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。

<ol>
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提交人没有宣布任何竞争性的金融利益或其他利益冲突。

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5,9,29. 与其他 BIBAC 向量相比, 与 BIBAC 的向量比较起来, 兴趣序列可以很容易地通过网关重组站点12来插入。修正后的向量克服了传统克隆策略中使用的 BIBAC 向量的一般问题: i) 有限数量的唯一限制点和 ii) 低 DNA 产量。网关重组站点使 BIBAC-GW 向量成为其他二进制载体的一个有吸引力的替代品, 用于生成转基因植物。
本文介绍了一系列的协议, 从产生的 BIBAC-GW 衍生物的利益序列, 植物转化, 和 DNA 印迹分析的数量和完整性的转基因序列。本文报告的几种协议, 即网关克隆、细菌电穿孔和植物转化, 是许多实验室的常见实践, 也可以进行轻微的修改。重要的是要知道, BIBAC 是一个单拷贝向量在大肠杆菌和. 农杆菌。因此, 在分离 DNA 时, 收率低;建议扩展隔离过程。
在转基因携带多个 T DNA 集成, 介绍转基因经常受到基因沉默1,2,4,30, 因此, 应避免大多数应用程序。为了确定转基因植物的单一, 完整的整合, 建议使用 DNA 印迹分析。虽然 dna 印迹以外的方法可用于确定转基因线 (分离分析、尾 pcr、定量 pcr (qPCR) 和数字滴 pcr) 的 t dna 拷贝数和完整性, 但人工密集, DNA 印迹往往是选择的方法。分离分析无法区分单个基因座上的多基因和单 T DNA 积分。尾 PCR 通常低于估计复制数, 特别是如果多个 T DNA 集成存在31, 并且 qPCR 需要对可靠结果进行精心优化31,32。如果所需的设备可用31, 则数字滴 PCR 是一种相当精确的拷贝数检测方法。DNA 印迹的额外好处是对截断的 t-dna 的简易检测, 在所有 PCR 技术中容易被遗漏。
在 DNA 印迹分析中, 印迹上的杂交片段需要在信号和大小上得到很好的识别。已知影响 DNA 印迹结果的几个因素。除了适当选择限制酶 (在图 4中指出的策略) 和大小标记之外, 还需要足够的高质量的 DNA。不到2µg 的基因组拟南芥DNA 不会产生良好的可识别片段。当处理更大的基因组时, 需要更多的 DNA。要获得足够数量的拟南芥DNA, 可以使用花组织或1周的老苗。在一个培养皿上生长的一批幼苗产生2–8µg 的 DNA。为了避免 DNA 在分离过程中降解, 应注意快速处理植物材料。此外, 基因组 DNA 应悬浮三 EDTA, 以减少其降解的核酸, 并储存在4°c 而不是-20 摄氏度, 以防止 DNA 偷由于反复冻融循环。如果不确定所有的 dna 样本完全消化, 建议 rehybridize dna 印迹与一个探针识别内源, 独特的基因组区域。选择用于识别转基因或内源序列的探针序列时, 只选择唯一序列是至关重要的。为了能够精确地确定杂交片段的大小, dna 凝胶槽和 dna 标记带的位置应该在透明 (图 5B) 上标记, 当在 UV 上可视化一溴溴染的凝胶 (图 5A) 时。transilluminator。如果标记序列不杂交探针 DNA, 或杂交是部分, 它是唯一的方法来跟踪的大小杂交片段。
一旦注意到获得良好的杂交信号和碎片大小的估计, 印迹结果的解释是直接的。当使用一个限制酶, 并与不同的探针杂交检测的左或右部分的 t dna, 检测到的碎片数量反映了 t dna 插入的数量。例如,图 7B、C使用图 7A中显示的策略显示相同的 DNA 印迹, 与不同的探针、 bar (图 7B) 和增强型黄色荧光蛋白 (eYFP) (图 7C) 进行杂交。所有车道, 除了 4, 在两个污点上显示相同数量的碎片: 6 行的两个碎片和所有其他行的单个片段。检测到的碎片数量是 T DNA 插入的数量。
当检测到的带探针的碎片数量与 t DNA 的左或右部分不同 (如图 7B、C、4行) 时, 不存在不完整的插入, 或者 t 脱氧核糖核酸已插入串联排列。串联插入显示一个 T DNA 片段的非随机片段长度 (图 4A, 右面板), 并可以通过比较混合片段大小与预期的基于限制策略来识别。可能需要额外的印迹策略来确认 T DNA 插入的串联排列。在4行 (图 7B、C) 中显示的示例中, 两个插入是按反向重复方向排列的。
当估计 T DNA 的完整性或部分时, 可以根据限制策略计算杂交片段的长度。任何与预期大小的偏差都表明插入不完整。例如, 在图 7D中的4条中, 杂交片段迁移到 8 kbp (而不是预期的 5.5 kbp), 表示由于缺少一个限制站点而导致片段大小增加。
BIBAC-GW 向量是在许多植物物种中生成单拷贝完整集成的优秀工具。这里报告的议定书提供了一个可靠的程序, 以确定植物的单一, 完整的整合的转基因的利益。

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) (图 2)10。总共有126个拟南芥植物被改造 (每壶9株, 14 盆)。这些植物的种子被汇集, 播种在托盘与土壤, 并允许生长两个星期之前, 膦铵溶液处理。只有表示bar基因的幼苗 (目前在 BIBAC-GW) 生存膦-铵处理 (图 3)。共11转基因转化为 pDM19, 相应的转化效率为0.02% 的种子分析。
对于11转基因的分离, dna 印迹被用来确定 T dna 积分的数量。为此目的, 用BglII 或ScaI (在图 4A上设计的策略) 切割基因组 DNA。这两种限制酶只切割一次到 T DNA 序列 (图 7A)。与探针杂交, 识别棒和 eYFP 编码区域允许检测各自 DNA 片段的数量。
在该污点上, 单个 dna 片段的数量可以用来估计记者行中的 T dna 插入数 (表 1)。单杂交片段与酒吧和 eYFP 探针表明存在一个单一的 T DNA 集成。从11转基因分析, 六进行单积分。平均积分数为1.2。
对于携带单个 T DNA 集成的6行, 插入的报告器构造的完整性是使用 DNA 印迹 (在图 4B中设计的策略) 进行测试的。基因组 DNA 是用BglII 和PciI 切开的, 释放一个 5.5 kb 的片段, 其中包含了 Bar 和 mTurquoise-eYFP 融合基因 (图 7A)。对 eYFP 的探针用于检测预期的碎片。所有被测试的植物运载了一个原封片断。请注意, 所检查的片段不包括左侧和右侧的 t dna 边界, 因此不检查整个 t dna 的完整性, 而只是包含感兴趣转基因的部分。
荧光报告基因的表达是在独立的单拷贝转基因线上确定的, 只有基因组定位的 T DNA。CaMV-35S 启动子驱动的 mTurquoise eYFP 记者的相对转录水平由 RT-qPCR 测量, 其四 DM19 的报告线携带完好, 单拷贝积分, 其中基因组位置确定为10。在报告基因表达水平之间的变化是次要的: mTurquoise-eYFP RNA 水平的最大差异是2倍 (图 8A)。
接下来, ODM 在这些记者行中进行。三在四独立的报告员线显示相当相似的 ODM 效率 (图 8B)。然而, 一条线, DM19 [4] 1, 产生了一个非常低的 ODM 效率比其他线。这些结果表明, ODM 受局部基因组上下文的影响。在什么方式 T 脱氧核糖核酸综合化的地方基因组上下文在 DM19 [4] 1 不同从那在其他线仍然将被辨认。非转基因植物基因组 T DNA 积分中活性和不活性染色质标记的可用数据分析没有提供应答10。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57295/57295fig1.jpg"> 图 1: pBIBAC 向量的函数映射.pBIBAC-GW 衍生物可用于膦 (bar) 的抗性或种子大衣 (DsRed) 中的 DsRed 荧光作为植物中的选择标记。对于这两种载体, 卡那霉素抗性基因是细菌的选择标记。网关ccdB盒显示在表示复合站点 "R1" 和 R2 的绿色箭头之间. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57295/57295fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57295/57295fig2.jpg"> 图 2: 突变报告器构造.mTurquoise-eYFP 的报告基因是由三十五年代-CaMV 助剂驱动的。mTurquoise 编码区域融合到一个 eYFP 编码区域, 在核苷酸位置120上携带 C 型突变, 导致过早的平移停止密码子 TAA, 并过早终止融合蛋白的翻译。3′ Nopaline 合酶 (3 ' nos) polyadenylation 信号用于终止构造22的转录。核定位信号 (NLS) 用于靶向细胞核的转化蛋白。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57295/57295fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57295/57295fig3.jpg"> 图 3: 在膦铵处理前后填充拟南芥幼苗的托盘.幼苗未表达的酒吧基因, 是存在于 pBIBAC-GW T DNA 死亡后, 喷出膦铵溶液。这些照片显示同一盘苗 (A) 之前喷洒膦铵, 播种后14天, (B) 10 天后, 喷两次。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57295/57295fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57295/57295fig4.jpg"> 图 4: 一般的 DNA 限制策略, 用于识别插入的完整性的数目和数量.(A) 在 t dna 中间的一个限制站点 (R) 允许对 t dna (绿色 R) 的左 (红 L) 和右部分进行独立探查。右边的卡通片显示, 依赖于单拷贝或多复制的 T dna 积分, 用 DNA 印迹获得不同的条纹图案。标记有 * 的带有一个定义的长度, 而其他波段的长度取决于侧翼基因组 DNA 中最近的限制点。单个插入:L 和 R 探针都给出一个独立的片段。预期的平均碎片大小可以根据基因组中限制点的频率来计算。最小大小是从限制站点到左边框 (磅) 或右边框 (RB) 的距离, 具体取决于正在探查的集成的末尾, 以及 T DNA 是否完好。串联重复:L 和 R 的探头给出两个碎片;对于每个探针, 其中一个片段包括侧翼基因组 DNA, 第二个片段有一个预期的大小, 并由两个探针识别。反转重复:根据集成卡带的方向性, 可以识别一个 l 和两个 r 片段, 或者两个 l 和一个 r。单个插入:结果是多个独立的片段, 并且片段的数量对应于集成的数量。(B) 在 T DNA 的四肢上的限制站点允许确定限制站点之间的片段的完整性。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57295/57295fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57295/57295fig5.jpg"> 图 5: 具有限制模式和匹配透明度的琼脂糖凝胶.(A) 在琼脂糖凝胶上, 显示了用EcoR消化的基因组 DNA。DNA 的正确消化是由离散的卫星波段的存在所说明的。(B) 在透明度上标记插槽和标记带的位置, 可以以后轻松地计算杂交片段的大小。这里, MRC 荷兰标记 (蓝色和红色) 被使用, 由 m.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57295/57295fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/57295/57295fig6.jpg"> 图 6: 毛细管印迹的设置.在毛细管印迹设置中, 滤纸放在塑料板上, 纸的两端悬挂在 20x SSC 缓冲器中。该纸被湿润的 20x SSC, 和一个琼脂糖凝胶放置在顶部, 其次是尼龙膜, 过滤纸, 和一堆组织。重量轻放在上面。注意去除凝胶、纸和膜之间的气泡。保鲜膜用于避免在安装过程中干燥。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57295/57295fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/57295/57295fig7.jpg"> 图 7: DNA 印迹策略和实验结果的示例.(A) dna 印迹策略来确定 T dna 积分的数量和完整性。在 T 脱氧核糖核酸之内选定的制约酵素的切口位置用垂直的酒吧表明。用于与已消化的基因组 DNA 杂交的eYFP和bar探头使用与 T DNA 下面的终端点线进行表示。(B-D)示例 DNA 印迹. 用ScaI 切割基因组 DNA, 并使用bar和eYFP探测器 (B和C) 探测印迹。用BglII 和PciI 切割基因组 DNA, 并用bar探针进行探测。完整的片段大小为 5.5 kbp (D)。请注意, D 中的样本集与 B 和 C 中显示的示例不同. * 表示预期的片段大小;M, 马克。在 B、C 和 D 中使用相同大小的标记。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57295/57295fig7large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/57295/57295fig8.jpg"> 图 8: 独立 mTurquoise eYFP 报告器行中的 mTurquoise eYFP 表达式级别和 ODM 效率.(A) 相对 mTurquoise-eYFP 记录级别, 由 DM19 记者行中的 RT qPCR 测量。使用肌动蛋白的规范化转录水平。(B) 在 DM19 报告器行中测量的 ODM 效率。对于 A 和 B, 条形表示至少五种生物复制的平均值。误差线表示 SEM.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57295/57295fig8large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td rowspan="3">T 型 DNA 轨迹类型</td>
			<td rowspan="3">T DNA 积分的 Nr</td>
			<td rowspan="3">记者行</td>
			<td colspan="4">检测到的片段数</td>
			<td>完整性</td>
		</tr>
<tr>
<td colspan="2">
Sca我</td>
			<td colspan="2">
Bgl二</td>
			<td>
Bgl二/PciI</td>
		</tr>
<tr>
<td>栏</td>
			<td>eYFP</td>
			<td>栏</td>
			<td>eYFP</td>
			<td>eYFP</td>
		</tr>
<tr>
<td rowspan="8">单轨迹积分</td>
			<td>1</td>
			<td>19 [2]-2</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>+</td>
		</tr>
<tr>
<td>1</td>
			<td>19 [2]-5</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>+</td>
		</tr>
<tr>
<td>1</td>
			<td>19 [2]-9</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>+</td>
		</tr>
<tr>
<td>1</td>
			<td>19 [2]-11</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>+</td>
		</tr>
<tr>
<td>1</td>
			<td>19 [4]-1</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>+</td>
		</tr>
<tr>
<td>1</td>
			<td>19 [4]-2</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>+</td>
		</tr>
<tr>
<td>2、倒置重复</td>
			<td>19 [2]-10</td>
			<td>2</td>
			<td>1</td>
			<td>1</td>
			<td>1</td>
			<td>+</td>
		</tr>
<tr>
<td>2、不完全集成</td>
			<td>19 [2]-3</td>
			<td>1</td>
			<td>2</td>
			<td>1</td>
			<td>1</td>
			<td>钕</td>
		</tr>
<tr>
<td rowspan="3">多个轨迹集成</td>
			<td>2</td>
			<td>19 [2]-6</td>
			<td>2</td>
			<td>2</td>
			<td>2</td>
			<td></td>
			<td>钕</td>
		</tr>
<tr>
<td>2</td>
			<td>19 [2]-7</td>
			<td>2</td>
			<td>2</td>
			<td>2</td>
			<td>2</td>
			<td>钕</td>
		</tr>
<tr>
<td>3/4</td>
			<td>19 [2]-1</td>
			<td>4</td>
			<td>3</td>
			<td>3</td>
			<td>3</td>
			<td>钕</td>
		</tr>
<tr>
<td>不确定。</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
</tbody></table>表 1:与 pDM19 转换后转基因分离的 DNA 印迹数据摘要.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Genetics

12.4K 次观看.  University of Amsterdam. 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。

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

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

The capacity to introduce exogenous proteins and express (or down-regulate) specific genes in plants provides a powerful tool for fundamental research as well as new applications in the field of plant biotechnology. Viable methods that currently exist for protein or gene transfer into plant cells, namely Agrobacterium and microprojectile bombardment, have disadvantages of low transformation frequency, limited host range, or a high cost of equipment and microcarriers. The following protocol outlines a simple and versatile method, which employs rationally-designed peptides as delivery agents for a variety of nucleic acid- and protein-based cargoes into plants. Peptides are selected as tools for development of the system due to their biodegradability, reduced size, diverse and tunable properties as well as the ability to gain intracellular/organellar access. The preparation, characterization and application of optimized formulations for each type of the wide range of delivered cargoes (plasmid DNA, double-stranded DNA or RNA, and protein) are described. Critical steps within the protocol, possible modifications and existing limitations of the method are also discussed.

Existing methods for the modification of plants have limited applicability. The novel peptide-derived technology proposed here promises both simplicity and efficiency in the introduction of exogenous protein or genes into the desired intracellular compartments of intact 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

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

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 which the gene of interest has been disrupted. A gene-disruption DNA construct containing a suitable antibiotic resistance marker gene is useful for the generation of gene-disrupted strains in bacteria. However, conventional construction methods, which require gene cloning steps, involve complex and time-consuming protocols. Here, a relatively facile, rapid, and cost-effective method for targeted gene disruption in Streptococcus mutans is described. The method utilizes a 2-step fusion polymerase chain reaction (PCR) to generate the disruption construct and electroporation for genetic transformation. This method does not require an enzymatic reaction, other than PCR, and additionally offers greater flexibility in terms of the design of the disruption construct. Employment of electroporation facilitates the preparation of competent cells and improves the transformation efficiency. The present method may be adapted for the generation of gene-disrupted strains of various species.

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

We describe a facile, rapid, and relatively inexpensive method for the generation of gene-disrupted Streptococcus mutans strains; this technique may be adapted for the generation of gene-disrupted strains of various species.

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

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 and mediating high levels of gene expression. However, their ability to integrate into the host cell genome enhances the risk of insertional mutagenicity and thus raises safety concerns and limits their usage in clinical settings. Further, the persistent expression of gene-editing components delivered by these integration-competent lentiviral vectors (ICLVs) increases the probability of promiscuous gene targeting. As an alternative, a new generation of integrase-deficient lentiviral vectors (IDLVs) has been developed that addresses many of these concerns. Here the production protocol of a new and improved IDLV platform for CRISPR-mediated gene editing and list the steps involved in the purification and concentration of such vectors is described and their transduction and gene-editing efficiency using HEK-293T cells was demonstrated. This protocol is easily scalable and can be used to generate high titer IDLVs that are capable of transducing cells in vitro and in vivo. Moreover, this protocol can be easily adapted for the production of ICLVs.

We describe the production strategy of integrase-deficient lentiviral vectors (IDLVs) as vehicles for delivering CRISPR/Cas9 to cells. With an ability to mediate quick and robust gene editing in cells, IDLVs present a safer and equally effective vector platform for gene delivery compared to integrase-competent vectors.

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

The jewel wasp Nasonia vitripennis has emerged as an effective model system for the study of processes including sex determination, haplo-diploid sex determination, venom synthesis, and host-symbiont interactions, among others. A major limitation of working with this organism is the lack of effective protocols to perform directed genome modifications. An important part of genome modification is delivery of editing reagents, including CRISPR/Cas9 molecules, into embryos through microinjection. While microinjection is well established in many model organisms, this technique is particularly challenging to perform in N. vitripennis primarily due to its small embryo size, and the fact that embryonic development occurs entirely within a parasitized blowfly pupa. The following procedure overcomes these significant challenges while demonstrating a streamlined, visual procedure for effectively removing wasp embryos from parasitized host pupae, microinjecting them, and carefully transplanting them back into the host for continuation and completion of development. This protocol will strongly enhance the capability of research groups to perform advanced genome modifications in this organism.

Embryo Microinjection and Transplantation Technique for Nasonia vitripennis Genome Manipulation

Microinjection of Nasonia vitripennis embryos is an essential method for generating heritable genome modifications. Described here is a detailed procedure for microinjection and transplantation of Nasonia vitripennis embryos, which will greatly facilitate future genome manipulation in this organism.

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

The clustered regularly interspersed palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) prokaryotic adaptive immune defense system has been co-opted as a powerful tool for precise eukaryotic genome engineering. Here, we present a rapid and simple method using chimeric single guide RNAs (sgRNA) and CRISPR-Cas9 Ribonucleoproteins (RNPs) for the efficient and precise generation of genomic point mutations in C. elegans. We describe a pipeline for sgRNA target selection, homology-directed repair (HDR) template design, CRISPR-Cas9-RNP complexing and delivery, and a genotyping strategy that enables the robust and rapid identification of correctly edited animals. Our approach not only permits the facile generation and identification of desired genomic point mutant animals, but also facilitates the detection of other complex indel alleles in approximately 4 - 5 days with high efficiency and a reduced screening workload.

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

Here, we present a method to engineer the genome of C. elegans using CRISPR-Cas9 ribonucleoproteins and homology dependent repair templates.

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

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 editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

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

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson&#8217;s disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications using hiPSCs and NPCs. The protocol is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

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

MicroRNAs (miRNAs) are 20- to 24-nucleotide (nt) endogenous small RNAs (sRNAs) extensively existing in plants and animals that play potent roles in regulating gene expression at the post-transcriptional level. Sequencing sRNA libraries by Next Generation Sequencing (NGS) methods has been widely employed to identify and analyze miRNA transcriptomes in the last decade, resulting in a rapid increase of miRNA discovery. However, two major challenges arise in plant miRNA annotation due to increasing depth of sequenced sRNA libraries as well as the size and complexity of plant genomes. First, many other types of sRNAs, in particular, short interfering RNAs (siRNAs) from sRNA libraries, are erroneously annotated as miRNAs by many computational tools. Second, it becomes an extremely time-consuming process for analyzing miRNA transcriptomes in plant species with large and complex genomes. To overcome these challenges, we recently upgraded miRDeep-P (a popular tool for miRNA transcriptome analyses) to miRDeep-P2 (miRDP2 for short) by employing a new filtering strategy, overhauling the scoring algorithm and incorporating newly updated plant miRNA annotation criteria. We tested miRDP2 against sequenced sRNA populations in five representative plants with increasing genomic complexity, including Arabidopsis, rice, tomato, maize and wheat. The results indicate that miRDP2 processed these tasks with very high efficiency. In addition, miRDP2 outperformed other prediction tools regarding sensitivity and accuracy. Taken together, our results demonstrate miRDP2 as a fast and accurate tool for analyzing plant miRNA transcriptomes, therefore a useful tool in helping the community better annotate miRNAs in plants.

A bioinformatics pipeline, namely miRDeep-P2 (miRDP2 for short), with updated plant miRNA criteria and an overhauled algorithm, could accurately and efficiently analyze microRNA transcriptomes in plants, especially for species with complex and large genomes.

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

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' lines that express the yeast transcription factor GAL4 in a tissue specific manner, with transgenic 'responder' lines carrying a candidate gene/RNA interference construct whose expression is controlled by Upstream Activation Sequences (UAS) that bind GAL4. In the ensuing progeny, the gene or silencing construct is thus expressed in a prescribed spatiotemporal manner, enabling the resultant phenotypes to be assayed and gene function inferred. The binary system enables flexibility in experimental approaches to screen phenotypes generated by transgene expression in multiple tissue-specific patterns, even if severe fitness costs are induced. We have adapted this system for Anopheles gambiae, the principal malaria vector in Africa.
In this article, we provide some of the common procedures used during GAL4-UAS analysis. We describe the An. gambiae GAL4-UAS lines already generated, as well as the cloning of new responder constructs for upregulation and RNAi knockdown. We specify a step by step guide for sexing of mosquito pupae to establish genetic crosses, that also includes screening progeny to follow inheritance of fluorescent gene markers that tag the driver and responder insertions. We also present a protocol for clearing An. gambiae embryos to study embryonic development. Finally, we introduce potential adaptions of the method to generate driver lines through CRISPR/Cas9 insertion of GAL4 downstream of target genes.

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

The bipartite GAL4-UAS system is a versatile tool for modification of gene expression in a controlled spatiotemporal manner which permits functional genetic analysis in Anopheles gambiae. The procedures described for using this system are a semi-standardized cloning strategy, sexing and screening of pupae for fluorescent protein markers and embryo fixation.

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' ...

Site-Directed &#966;C31-Mediated Integration and Cassette Exchange in Anopheles Vectors of Malaria

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the genome of Anopheles mosquitoes. Amongst these methods, the &#966;C31 system allows precise and stable site-directed integration of transgenes, or the substitution of integrated transgenic cassettes via recombinase-mediated cassette exchange (RMCE). This method relies on the action of the Streptomyces &#966;C31 bacteriophage integrase to catalyze recombination between two specific attachment sites designated attP (derived from the phage) and attB (derived from the host bacterium). The system uses one or two attP sites that have been integrated previously into the mosquito genome and attB site(s) in the donor template DNA. Here we illustrate how to stably modify the genome of attP-bearing Anopheles docking lines using two plasmids: an attB-tagged donor carrying the integration or exchange template and a helper plasmid encoding the &#966;C31 integrase. We report two representative results of &#966;C31-mediated site-directed modification: the single integration of a transgenic cassette in An. stephensi and RMCE in An. gambiae mosquitoes. &#966;C31-mediated genome manipulation offers the advantage of reproducible transgene expression from validated, fitness neutral genomic sites, allowing comparative qualitative and quantitative analyses of phenotypes. The site-directed nature of the integration also substantially simplifies the validation of the single insertion site and the mating scheme to obtain a stable transgenic line. These and other characteristics make the &#966;C31 system an essential component of the genetic toolkit for the transgenic manipulation of malaria mosquitoes and other insect vectors.

The protocol describes how to achieve site-directed modifications in the genome of Anopheles malaria mosquitoes using the &#966;C31 system. Modifications described include both the integration and the exchange of transgenic cassettes in the genome of attP-bearing docking lines.

位点导向 的φC31介导的疟疾 按蚊 载体中的整合和盒式交换

Functional genomic analysis and related strategies for genetic control of malaria rely on validated and reproducible methods to accurately modify the ...