plant receptor proteins of Arabidopsis thaliana. Two of the expressed plant proteins, the extracellular domains of Arabidopsis TDR and PRK3 plasma membrane receptors, were crystallized for X-ray crystallographic studies. The modified vector system is an improved tool that can potentially be used for the expression of recombinant secretory proteins in the animal kingdom as well.

Here, we present the protocols for utilizing the insect cell and baculovirus protein expression system to produce large quantities of plant secreted proteins for protein crystallization. A baculovirus expression vector has been modified with either GP67 or insect hemolin signal peptide for plant protein secretion expression in insect cells.

Modificar vetores de expressão de Baculovirus para produzir secretado proteínas vegetais em células de inseto

It has been a challenge for scientists to express recombinant secretory eukaryotic proteins for structural and biochemical studies. The ...

Bioquímica

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Inducible, expressão de tipo específico de célula em Arabidopsis thaliana mediada através de LhGR Trans-ativação

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

Genética

Rapid Assembly of Multi-Gene Constructs using Modular Golden Gate Cloning

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that cut outside of their recognition sites and create a short overhang. This modular cloning (MoClo) system uses a hierarchical workflow in which different DNA parts, such as promoters, coding sequences (CDS), and terminators, are first cloned into an entry vector. Multiple entry vectors then assemble into transcription units. Several transcription units then connect into a multi-gene plasmid. The Golden Gate cloning strategy is of tremendous advantage because it allows scar-less, directional, and modular assembly in a one-pot reaction. The hierarchical workflow typically enables the facile cloning of a large variety of multi-gene constructs with no need for sequencing beyond entry vectors. The use of fluorescent protein dropouts enables easy visual screening. This work provides a detailed, step-by-step protocol for assembling multi-gene plasmids using the yeast modular cloning (MoClo) kit. We show optimal and suboptimal results of multi-gene plasmid assembly and provide a guide for screening for colonies. This cloning strategy is highly applicable for yeast metabolic engineering and other situations in which multi-gene plasmid cloning is required.

The goal of this protocol is to provide a detailed, step-by-step guide for assembling multi-gene constructs using the modular cloning system based on Golden Gate cloning. It also gives recommendations on critical steps to ensure optimal assembly based on our experiences.

Montagem rápida de construções multigênicas usando clonagem modular de golden gate

The Golden Gate cloning method enables the rapid assembly of multiple genes in any user-defined arrangement. It utilizes type IIS restriction enzymes that ...

Bioengenharia

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

A Plataforma de Produção de Proteína Complexo MultiBac no EMBL

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Biologia

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most commonly used agent for agroinfiltration is Agrobacterium tumefaciens, a pathogen of many dicot plant species. This implies that agroinfiltration can be applied to many plant species. Here, we present our protocols and expected results when applying these methods to the potato (Solanum tuberosum), its related wild tuber-bearing Solanum species (Solanum section Petota) and the model plant Nicotiana benthamiana. In addition to functional analysis of single genes, such as resistance (R) or avirulence (Avr) genes, the agroinfiltration assay is very suitable for recapitulating the R-AVR interactions associated with specific host pathogen interactions by simply delivering R and Avr transgenes into the same cell. However, some plant genotypes can raise nonspecific defense responses to Agrobacterium, as we observed for example for several potato genotypes. Compared to agroinfiltration, detection of AVR activity with PVX agroinfection is more sensitive, more high-throughput in functional screens and less sensitive to nonspecific defense responses to Agrobacterium. However, nonspecific defense to PVX can occur and there is a risk to miss responses due to virus-induced extreme resistance. Despite such limitations, in our experience, agroinfiltration and PVX agroinfection are both suitable and complementary assays that can be used simultaneously to confirm each other&#39;s results.

Agroinfiltration and PVX Agroinfection in Potato and Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are routine functional assays for transient ectopic expression of genes in plants. These methods are efficient assays in effectoromics strategies (rapid resistance and avirulence gene discovery) and crucial to modern research in molecular plant pathology. They meet the demand for robust high-throughput functional analysis in plants.

Agroinfiltração e Agroinfecção PVX em Batata e Nicotiana benthamiana

Agroinfiltration and PVX agroinfection are two efficient transient expression assays for functional analysis of candidate genes in plants. The most ...

<meta charset="utf8"></head><body>Edição eficiente de genoma baseada em CRISPR para amido resistente no melhoramento de arroz

Breeding by Design for Functional Rice with Genome Editing Technologies

The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and mutation breeding, face challenges in efficiently introducing targeted traits and generating diverse plant populations. Conversely, the emergence of genome editing technologies has ushered in a paradigm shift, enabling the precise and expedited manipulation of plant genomes to intentionally introduce desired characteristics. One of the most widespread editing tools is the CRISPR/Cas system, which has been used by researchers to study important biology-related problems. However, the precise and effective workflow of genome editing has not been well-defined in crop breeding. In this study, we demonstrated the entire process of breeding rice varieties enriched with high levels of resistant starch (RS), a functional trait that plays a crucial role in preventing diseases such as diabetes and obesity. The workflow encompassed several key steps, such as the selection of functional SBEIIb gene, designing the single-guide RNA (sgRNA), selecting an appropriate genome editing vector, determining the vector delivery method, conducting plant tissue culture, genotyping mutation and phenotypic analysis. Additionally, the time frame necessary for each stage of the process has been clearly demonstrated. This protocol not only streamlines the breeding process but also enhances the accuracy and efficiency of trait introduction, thereby accelerating the development of functional rice varieties.

<meta charset="utf8"></head><body>Autor em destaque: Simplificando o melhoramento de arroz com CRISPR / Cas para obter características fenotípicas e agronômicas ideais

The protocol describes breeding resistant starch rice varieties by design using genome editing technologies in a precise, efficient, and technically simple way.

Melhoramento por Design para Arroz Funcional com Tecnologias de Edição de Genoma

The conventional approaches to crop breeding, which rely predominantly on time-consuming and labor-intensive methods such as traditional hybridization and ...

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.
In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.

Floral-Dip Transformation of Flax Linum usitatissimum to Generate Transgenic Progenies with a High Transformation Rate

Here, we present a protocol to transform flax using Agrobacterium-mediated plant transformation via floral-dip. This protocol is simple to perform and inexpensive, yet yields a higher transformation rate than the current available methods for flax transformation.

Floral-Dip Transformação de linho (<em&gt; Linum usitatissimum</em&gt;) Para gerar Transgênicos Progênies com uma alta taxa de Transformação

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be ...

Geração de plantas transgênicas com inserções de cópia única usando vetor binário BIBAC-GW

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