Name: generating transgenic plants with single-copy insertions using bibac-gw binary vector
Uploaded: 2018-03-28T14:30:00
Description: Watch this Scientific Journal Video about Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector at JoVE.com

Denne forskning er støttet af den hollandske teknologi Foundation STW (12385), som er en del af den nederlandske organisation for videnskabelig forskning (NWO), og som er delvis finansieret af Ministeriet for økonomiske anliggender (OTP Grant 12385 til MS).  Vi takker Carol M. Hamilton (Cornell University, USA) for at give pCH20, rygraden i BIBAC-GW vektorer.

1. indsætte sekvenser af interesse i binære vektor
<ol><li>
Forberede Gateway indrejse og binære vektorer.
	<ol>
<li>Isolere Gateway post vektor indeholdende en DNA fragmenter eller gen af interesse ved hjælp af en mini prep kit efter forslag af leverandøren. Bemærk: BIBAC-GW vektorer kræver brug af kanamycin (Km) til valg i bakterier, derfor, bruge en løsning vektor med en anden modstand markør i stedet for kanamycin. For eksempel, er pENTR-gm vektor, bærer en Gentamicin resistens gen, et...

<ol>
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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>

Kritisk til at generere transgenics med enkelt, intakt integrationer af en transgen er valget af den binære vektor, der anvendes. BIBAC familie vektorer har været brugt til at levere sekvenser af interesser mange plante arter23,24,25,26,27,28. BIBAC vektorer, herunder BIBAC-GW, give enkelt-kopi integrationer med høj effekt...

Når du genererer transgene planter, er normalt målet at have integreret transgeners stabilt udtrykt. Dette kan opnås ved intakt enkelt kopi integrationer af en transgenet. Flere integrationer kan føre til øget udtryk for en transgen, men også til genhæmning. Hæmning af transgener er mere sandsynlige, hvis indsatte sekvenser er arrangeret i tandem eller omvendt gentagelser1,2,3,4. Binære vektorer bruges som transport i Agrobacterium-medieret transformation eksperimenter for at levere sekvenser af interesse i anlægget genomer. Antallet af integrationer i en plante genom er afhængig af kopi antallet af den binære vektor i Agrobacterium tumefaciens5,6. Mange almindeligt anvendte binær vektorer er høj kopi vektorer, og derfor give en høj gennemsnitlig transgen kopi nummer: 3.3 til 4,9 eksemplarer i Arabidopsis5.
Antallet af T-DNA integrationer kan sænkes ved hjælp af binær vektorer, der har en lav-kopi nummer i A. tumefaciens, såsom BIBAC7, eller ved at lancere en T-DNA fra A. tumefaciens kromosom5. Det gennemsnitlige antal af transgenet integrationer i sådanne tilfælde er under 25,8,9,10. På grund af er enkelt-kopi i A. tumefaciens, og også i Escherichia coli, BIBAC-derivater kan vedligeholde og levere konstruktioner så stor som 150 kb11.
GW-kompatible BIBAC vektorer10,12 tillader nem indførelsen af gener af interesse vektor bruger Gateway kloning. Brugen af Gateway teknologi forenkler proceduren for kloning, men også overvinder almindelige problemer i forbindelse med store lav-kopi-nummer vektorer13,14, såsom et lavt DNA udbytte og et begrænset udvalg af unikke begrænsning sites tilgængelige for kloning7,11. PBIBAC-GW derivater er tilgængelige med enten resistens over for ammoniumglufosinat (pBIBAC-BAR-GW) eller DsRed fluorescens i frø frakker (pBIBAC-RFP-GW) for udvalg i planter (figur 1)10,12. For begge vektorer bruges et kanamycin resistens gen som udvalg markør i bakterier.
PBIBAC-GW vektorer kombinere: (1) let design og genmanipulation i E. coli, (2) intakt enkelt-kopi integrationer og planta på høj effektivitet. PBIBAC-GW vektorer udbytte på gennemsnit 1,7 integrationer i Arabidopsis med ca. halvdelen af de transgene planter bærer en enkelt integreret T-DNA10.
Stabil udtryk af transgener er et krav for de fleste transgenics genereret. Stabil transgen udtryk kan opnås ved intakt, enkelt-kopi integrationer. Arbejde med transgene planter bærer intakt, enkelt-kopi integrationer er imidlertid endnu vigtigere, hvis for eksempel, formålet er at undersøge effektiviteten af kromatin-baserede processer, såsom mutagenese, rekombination, eller reparation og afhængighed af disse processer på lokationen genomisk og kromatin struktur på indsættelsesstedet. For vores interesse for at studere afhængighed af oligonukleotid instrueret mutagenese (ODM) på den lokale genomiske kontekst, blev et sæt reporter linjer med intakt, enkelt-kopi integrationer af en mutagenese reporter gen genereret (figur 2)10. Bruger dette sæt af linjer, blev det vist, at ODM effektivitet varierer mellem transgene loci integreret på forskellige genomisk steder, trods de transgene udtryk niveauer er temmelig lignende.

<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 ccdB Survival 2 T1R Competent Cells (A10460) by Invitrogen or any other ccdB resistant E. coli strain can be used instead&#160;&#160;</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&#160;</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&#160;</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&#160;</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 &#8216;Blauw&#8217;; DNA ladder.</td>
			<td>MRC Holland</td>
			<td>MCT8070</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA marker &#8216;Rood&#8217;; 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

Når du genererer transgene planter, generelt er målet at have stabile udtryk for en transgen. Dette kræver en enkelt, intakt integration af transgenet, som multi kopi integrationer udsættes ofte for genhæmning. Gateway-kompatible binære vektor baseret på bakteriel kunstige kromosomer (pBIBAC-GW), ligesom andre pBIBAC derivater, giver mulighed for isætning af enkelt-kopi transgener med høj effektivitet. Som en forbedring af den oprindelige pBIBAC, er en Gateway kassette blevet klonet i pBIBAC-GW, så at sekvenser af interesse kan nu let integreres i vektor overførsel DNA (T-DNA) af Gateway kloning. Almindeligt, transformation med pBIBAC-GW resulterer i en effektivitet på 0,2-0,5%, hvorved halvdelen af transgenics bære en intakt enkelt-kopi integration af T-DNA. PBIBAC-GW vektorer fås med resistens over for ammoniumglufosinat eller DsRed fluorescens i frø frakker til udvalg i planter og modstand mod kanamycin som en markering i bakterier. Her, en række protokoller er præsenteret som guide læseren gennem processen til oprettelse af transgene planter ved hjælp af pBIBAC-GW: start fra rekombinerer sekvenser af interesse i pBIBAC-GW vektor af valg, at plante transformation med Agrobacterium, udvalg af transgenics, og test planter til handelsformer og kopier række indsætter ved hjælp af DNA blotting. Opmærksomhed er givet til at designe en DNA blotting strategi at genkende single og multi kopi integrationer på enkelt- og flersidede loci.

Ved hjælp af BIBAC-GW system, blev reporter konstruktioner for at studere ODM i planterne genereret10. Konstruktioner blev designet i Gateway post vektor pENTR-gm12 og indsat i pBIBAC-BAR-GW (figur 1) ved hjælp af Gateway LR rekombination reaktion.
Arabidopsis blev omdannet med pDM19, en BIBAC-BAR-GW plasmid med en mTurquoise-eYFP reporter transporte...

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

Ved hjælp af en pBIBAC-GW binære vektor gør skabe transgene planter med intakt enkelt-kopi indsættelser, en nem proces. Her, præsenteres en række protokoller som guide læseren gennem processen med at generere gensplejsede Arabidopsis planter, og test planter til handelsformer, og kopierer antallet af indsætter.

Generering af transgene planter med enkelt-kopi indrykninger ved hjælp af BIBAC-GW binære vektor

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

The overall goal of this methodology is to generate transgenic plants carrying intact, single-copy insertions of a transfer DNA, or T-DNA, in an efficient manner. The BIBAC-Gateway vector is a valuable tool for generating transgenic plants that carry intact, single-copy insertions of sequences of interest that show stable gene expression. With the BIBAC-Gateway vector, one can use Gateway recombination technology to insert sequences of interest with minimal effort.All BIBAC derivatives, including the BIBAC-Gateway vectors, are suitable for transferring large, transgenic DNA fragments into plant genomes. The BIBAC-GW vectors can be used to insert sequences of interest into many plant systems, such as maize, rice, and tomato. There are two BIBAC-GW plasmids available.The BIBAC-RFP-GW plasmid contains a DsRed gene that is expressed in seed coats. The BIBAC-BAR-GW plasmid contains a gene providing resistance to glufosinate. Both vectors contain a kanamycin-resistant gene as a selection marker in bacteria.To insert the sequences of interest into the binary vector, first prepare the Gateway Entry and binary vectors, as described in the text protocol. To carry out a Gateway reaction, prepare the LR recombination reaction according to the supplier's suggestions in a 1.5-milliliter microcentrifuge tube at room temperature. Mix 100 to 300 nanograms of the supercoiled Entry clone and 300 nanograms of the BIBAC-Gateway vector.Adjust the volume of the mixture to 16 microliters with TE.Finally, add four microliters of LR Clonase enzyme mix, and mix by vortexing. Incubate the mixture at 25 degrees Celsius for one hour. Terminate the LR reaction by adding two microliters of Proteinase K solution to the mixture.Mix and incubate at 37 degrees Celsius for 10 minutes before proceeding with transformation into E.coli, as described in the text protocol. To prepare the Arabidopsis plants for transformation, grow Arabidopsis plants in a greenhouse or climate-controlled growth chamber until they are flowering. Clip the first bolts to allow more secondary bolts to emerge.Plants are ready for dipping four to six days after clipping, when the plants have many immature flower heads and not many fertilized siliques. To perform floral dipping, dip inflorescences for five to 10 seconds in Agrobacterium suspension. Use gentle agitation.Wrap the above-ground parts of the plants in cling film to keep the humidity high, and cover the plant pots with a box to keep the plants in the dark. Incubate the plants for two days in a greenhouse or growth chamber. After two days, remove the box and the cling film and grow the plants to maturity in a greenhouse or growth chamber.To increase the efficiency of transformation, the same plants can be re-dipped seven days after the first dipping. Next, when the transformed plants are mature and dry, harvest the seeds. Pool and analyze the seeds of plants transformed with the same construct as a single set.In case plants are transformed with a BIBAC-RFP-Gateway plasmid derivative, identify transgenic plants by analyzing the seeds using fluorescence microscopy. To detect DsRed expression in seed coats, image the seeds at an excitation of 560 nanometers and emission of 600 to 650 nanometers. Separate the fluorescent seeds from non-fluorescent counterparts using forceps.To screen for transgenic plants transformed with a BIBAC-BAR-Gateway plasmid derivative, sow the seeds in trays filled with soil. Ensure an even spreading of seeds over trays by suspending seeds in 0.1%agar in 0.5x MS medium, and spread the seeds using a one-milliliter pipette. Stimulate the seeds to germinate in a synchronous manner by incubating the seeds for at least two days at four degrees Celsius.This can be done before or after sowing the seeds. Two and three weeks after sowing the seeds, spray the seedlings with 0.5%glufosinate-ammonium solution. Use 500 milliliters of glufosinate-ammonium solution per one square meter.Transfer surviving seedlings to pots. Analyze the glufosinate-ammonium-resistant plants by PCR for the presence of the construct of interest. Determine the number of T-DNA integrations and their integrity by DNA blotting using restriction enzymes.This method allows identification of single as well as repeated integrations at the same or different loci in the genome. Use a series of restriction digestions to identify the different integration patterns possible. Select an enzyme that cuts once in the middle of the T-DNA, to be able to independently probe sequences upstream and downstream of the restriction site.Also, select an enzyme or combination of enzymes cutting out the entire sequence of interest at once. Take care to use only restriction enzymes that are not sensitive to cytosine methylation. After performing DNA blotting, as detailed in the text protocol, perform blot analysis.The analysis depends on the restriction strategy used. When analyzing the blot using the strategy with one restriction site in the middle of the T-DNA, count the number of the fragments detected. In this strategy, the number of hybridized fragments suggest the number of T-DNA integrations.Compare the number of the fragments detected with a probe for the left and the right side of the T-DNA. If a different number of hybridizing fragments is detected with the two probes, then either multiple T-DNA integrations in an inverted repeat or incompletely integrated T-DNAs are present. To identify tandem and inverted arrangements of the T-DNAs, estimate the sizes of the hybridizing fragments on the blot based on the size of the marker bands, and compare the estimated sizes with the expected fragment sizes calculated based on the restriction strategy.To examine if the transgenic sequence of interest is intact, analyze the blot prepared according to the strategy with restriction sites at the extremities of the sequence of interest. Estimate the size of the hybridizing fragments on the blot based on the size of the marker bands, and compare it with the expected size. An intact insertion yields a single fragment with a defined length.Any deviation from the expected length indicates incomplete integration. The DNA restriction and blotting strategy to determine the number and intactness of the T-DNA integrations is shown. The ScaI restriction site divides the reporter sequence into the left border proximal parts that are detected when using a probe for bar and the right border proximal parts that are detected when using a probe for eYFP.The BglII and PciI restriction sites are positioned within the T-DNA, outside the reporter construct. A double digestion results in a fragment with a defined size, and any deviation is an indication for incomplete insertions. The number of hybridizing fragments identified reflects the number of T-DNA insertions.One blot containing genomic DNA of nine transgenics digested with ScaI was hybridized with probes for bar and eYFP. Several lanes indicate transgenics with single T-DNA integrations. Lane four displays two fragments with bar and one with eYFP, indicating either a truncation of the eYFP sequence or inverted repeat around the right border.Lane six displays two fragments with bar and eYFP. One fragment has a weak intensity with the bar probe. The other fragment has a high intensity, indicating two T-DNA insertions, one of which is truncated.After watching this video, you should have a good understanding of how to incorporate your DNA sequence of interest into a BIBAC-GW binary vector using Gateway recombination, followed by Arabidopsis transformation, screening, and assessment of the number of T-DNA integrations and their integrity. Generating and characterizing transgenic plants is time-consuming. When using BIBAC-Gateway as a binary vector, the process of identifying transgenics with intact, single-copy integrations can be shortened.About half of the transgenics generated with BIBAC derivatives carry intact, single-copy integrations. Generally, individuals do not realize that many transgenic plants generated using common binary vectors carry truncated copies of a transgene or transgenes that show gene silencing. With BIBAC derivatives, most of the transgenic plants generated carry intact, single integrations of a transgene that show seldom gene silencing.When using BIBAC-GW for generating transgenics, it is important to transform a sufficient number of plant materials, as the overall transformation efficiency of BIBAC derivatives is lower than that of commonly used binary vectors. The transformation efficiency of BIBAC-GW is generally between 0.2%and 0.5%After identifying transgenic plants carrying intact, single-copy T-DNA integrations, if needed, further analysis can be performed to define the precise genomic location of the T-DNA insertion. Transgenics with intact, single-copy integrations show comparable expression levels of the transgenes.This indicates that integration position of the T-DNA in the genome has little effect on the expression level of the transgene.

Research

JoVE Journal

Genetics

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