LV是支持由美国国家科学基金会（内部监督办公室1002837）

1。使原生质体<ol><li>加入9 mL的8％甘露醇培养皿。 </li><li>使用压舌板，放2-3 PPNH4板5 - 7天的老苔培养皿中含有甘露醇。 </li><li>加入3毫升2％Driselase D9515 - 25G（Sigma公司）的培养皿中。 </li><li>轻轻摇晃1小时的培养皿中，在室温下孵育。 </li><li>原生质体悬浮液过滤器通过一个100微米的目（屋宇署猎鹰352350）。 </li><li>旋转250克5分钟，过滤悬浮。去除上清。 </li><li>重悬原生质体轻轻8％甘露醇500μL。 </li><li>加入9.5 mL的培养管的8％甘露醇。确保原生质体是完全暂停。 </li><li>重复过滤和再悬浮的步骤（1.6，1.7和1.8）两次。 </li><li>取10μL的原生质体解决方案，并在一个血球计数原生质体。 </li><li> 10,000乘以原生质体在一个16平方米的面积获得每毫升的原生质体的数量（见图1）。 </li></ol> 2。转型<ol><li>原生质体悬浮液旋转250克5分钟。去除上清。 </li><li>再暂停160万原生质体/ ml的浓度在MMG的解决方案的原生质体。 </li><li>原生质体悬浮液在室温下孵育20分钟。 </li><li> 600原生质体悬浮液加入含有60微克的DNA成为一种文化管。轻轻涡流管。 </li><li>原生质体/ DNA混合物中加入700μLPEG / CA解决方案。管轻轻摇动，直到整个混合物均匀。 </li><li>在室温下孵育30分钟的混合物。 </li><li>在此等待期间，用玻璃纸覆盖PRM - B板（80毫米直径的圆圈，AA包装有限公司，英国）;玻璃纸水合物板面允许至少5分钟。 </li><li>用抹刀，删除任何空气被困玻璃纸和板块之间的气泡。 </li><li> W5的解决方案与3毫升稀释的混合物。 </li><li>旋转250克5分钟的混合物。去除上清。 </li><li>在融化的PRM - T的2毫升重新暂停原生质体（将它添加到该解决方案可以保持在42 ° C的液体原生质体之前，）。板1毫升每玻璃纸覆盖的PRM - B板重新挂起原生质体。包装板微孔（3M公司，保健）。在生长室中，保持在25 ° C的板。 </li><li>此外，原生质体可重新悬浮于1毫升液体电镀液和电镀0.5毫升用玻璃纸覆盖了PRM - B板。这将允许更快的再生，但再生植物的数量较低。 </li><li>生长室设置了16小时。光照和8小时。暗周期。 </li><li>移动到4天，改造后的新鲜选择板的玻璃纸。 </li></ol> 3。代表性的成果： 转型的一个例子是如图2所示。在这个例子中使用的DNA是一个超螺旋的7.8 kb的质粒携带潮霉素抗性卡带。镀原生质体的数量减少到50％摄影。照片拍摄两周后，被转移到植物的选择板。转化效率是不影响DNA浓度高达60微克。高浓度的DNA转化效率是不利的。数据表明，该方法提供了较广泛的DNA金额一致的结果。这种方法也可以产生稳定的转化，有效，如表1所示，我们可以得到约4％微克的线性DNA，这是远远比以前 14报道的稳定转化。 我们还测试了一个超螺旋质粒携带一个荧光蛋白作为标记基因转化的热休克转化效率的影响。热休克进行了室温孵育10分钟后（在步骤2.6）在45 ° C为3分钟的水浴孵育混合物。混合物在室温水浴孵育后立即热休克17分钟。结果改造后的22个小时。获得的转化和未经热休克的比率非常相似（〜25％），但我们观察到原生质体（数据未显示）的可行性热休克的不利影响。 <img src="/files/ftp_upload/2560/2560fig1.jpg" alt="图1" /> 图1。血球原生质体 ，原生质体计数所需的部分是由一个红色正方形表示。注意计数原生质体（见1.11）所需的16个广场;的图像显示了24原生质体数（24万细胞/ mL）。 <img src="/files/ftp_upload/2560/2560fig2.jpg" alt="图2" /> 图2。 18日龄的瞬态转化的图片。原生质体转运形成超螺旋质粒表示。答：15μg; B：30μg，C：60μg，D：120μg。 <table border="1"><tbody><tr><td> DNA的量（微克） </td><td> 效率（tranformants /微克DNA） </td></tr><tr><td> 15，超螺旋质粒</td><td> 120 ± 24 </td></tr><tr><td> 30，超螺旋质粒</td><td> 145 ± 54 </td></tr><tr><td> 60，超螺旋质粒</td><td> 121 ± 60 </td></tr><tr><td> 120，超螺旋质粒</td><td> 71 ± 38 </td></tr><tr><td> 60，线性质粒</td><td> 4 ± 1 </td></tr></tbody></table> 表1：在不同的DNA量的转化效率。

<ol>
	<li>Abel, S., Theologis, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+transformation+of+Arabidopsis+leaf+protoplasts:+a+versatile+experimental+system+to+study+gene+expression.">Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression.</a> Plant J. 5, 421-427 (1994).</li><li>Wood, A. J., Oliver, M. J., Cove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bryophytes+as+model+systems.">Bryophytes as model systems.</a> Bryologist. 103, 128-133 (2000).</li><li>Schaefer, D. G., Zr&#255;d, J. -. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+targeting+in+the+moss+Physcomitrella+patens.">Efficient gene targeting in the moss Physcomitrella patens.</a> Plant J. 11, 1195-1206 (1997).</li><li>Schaefer, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+targeting+in+Physcomitrella+patens.">Gene targeting in Physcomitrella patens.</a> Curr. Opin. Plant Biol. 4, 143-150 (2001).</li><li>Kamisugi, Y., Cuming, A. C., Cove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parameters+determining+the+efficiency+of+gene+targeting+in+the+moss+Physcomitrella+patens.">Parameters determining the efficiency of gene targeting in the moss Physcomitrella patens.</a> Nucleic Acids Res. 33, e173-e173 (2005).</li><li>Cove, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+moss+Physcomitrella+patens.">The moss Physcomitrella patens.</a> Annu. Rev. Genet. 39, 339-358 (2005).</li><li>Kamisugi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mechanism+of+gene+targeting+in+Physcomitrella+patens:+homologous+recombination,+concatenation+and+multiple+integration.">The mechanism of gene targeting in Physcomitrella patens: homologous recombination, concatenation and multiple integration.</a> Nucleic Acids Res. 34, 6205-6215 (2006).</li><li>Vidali, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+formin-mediated+actin-filament+elongation+is+essential+for+polarized+plant+cell+growth.">Rapid formin-mediated actin-filament elongation is essential for polarized plant cell growth.</a> Proc. Natl. Acad. Sci. U.S.A. , (2009).</li><li>Sawahel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+foreign+DNA+into+Physcomitrella+protonemal+tissue+by+using+the+gene+gun.">Transfer of foreign DNA into Physcomitrella protonemal tissue by using the gene gun.</a> Plant Mol. Biol. 10, 315-316 (1992).</li><li>Schaefer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+transformation+of+the+moss+Physcomitrella+patens.">Stable transformation of the moss Physcomitrella patens.</a> Mol. Gen. Genet. 226, 418-424 (1991).</li><li>Cho, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Particle+bombardment+mediated+transformation+and+GFP+expression+in+the+moss+Physcomitrella+patens.">Particle bombardment mediated transformation and GFP expression in the moss Physcomitrella patens.</a> Mol. Cells. 9, 14-19 (1999).</li><li>Ashton, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+bryophyte+Physcomitrella+patens+replicates+extrachromosomal+transgenic+elements.">The bryophyte Physcomitrella patens replicates extrachromosomal transgenic elements.</a> New Phytol. 146, 391-402 (2000).</li><li>Hohe, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+and+highly+standardized+transformation+procedure+allows+efficient+production+of+single+and+multiple+targeted+gene-knockouts+in+a+moss,+Physcomitrella+patens.">An improved and highly standardized transformation procedure allows efficient production of single and multiple targeted gene-knockouts in a moss, Physcomitrella patens.</a> Curr. Genet. 44, 339-347 (2004).</li><li>Vidali, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profilin+is+essential+for+tip+growth+in+the+moss+Physcomitrella+patens.">Profilin is essential for tip growth in the moss Physcomitrella patens.</a> Plant Cell. 19, 3705-3722 (2007).</li></ol>

这里介绍的方法可以直接和一致的DNA转化成小立碗藓原生质体。该方法最初用于拟南芥 1，但在这里，我们表明，它执行与小立碗藓 ，一个简单的和不同的植物原生质体。因此，这种方法可以应用到其他植物物种，稍作修改。优化的可能的修改包括原生质体的浓度在MMG，MMG和PEG / CA解决方案的孵化时间。我们选择在我们日常的实验中使用60微克的DNA，因为转化与DNA浓度呈线性增加，直到这一点（见表1）。我们可以得到约7000瞬态转化一个单一的60微克DNA转化，这为进一步筛选和分析人口众多的植物，或200个稳定转化。 转化原生质体可以是液体电镀中等或PRM - T的蔓延。虽然再生效率较低时使用液体电镀液中，我们选择这种方法时，原生质体与超螺旋质粒转化。如表1显示，超螺旋质粒DNA的转换生成更多的转化，因此，我们仍然可以用液体电镀液中获得大量的植物。此外，传播与液体电镀液的原生质体，允许更快的再生，这是非常重要的实验涉及瞬态的表型，如RNA 干扰 ，击倒实验9,15的。此外，高层琼脂的情况下允许免疫组化和RT - PCR检测9,15容易植物隔离。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 成分 </td></tr><tr><td> PPNH 4 </td><td> 1.84毫米KH 2 PO 4 3.4毫米的Ca（NO 3）2 1毫米硫酸镁4 2.72 mM的二铵酒石酸 54微米FESO 4 .7 H 2 O 9.93μMH 3 BO 3 1.97μMMnCl 2 .4 H 2 O 0.23μMCoCl 2 .6 H 2 O 硫酸锌0.19μM。 7H 2 O 0.22微米硫酸铜4。 5H 2 O 0.10μM娜2的MoO 4 .2 H 2 O 0.168μM碘化钾 添加0.7％琼​​脂固体培养基。 </td></tr><tr><td> PRM - B </td><td> PPNH 4，6％甘露醇和0.8％琼脂。浇筑前板，添加10毫升1M 氯化钙 2至1 L 。 </td></tr><tr><td> PRM - T </td><td> PPNH 4，6％甘露醇和0.6％的琼脂。添加0.5毫升1M氯化钙使用前的2至50毫升。 </td></tr><tr><td>液体电镀中等</td><td> PPNH 4和8.5％，无琼脂甘露醇。添加0.5毫升1M氯化钙使用前的2至50毫升。 </td></tr><tr><td> 2％Driselase </td><td>添加4克Driselase D9515 - 25G（Sigma公司）的8％甘露醇200毫升。轻轻搅拌30分钟。在室温下孵育30分钟。在4 ° C，并轻轻搅拌5分钟。在室温下。 10分钟旋转2500克，丢弃沉淀。与0.22微米，10毫升分装冷冻储存过滤。使用前在室温水浴解冻。 </td></tr><tr><td> MMG </td><td> 0.4中号甘露醇 15毫米氯化镁2 4毫米的MES（pH值5.7） </td></tr><tr><td> PEG / CA </td><td> 4克PEG4000 3 mL的H 2 O 2.5毫升0.8甘露醇中号 1毫升1M 氯化钙 </td></tr><tr><td> W5号</td><td> 154 mM氯化钠 125毫米氯化钙2 5 mM的氯化钾 2毫米的MES（pH值5.7） </td></tr></tbody></table>

efficient polyethylene glycol (peg) mediated transformation of the moss physcomitrella patens

<p class="jove_content"&gt;一个简单而有效的方法来改造<em&gt;小立碗pantens</em&gt;原生质体的描述。这种方法是改编自协议<em&gt; Physocmitrella</em&gt; protonemal原生质体和<em&gt;拟南芥</em&gt;叶肉原生质体转化<sup&gt; 1</sup&gt;。由于其容量，进行高效的有丝分裂，同源重组<em&gt;，小立碗藓</em&gt;已经成为一个重要的模型系统近年来<sup&gt; 2</sup&gt;。这种能力允许高频率的基因定位<sup&gt; 3-9</sup&gt;，这是没有看到在其他模式植物，如<em&gt;拟南芥</em&gt;。要充分利用本系统，我们需要一种有效，简便的方法来传递到苔细胞的DNA。最常见的方法来改造这个苔粒子轰击<sup&gt; 10</sup&gt;和PEG介导的DNA摄取<sup&gt; 11</sup&gt;。虽然粒子轰击可以生产出高转换效率<sup&gt; 12</sup&gt;，基因枪是没有现成的许多实验室和协议是难以标准化。另一方面，PEG介导的转化并不需要专门的设备，并且可以在任何实验室用无菌罩。在这里，我们展示一个简单高效的方法，苔原生质体转化。这种方法可以产生每超过120微克的DNA，这是一个从先前公布的最有效的协议的改善瞬态转化<sup&gt; 13</sup&gt;。由于它的简单性，效率和可重复性，这种方法可以应用以及需要大量转化为日常改造项目。</p

Watch this Scientific Journal Video about Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens at JoVE.com

Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens

一个简单而有效的方法来改造<em&gt;小立碗pantens</em&gt;原生质体的描述。这种方法是改编自协议<em&gt; Physocmitrella</em&gt; protonemal原生质体和<em&gt;拟南芥</em&gt;叶肉原生质体转化<sup&gt; 1</sup&gt;。

高效的聚乙二醇（PEG）介导的转化苔小立碗藓</em

A simple and efficient method to transform Physcomitrella pantens protoplasts is described. This method is adapted from protocols for Physocmitrella ...

efficient-polyethylene-glycol-peg-mediated-transformation-moss

Research

JoVE Journal

Biology

Efficient Polyethylene Glycol (PEG) Mediated Transformation of the Moss Physcomitrella patens

40.3K 次观看.  Worcester Polytechnic Institute- WPI. 一个简单而有效的方法来改造小立碗pantens原生质体的描述。这种方法是改编自协议 Physocmitrella protonemal原生质体和拟南芥叶肉原生质体转化 1。

视频: Efficient Polyethylene Glycol PEG Mediated Transformation of the Moss Physcomitrella patens

Environmentally Induced Heritable Changes in Flax

Some flax varieties respond to nutrient stress by modifying their genome and these modifications can be inherited through many generations. Also associated with these genomic changes are heritable phenotypic variations 1,2. The flax variety Stormont Cirrus (Pl) when grown under three different nutrient conditions can either remain inducible (under the control conditions), or become stably modified to either the large or small genotroph by growth under high or low nutrient conditions respectively. The lines resulting from the initial growth under each of these conditions appear to grow better when grown under the same conditions in subsequent generations, notably the Pl line grows best under the control treatment indicating that the plants growing under both the high and low nutrients are under stress. One of the genomic changes that are associated with the induction of heritable changes is the appearance of an insertion element (LIS-1) 3, 4 while the plants are growing under the nutrient stress. With respect to this insertion event, the flax variety Stormont Cirrus (Pl) when grown under three different nutrient conditions can either remain unchanged (under the control conditions), have the insertion appear in all the plants (under low nutrients) and have this transmitted to the next generation, or have the insertion (or parts of it) appear but not be transmitted through generations (under high nutrients) 4. The frequency of the appearance of this insertion indicates that it is under positive selection, which is also consistent with the growth response in subsequent generations. Leaves or meristems harvested at various stages of growth are used for DNA and RNA isolation. The RNA is used to identify variation in expression associated with the various growth environments and/or t he presence/absence of LIS-1. The isolated DNA is used to identify those plants in which the insertion has occurred.

Growing some flax varieties under nutrient stress results in genomic variation within a subset of the genome and phenotypic variation. A complex insertion at a specific site is associated with growth under various nutrient regimes and with changes in gene expression around this site.

环境引起的遗传亚麻的变化

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

科研

生物学

Cryopreservation of Mouse Embryos by Ethylene Glycol-Based Vitrification

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic modifications and the number is consistently increasing year by year. Its technical development started with slow freezing methods in the 1970s1, then followed by vitrification methods developed in the late 1980s2. Generally, the latter technique is advantageous in its quickness, simplicity, and high survivability of recovered embryos. However, the cryoprotectants contained are highly toxic and may affect subsequent embryo development. Therefore, the technique was not applicable to certain strains of mice, even when the solutions are cooled to 4&deg;C to mitigate the toxic effect during embryo handling. At the RIKEN BioResource Center, more than 5000 mouse strains with different genetic backgrounds and phenotypes are maintained3, and therefore we have optimized a vitrification technique with which we can cryopreserve embryos from many different strains of mice, with the benefits of high embryo survival after vitrifying and thawing (or liquefying, more precisely) at the ambient temperature4.
Here, we present a vitrification method for mouse embryos that has been successfully used at our center. The cryopreservation solution contains ethylene glycol instead of DMSO to minimize the toxicity to embryos5. It also contains Ficoll and sucrose for prevention of devitrification and osmotic adjustment, respectively. Embryos can be handled at room temperature and transferred into liquid nitrogen within 5 min. Because the original method was optimized for plastic straws as containers, we have slightly modified the protocol for cryotubes, which are more easily accessible in laboratories and more resistant to physical damages. We also describe the procedure of thawing vitrified embryos in detail because it is a critical step for efficient recovery of live mice. These methodologies would be helpful to researchers and technicians who need preservation of mouse strains for later use in a safe and cost-effective manner.

An ethylene glycol-based vitrification method for mouse embryos is described. It is advantageous to other methods in its simplicity and low embryonic toxicity, and therefore can be broadly applicable to many strains of mice, including inbred and gene-modified mice.

基于乙二醇玻璃化冷冻小鼠胚胎的超低温保存

Cryopreservation of mouse embryos is a technological basis that supports biomedical sciences, because many strains of mice have been produced by genetic ...

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic redundancy affecting simple genetics in higher plants. The novel model organism Ostreococcus tauri is the smallest free-living eukaryote known to date, and possesses a greatly reduced genome size and cellular complexity1,2, manifested by the presence of just one of most organelles (mitochondrion, chloroplast, golgi stack) per cell, and a genome containing only ~8000 genes. Furthermore, the combination of unicellularity and easy culture provides a platform amenable to chemical biology approaches. Recently, Ostreococcus has been successfully employed to study basic mechanisms underlying circadian timekeeping3-6. Results from this model organism have impacted not only plant science, but also mammalian biology7. This example highlights how rapid experimentation in a simple eukaryote from the green lineage can accelerate research in more complex organisms by generating testable hypotheses using methods technically feasible only in this background of reduced complexity. Knowledge of a genome and the possibility to modify genes are essential tools in any model species. Genomic1, Transcriptomic8, and Proteomic9 information for this species is freely available, whereas the previously reported methods6,10 to genetically transform Ostreococcus are known to few laboratories worldwide.
In this article, the experimental methods to genetically transform this novel model organism with an overexpression construct by means of electroporation are outlined in detail, as well as the method of inclusion of transformed cells in low percentage agarose to allow selection of transformed lines originating from a single transformed cell. Following the successful application of Ostreococcus to circadian research, growing interest in Ostreococcus can be expected from diverse research areas within and outside plant sciences, including biotechnological areas. Researchers from a broad range of biological and medical sciences that work on conserved biochemical pathways may consider pursuing research in Ostreococcus, free from the genomic and organismal complexity of larger model species.

Genomic Transformation of the Picoeukaryote Ostreococcus tauri

This article describes genetic transformation of the unicellular marine alga Ostreococcus tauri by electroporation. This eukaryotic organism is an effective model platform for higher plants, possesing greatly reduced genomic and cellular complexity and being readily amenable to both cell culture and chemical biology.

基因改造的Picoeukaryote<em&gt; Ostreococcus金牛</em

Common problems hindering rapid progress in Plant Sciences include cellular, tissue and whole organism complexity, and notably the high level of genomic ...

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) and Wuschel2 (Wus2). Bbm is regulated by the maize phospholipid transferase gene (Pltp) promoter, and Wus2 is under the control of a maize auxin-inducible (Axig1) promoter. An Agrobacterium strain carrying these morphogenic genes on transfer DNA (T-DNA) and extra copies of Agrobacterium virulence (vir) genes are used to infect maize immature embryo explants. Somatic embryos form on the scutella of infected embryos and can be selected by herbicide resistance and germinated into plants. A heat-activated cre/loxP recombination system built into the DNA construct allows for removal of morphogenic genes from the maize genome during an early stage of the transformation process. Transformation frequencies of approximately 14%, 4%, and 4% (numbers of independent transgenic events per 100 infected embryos) can be achieved for W22, B73, and Mo17, respectively, using this protocol.

Agrobacterium-Mediated Immature Embryo Transformation of Recalcitrant Maize Inbred Lines Using Morphogenic Genes

Plant morphogenic genes can be used to improve genetic transformation of recalcitrant genotypes. Described here is an Agrobacterium-mediated genetic transformation (QuickCorn) protocol for three important public maize inbred lines.

使用形态基因的农业细菌 -顽固玉米近亲繁殖系的介导未成熟胚胎转化

Demonstrated here is a detailed protocol for Agrobacterium-mediated genetic transformation of maize inbred lines using morphogenic genes Baby boom (Bbm) ...

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by filarial nematodes such as canine heartworm and elephantasis. Recently, CRISPR/Cas9 genome editing has been used to induce site-directed mutations by injecting a Cas9 protein that has been complexed with a guide RNA (gRNA) into freshly laid embryos of several insect species, including mosquitoes that belong to the genera Anopheles and Aedes. Manipulating and injecting Culex mosquitoes is slightly more difficult as these mosquitoes lay their eggs upright in rafts rather than individually like other species of mosquitoes. Here we describe how to design gRNAs, complex them with Cas9 protein, induce female mosquitoes of Culex pipiens to lay eggs, and how to prepare and inject newly laid embryos for microinjection with Cas9/gRNA. We also describe how to rear and screen injected mosquitoes for the desired mutation. The representative results demonstrate that this technique can be used to induce site-directed mutations in the genome of Culex mosquitoes and, with slight modifications, can be used to generate null-mutants in other mosquito species as well.&#160;

Preparing and Injecting Embryos of Culex Mosquitoes to Generate Null Mutations using CRISPR/Cas9

CRISPR/Cas9 is increasingly used to characterize gene function in non-model organisms. This protocol describes how to generate knock-out lines of Culex pipiens, from preparing injection mixes, to obtaining and injecting mosquito embryos, as well as how to rear, cross, and screen injected mosquitoes and their progeny for desired mutations.

准备和注射 Culex 蚊子的胚胎，使用CRISPR/Cas9产生空突变

Culex mosquitoes are the major vectors of several diseases that negatively impact human and animal health including West Nile virus and diseases caused by ...

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2&#8211;3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice

This work describes the use of CRISPR-Cas9 genome editing technology to knockout endogenous gene OsABCG15 followed by a modified Agrobacterium-mediated transformation protocol to produce a stable male-sterile line in rice.

农业细菌-中生菌遗传转化、转基因生产及其在水稻雄性生殖发育研究中的应用

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive ...

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic pest management strategies. The most used transposable element in insect transformation is piggyBac, and piggyBac-based germline transformation has been successfully conducted in model insects. However, it is still challenging to employ this technology in non-model insects that include agricultural pests. This paper reports on germline transformation of a global agricultural pest, the fall armyworm (FAW), Spodoptera frugiperda, using the hyperactive piggyBac transposase (hyPBase).
In this work, the hyPBase mRNA was produced and used in place of helper plasmid in embryo microinjections. This change led to the successful generation of transgenic FAW. Furthermore, the methods of screening transgenic animals, PCR-based rapid detection of transgene insertion, and thermal asymmetric interlaced PCR (TAIL-PCR)-based determination of the integration site, are also described. Thus, this paper presents a protocol to produce transgenic FAW, which will facilitate piggyBac-based transgenesis in FAW and other lepidopteran insects.

Hyperactive piggyBac Transposase-mediated Germline Transformation in the Fall Armyworm, Spodoptera frugiperda

Successful germline transformation in the fall armyworm, Spodoptera frugiperda, was achieved using mRNA of hyperactive piggyBac transposase.

多活性猪猪Bac转座酶介导的秋粘虫，蜉蝣属果蝇蛆中的种系转化

Stable insertion of genetic cargo into insect genomes using transposable elements is a powerful tool for functional genomic studies and developing genetic ...

Agrobacterium tumefaciens-Mediated Genetic Transformation of Narrowleaf Plantain

Species in the genus Plantago have several unique traits that have led to them being adapted as model plants in various fields of study. However, the lack of a genetic manipulation system prevents in-depth investigation of gene function, limiting the versatility of this genus as a model. Here, a transformation protocol is presented for Plantago lanceolata, the most commonly studied Plantago species. Using Agrobacterium tumefaciens-mediated transformation, 3 week-old roots of aseptically grown P. lanceolata plants were infected with bacteria, incubated for 2-3 days, and then transferred to a shoot induction medium with appropriate antibiotic selection. Shoots typically emerged from the medium after 1 month, and roots developed 1-4 weeks after the shoots were transferred to the root induction medium. The plants were then acclimated to a soil environment and tested for the presence of a transgene using the &#946;-glucuronidase (GUS) reporter assay. The transformation efficiency of the current method is ~20%, with two transgenic plants emerging per 10 root tissues transformed. Establishing a transformation protocol for narrowleaf plantain will facilitate the adoption of this plant as a new model species in various areas.

Agrobacterium tumefaciens-Mediated Genetic Transformation of Narrowleaf Plantain

Because of its versatile application as a model species in various fields of study, there is a need for a genetic transformation toolkit in narrowleaf plantain (Plantago lanceolata). Here, using Agrobacterium tumefaciens-mediated transformation, a protocol is presented that results in stable transgenic lines with a transformation efficiency of 20%.

根癌农杆菌介导的窄叶车前草遗传转化

Species in the genus Plantago have several unique traits that have led to them being adapted as model plants in various fields of study. However, the lack ...

一步法毛状根转化方法推进根相关生物学

An Efficient and Reproducible Method for Producing Composite Plants by Agrobacterium rhizogenes-Based Hairy Root Transformation

Producing composite plants with transgenic roots and nontransgenic stems and buds using Agrobacterium rhizogenes-mediated hairy root transformation is a powerful tool to study root-related biology. Hairy root transformation is established in a wide range of dicotyledons and in several monocotyledon species and is almost independent of the genotype. The traditional method of hypocotyl injection with A. rhizogenes to obtain composite plants is inefficient, time-consuming, laborious, and frequently causes the death of tender and tiny hypocotyl plants. A highly efficient, one-step hairy root transformation mediated by A. rhizogenes was established previously, which eliminates the need for transplanting after producing hairy roots. In this study, a partial hypocotyl and primary root were removed, the hypocotyl incision site was coated with A. rhizogenes, and&#160;then hypocotyls were planted in sterile vermiculite. After 12 days of cultivation, the hypocotyl incision expanded and new hairy roots were induced. This article provides the detailed protocol of a one-step transformation method mediated by A. rhizogenes, with its effectiveness demonstrated by producing composite plants of wild soybean, Solanum americanum, and pumpkin.

作者聚焦：通过根瘤农杆菌介导的毛状根转化简化复合植物生产 

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

一种基于根 茎农杆菌毛根转化生产复合植物的高效且可重复的方法

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