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

Это исследование поддерживается голландской технологии фонд STW (12385), которая является частью Нидерландской организации научных исследований (НВО), и которая частично финансируется министерства экономических дел (Грант 12385 OTP для MS).  Мы благодарим Кэрол м. Гамильтон (Корнельский университет, Соединенные Штаты) за предоставление pCH20, костяк BIBAC-GW векторов.

1. Вставка последовательности интерес в бинарный вектор
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Подготовка записи шлюза и бинарных векторов.
	<ol>
<li>Изолируйте запись шлюза вектор, содержащий фрагмент ДНК или гена интереса, используя набор мини-prep согласно предложения поставщика. Примечание: BIBAC-GW векторов тре?...

<ol>
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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. 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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>

Решающее значение для генерации мутация с одной, нетронутыми интеграций трансген – это выбор бинарный вектор, используемый. BIBAC семьи векторов были использованы для доставки последовательности интересов многих растений видов23,24,25,...

При генерации трансгенных растений, обычно цель заключается в том, чтобы иметь комплексный transgene(s) стабильно выразил. Это достигается за счет нетронутыми Поэкземплярная интеграция трансген. Несколько интеграции может привести к увеличению выражение трансген, но и для подавления экспрессии гена. Глушителей трансгенов является более вероятным, если вставленный последовательности расположены в тандеме или перевернутый повторяется1,2,3,4. Бинарных векторов используются как Трансфер в Agrobacterium-опосредованной преобразования экспериментов, чтобы доставить последовательности интерес в геномах растений. Количество внедрений в геном растения зависит количество копию бинарный вектор. Agrobacterium tumefaciens5,6 Многие часто используемые бинарных векторов высокой копии векторы и поэтому дают высокий средний трансген копии номер: 3.3 до 4,9 копий в проростках Arabidopsis5.
Количество внедрений T-ДНК может быть снижен с помощью бинарных векторов, которые имеют низкий копия число в A. tumefaciens, например BIBAC7, или путем запуска T-ДНК от A. tumefaciens хромосомы5. Среднее количество внедрений трансген в таких случаях является ниже 25,8,9,10. Из-за одной копии в A. tumefaciens, а также кишечная палочка, BIBAC-производные может поддерживать и доставить конструкции, как большой, как11150 КБ.
GW-совместимых BIBAC векторов10,12 позволяют легко введение в vector с использованием шлюза клонирования генов интерес. Использование шлюза технологии значительно упрощает процесс клонирования, но также преодолевает общие проблемы, связанные с большими низким копии число векторов13,14, таких как низкая доходность ДНК и ограниченный выбор уникальных ограничений сайты, доступные для клонирования7,11. PBIBAC-GW производные доступны либо сопротивление к глюфосинату аммония (pBIBAC бар-GW) или DsRed флуоресценции в пальто (pBIBAC ЗП-GW) семян для выбора растений (рис. 1)10,12. Для обоих векторов канамицин сопротивления ген используется в качестве маркера выделения бактерий.
PBIBAC-GW векторов объединить: (1) легкий дизайн и генетических манипуляций в E. coliи (2) нетронутыми поштучными внедрений в planta с высокой эффективностью. PBIBAC-GW векторов урожайность в среднем 1,7 внедрений в проростках Arabidopsis с примерно половина трансгенных растений, перевозящих один интегрированный10T-ДНК.
Стабильные выражение трансгенов является требованием для большинства мутация генерируется. Стабильная трансген выражение может быть достиган нетронутыми, одной копии интеграций. Работа с трансгенных растений, перевозящих нетронутыми, одной копии интеграции является, однако, еще более важно, если например, целью является изучение эффективности процессов, основанных на хроматина, например мутагенеза, рекомбинации, или ремонт и зависимость от этих процессы на геномной расположение и структура хроматина в месте вставки. Для наших интересов изучать зависимость олигонуклеотида Направленный мутагенез (ODM) в контексте местных геномной, набор репортер линий с неповрежденными, одной копии интеграций мутагенеза Репортер ген был сгенерированный (рис. 2)10. С помощью этого набора строк, было показано, что эффективность ODM колеблется от трансгенных локусов, интегрированный в разных местах генома, несмотря на довольно аналогичные уровни выражения трансген.

<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

При генерации трансгенных растений, обычно цель заключается в том, чтобы иметь стабильные выражение трансген. Это требует единого, нетронутыми интеграции трансген, интеграции нескольких копирования часто подвергаются сайленсинга генов. Шлюз совместимых бинарный вектор, основанные на бактериальных искусственных хромосом (pBIBAC-GW), как и другие производные pBIBAC, позволяет вставки одной копии трансгенов с высокой эффективностью. Как улучшение в оригинальной pBIBAC шлюз кассету был клонирован в pBIBAC-GW, так что последовательности интерес может теперь быть легко включены в вектор передачи ДНК (T-ДНК) путем клонирования шлюза. Обычно, преобразования с pBIBAC-GW приводит к эффективности 0,2 – 0,5%, при котором половина мутация нести нетронутыми поштучными интеграции T-ДНК. PBIBAC-GW векторов доступны с устойчивость к глюфосинату аммония или DsRed флуоресценции в пальто семян для отбора в растениях и устойчивость к канамицину как выделение бактерий. Здесь, что руководство читателя через процесс создания трансгенных растений, с помощью pBIBAC-GW представлены серии протоколов: начиная от комбинирование последовательности интерес в pBIBAC-GW вектора выбора, к заводе преобразования с Agrobacterium, выбор мутация и тестирования растений для целостности и копировать количество вставок, используя ДНК blotting. Внимание уделяется разработке стратегии переноса ДНК признать одним и несколькими копиями внедрений в одной системе и нескольких локусов.

С помощью системы BIBAC-GW, репортер конструкции для изучения ODM в растениях были созданные10. Конструкции были разработаны в pENTR-gm вектора входа шлюза12 и вставляется в pBIBAC бар-GW (рис. 1) с использованием реакции рекомбинации шлюза LR.<...

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

С помощью бинарный вектор pBIBAC-GW делает создания трансгенных растений с нетронутыми одной копии вставки, легкий процесс. Здесь ряд протоколов представлены Путеводитель читателя через процесс создания трансгенных растениях Arabidopsis и тестирования растений для целостности и копировать количество вставок.

Создание трансгенных растений с одной копии вставки с помощью бинарный вектор 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 ...

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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