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

这项研究得到荷兰技术基金会 STW (12385) 的支持, 该基金是荷兰科学研究组织 (NWO) 的一部分, 由经济事务部 (检察官办公室赠款12385至 MS) 部分资助。 我们感谢卡罗尔 m. 汉密尔顿 (美国康奈尔大学) 提供 pCH20, BIBAC 的主干。

1. 将感兴趣的序列插入二进制向量中
<ol><li>
准备网关条目和二进制向量。
	<ol>
<li>根据供应商的建议, 将包含 DNA 片段或感兴趣基因的网关入口向量隔离。 注: BIBAC-GW 载体需要使用卡那霉素 (公里) 来选择细菌, 因此, 使用一个输入向量与另一种抵抗标记而不是卡那霉素。例如, 携带庆大霉素抗性基因的 pENTR 转基因载体是一个很好的选择12。</li>
		<li>传播和隔离 BIBAC ...

<ol>
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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. 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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. 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对产生转基因的关键是单一的, 完整的整合的转基因是使用二进制向量的选择。BIBAC 家族载体已被用来交付许多植物物种的利益序列23,24,25,26,27,28。BIBAC 向量, 包括 BIBAC, 具有高效率的单拷贝集成: 每行插入的平均数量是1.5 到 2, 而最常用的二进制向量...

在生成转基因植物时, 通常的目的是使综合转基因 (s) 稳定表达。这可以通过转基因的完整的单拷贝集成来实现。多重整合可以导致基因表达的增加, 同时也会使转基因沉默。如果插入的序列排列在串联或反转重复的1、2、3、4中,则更有可能转基因沉默。在农杆菌介导的转化实验中, 二进制向量被用作航天飞机, 将感兴趣的序列传递到植物基因组中。集成到植物基因组的数量依赖于农杆菌5、6中二进制向量的拷贝号.许多常用的二进制向量都是高拷贝向量, 因此产生高平均的转基因拷贝数:拟南芥5中的3.3 到4.9 个拷贝。
通过使用.BIBAC 中具有低拷贝数的二进制向量 (如7) 或从a. 农杆菌染色体5启动 t dna, 可以降低 t dna 积分的数量。在这种情况下, 转基因集成的平均数量低于 25,8,9,10。由于在A. 农杆菌和大肠杆菌中都是单拷贝, BIBAC 衍生物可以维护和交付 150 kb11大的构造。
与 GW 兼容的 BIBAC 矢量10,12允许使用网关克隆轻松地将感兴趣的基因引入到向量中。网关技术的使用大大简化了克隆过程, 也克服了与大型低拷贝数向量13、14相关的常见问题, 如低 DNA 产量和有限的独特限制选择。可用于克隆7、11的站点。在植物 (图 1)10,12中, pBIBAC 的衍生物可在种子大衣 (pBIBAC-RFP) 中对膦铵 (pBIBAC 棒) 或 DsRed 荧光进行抗性。对于这两种载体, 卡那霉素抗性基因被用作细菌的选择标记。
pBIBAC 的向量结合: (1) 易于设计和遗传操作在大肠杆菌和 (2) 完整的单拷贝集成在 planta高效率。在拟南芥中, pBIBAC 的向量平均1.7 积分, 其中大约一半的转基因植株携带着单个集成的 T 型 DNA10。
转基因的稳定表达是大多数转基因产生的必要条件。稳定的转基因表达可以通过完整的单拷贝集成实现。然而, 与转基因植物进行完整的, 单拷贝集成是更重要的, 如果例如, 目的是研究的效率, 染色质的过程, 如诱变, 重组, 或修复, 并依赖这些在插入部位的基因组位置和染色质结构的过程。为了我们的兴趣, 研究寡核苷酸定向诱变 (ODM) 对局部基因组上下文的依赖性, 生成了一组完整的、单拷贝的突变报告基因集成的报告 (图 2)10。使用这组线, 结果表明, 在不同基因组位置集成的转基因基因座的 ODM 效率不同, 尽管转基因表达水平相当相似。

<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

在生成转基因植物时, 一般的目的是要有稳定的转基因表达。这需要一个单一的, 完整的整合的转基因, 因为多拷贝集成往往受到基因沉默。与其他 pBIBAC 衍生物一样, 基于细菌人工染色体 (pBIBAC) 的网关兼容二进制向量允许单拷贝转基因的高效插入。作为对原 pBIBAC 的改进, 一个网关盒已被克隆成 pBIBAC, 因此, 现在的兴趣序列可以很容易地纳入矢量转移 dna (T DNA) 的网关克隆。通常情况下, 与 pBIBAC 的转换结果是0.2–0.5% 的效率, 即转基因的一半携带完整的单拷贝集成的 T DNA。pBIBAC 的载体是可利用的抗膦铵或 DsRed 荧光的种子大衣, 以选择在植物, 和抗卡那霉素作为选择的细菌。在这里, 提出了一系列的协议, 引导读者通过使用 pBIBAC 生成转基因植物的过程: 从将兴趣序列重新结合到 pBIBAC 的选择向量, 到植物转化与农杆菌, 选择转基因, 并测试植物的完整性和复制数量的插入使用 DNA 印迹。注意设计一种 DNA 印迹策略, 以识别单个和多个位点的单拷贝和多复制集成。

利用 BIBAC 系统, 生成了用于研究植物 ODM 的记者构造10。构造是在网关条目向量 pENTR-gm12中设计的, 并使用网关 LR 复合反应插入 pBIBAC BAR (图 1)。
拟南芥是与 pDM19, 一个 BIBAC-GW 质粒与 mTurquoise eYFP 的记者, 携带一个平移停止密码子在 eYFP 阅读框架的位置 120 (mTurquoise2-eYFP*40) ...

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

使用 pBIBAC 的二进制向量, 可以生成具有完整单拷贝插入的转基因植物, 这是一个简单的过程。在这里, 提出了一系列的协议, 指导读者通过生成转基因拟南芥植物的过程, 并测试植物的完整性和复制数量的插入。

用 BIBAC 二元矢量生成单拷贝插入的转基因植物

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

Research

JoVE Journal

Genetics

Peptide-derived Method to Transport Genes and Proteins Across Cellular and Organellar Barriers in Plants

肽衍生的方法在运输跨越植物细胞的基因和蛋白质和细胞器障碍

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as ...

科研

遗传学

Single-cell Gene Expression Profiling Using FACS and qPCR with Internal Standards

单细胞基因表达谱使用流式细胞仪和qPCR内部标准

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those ...

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

Generation of a Gene-disrupted Streptococcus mutans Strain Without Gene Cloning

无基因克隆的基因破坏的变形链球菌菌株的生成

Typical methods for the elucidation of the function of a particular gene involve comparative phenotypic analyses of the wild-type strain and a strain in ...

A Protocol for the Production of Integrase-deficient Lentiviral Vectors for CRISPR/Cas9-mediated Gene Knockout in Dividing Cells

整合酶缺陷慢病毒载体载体的制备及其在细胞 CRISPR/Cas9-mediated 基因敲除中的表达

Lentiviral vectors are an ideal choice for delivering gene-editing components to cells due to their capacity for stably transducing a broad range of cells ...

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

金小蜂金小蜂基因组操作的胚胎显微注射和移植技术

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex ...

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

利用 CRISPR/Cas9 Ribonucleoproteins 快速简便地生成线虫基因组点突变体的管道

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

利用南方印迹和聚合酶链反应鉴定小鼠胚胎干细胞的同源重组事件

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

慢病毒载体平台, 用于将表观基因编辑工具高效地传递到人类诱导的多能干细胞衍生疾病模型中

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the ...

A Bioinformatics Pipeline to Accurately and Efficiently Analyze the MicroRNA Transcriptomes in Plants

生物信息学管道，用于准确、高效地分析植物中的微RNA转录

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in ...

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae

Using the GAL4-UAS System for Functional Genetics in Anopheles gambiae

使用GAL4-UAS系统在冈比亚按蚊的功能遗传学中

The bipartite GAL4-UAS system is a versatile and powerful tool for functional genetic analysis. The essence of the system is to cross transgenic 'driver' ...