作者感谢全南 (美国国立大学) 的宝贵意见和对我们手稿的透彻阅读。这项工作得到了于忠清国立大学研究基金的支持。

1. 转基因亚克隆
<ol><li>在 pEUI (+) 的 MCS 中选择适当的限制酶识别站点 (或站点), 并亚克隆所需方向的感兴趣基因 (图1)。 注意: EGFP或标志位标记的锚蛋白重复域 13A (ANKRD13A) 转基因在 EcoRI (+) 向量 pEUI 站点 subcloned, 使用 T4 DNA 聚合酶在序列和结扎无关的克隆方法 19.</li>
	<li>进行线性化 pEUI (+), EcoRI 1 小时, 37 摄氏度。将50µL 线性化 pEUI (+) 与 PCR 扩增的EGFP或 ANKRD13A 的 40 ng/µL 混合在一起,并与 T4 DNA 聚合酶 (1 U) 在室温下孵化前1分钟 (RT) 反应10分钟. 将反应混合物的10µL 直接添加到100µLDH10B 胜任细胞进行以下改造实验。 注意: 虽然在 pEUI (+) 的 MCS 中添加了几个限制酶识别站点 (图 1B), 但我们建议将目标基因亚克隆到 EcoRI 站点, 使用结扎无关的克隆技术 19, 20.</li>
	<li>通过测序验证插入, 然后使用适合转染的 dna 纯化试剂盒纯化 dna。</li>
</ol>2. 瞬变转染和 Teb 处理
<ol><li>准备四细胞培养皿 (60 毫米直径), 含有新鲜的传代 HEK293 细胞在5毫升 Dulbecco 的改良鹰的培养基 (DMEM) 补充 5-10% 胎牛血清 (血清) 和抗生素 (100 U/毫升青霉素, 100 µg/毫升链霉素)。此后, "细胞培养培养基" 或简单的 "培养基" 代表 DMEM 辅以血清和抗生素。保持 HEK293 细胞在37摄氏度。</li>
	<li>用3µL 转染试剂将纯化 DNA 的1µg 染 50-60% 密度的 HEK293 细胞中。添加转染试剂到200µL 稀释剂 (DMEM 没有血清和抗生素) 含有 DNA。在30分钟的反应混合物孵化后, 在培养皿中加入 HEK-293 细胞的混合物。使用两个盘子进行转染, 并让其他两个不治疗作为控制。</li>
	<li>在转染 12-24 小时后, 将 Teb (最终浓度, 0.2-10 µmol) 添加到已染成的样本之一中, 并将其加入到一条未染的控制中。将其他两个样本保留为实验性控制。 注: Teb 库存溶液 (50 毫米溶解在二甲基亚砜) 可以稀释在细胞培养基, DMEM, 或磷酸盐缓冲盐水 (PBS) 在适当的浓度, 然后添加到培养细胞根据实验参数。但是, 由于 Teb 在水溶液中溶解度低, 避免了在培养基中制备高浓度 Teb 的主混合;取而代之的是, 用含有 Teb 的新鲜培养基取代完整的细胞培养培养基, 以达到预期的浓度。</li>
	<li>培养 Teb 治疗和未经治疗的细胞为 12-48 小时在37摄氏度。</li>
	<li>如果使用EGFP插入, 请在荧光显微镜下分析 EGFP 表达式 (图 2)。否则, 收获细胞的免疫印迹法化验。<ol>
<li>对于免疫印迹法化验, 用冷的 PBS 冲洗两次细胞, 然后用一个细胞刮刀将它们刮出来。</li>
		<li>对于堆积细胞, 在 2.1万 x g (RT) 的2分钟内, 在15毫升圆锥管中的收成下旋转。</li>
		<li>完全丢弃 PBS 后, 添加0.5 毫升的冰-冷哺乳动物蛋白萃取试剂 (M 每), 连同蛋白酶抑制剂 (5 µL 的100x 蛋白酶抑制剂鸡尾酒), 如果细胞培养在60毫米菜。</li>
		<li>用几轮吹打重新悬浮细胞, 然后在 18-25 摄氏度 (RT) 上孵化悬浮细胞15分钟。</li>
		<li>旋转细胞裂解液在 2.1万 x g 10 分钟在4摄氏度。</li>
		<li>将水中清液转化为新鲜的1.5 毫升离心管。</li>
		<li>用适当的抗体免疫印迹法的标准协议, 分析上清液。</li>
	</ol>
</li>
</ol>3. pEUI (+) 基因开关的基因组积分生成稳定的 HEK293 细胞系
<ol><li>染 5-10 µg 的纯化 pEUI (+) 与转基因成 HEK293 细胞, 50-60% 汇合在90毫米直径细胞培养皿, 使用 15-30 µL 的转染试剂。添加转染试剂到500µL 稀释剂 (DMEM 没有血清和抗生素) 含有 DNA。在30分钟的反应混合物孵化后, 在培养皿中加入 HEK-293 细胞的混合物。</li>
	<li>转染后, 允许 transfectants 在非选择性培养基 (不嘌呤霉素) 下生长和表达嘌呤霉素抗性基因产物, 24-48 小时37摄氏度。</li>
	<li>用 trypsinization 将细胞从培养皿中分离出来。丢弃培养基后, 用预热后的 PBS (37 °c) 冲洗 transfectants, 加入1毫升0.25% 胰蛋白酶/乙二胺 (EDTA), 并在37摄氏度孵育1分钟。在通过加入预热培养基 (10 毫升, 37 °c) 淬火胰蛋白酶/EDTA 后, 使用细胞刮刀收割细胞, 然后在15毫升圆锥管中离心, 在18-25°C 的 2.1万 x g 处为2分钟。</li>
	<li>在50毫升预热培养基 (37 °c) 中重新悬浮 transfectants, 将10毫升的细胞转移到90毫米新鲜细胞培养皿中。培养细胞在37°c 直到 50-60% 融合在嘌呤霉素治疗之前 (一般来说, 这需要大约 24-48 h)。</li>
	<li>培养 transfectants 在37°c 在细胞培养培养基补充3µg/毫升嘌呤霉素 3-4 周。在选择期间, 每 2-3 天, 改变含有嘌呤霉素 (3 µg/毫升) 的细胞培养基, 以保持选择压力。当细胞完全汇合时, 按照步骤 3.3-3.4 中的过程划分它们, 其中含有嘌呤霉素的培养基。如果不需要, 请丢弃区域性介质中的其余单元格。 注意: 必须为特定的细胞类型建立实验性的嘌呤霉素选择条件。我们在细胞培养培养基中使用了3µg/毫升嘌呤霉素;这足以诱发非转染 HEK293 细胞死亡。</li>
	<li>使用克隆圆柱体从单个菌落中收获细胞, 并按照制造商的说明进行操作。<ol>
<li>丢弃细胞培养基。用预热后的 (37 °c) PBS 冲洗两次盘子, 以去除漂浮的细胞。</li>
		<li>使用无菌钳, 将克隆筒放在各个菌落上。</li>
		<li>添加0.2 毫升0.35% 胰蛋白酶/EDTA 到每个气缸。</li>
		<li>孵化的菜在37°c 1 分钟, 直到细胞分离, 然后添加几滴预预热 (37 °c) 培养基到气缸。</li>
		<li>将每个气缸的细胞转移到一个6井板的单个井中, 每个井中有2毫升培养基和1µg/毫升嘌呤霉素。 注意: 当细胞汇合时, 放大文化。现在可以建立克隆。</li>
	</ol>
</li>
	<li>维持细胞培养培养基中的单个无性系, 辅以1µg/毫升嘌呤霉素。</li>
	<li>通过测试其对 Teb 治疗 (最终浓度, 10 µM) 的敏感性, 验证各自的菌落数 24 h. 通过分析转基因与免疫印迹法的表达水平, 测试克隆对 Teb 的响应性。选择所需的克隆 (或克隆)。在对转基因的诱导水平进行分析之前, 在 12-48 小时的培养基上培养 Teb 处理的和未经治疗的样品。转基因的表达水平可以通过免疫印迹法测量, 遵循步骤 2.5.1 2.5.7。</li>
</ol>4. Teb 的损耗
<ol><li>用 Teb 游离培养基取代含有细胞培养培养基的 Teb, Teb 刺激。 注意: 建议在不首先补种细胞进入新的培养皿的情况下更换培养基。残留的 Teb 似乎附着在塑料盘子上, 并会引起转基因的强烈背景表达。在将细胞补种到新的培养板上之前, 用培养基或 PBS 多次冲洗收获细胞。<ol>
<li>用 trypsinization 将细胞从培养皿中分离出来。在丢弃含有培养基的 Teb 后, 用预热后的 PBS (37 °c) 冲洗两次 Teb 刺激细胞, 加入1毫升0.25% 胰蛋白酶/EDTA, 在37摄氏度时孵育1分钟。在通过加入预热的 Teb 培养基 (10 毫升, 37 °c) 淬火胰蛋白酶/EDTA 后, 用一个细胞刮刀收割细胞, 然后在15毫升圆锥管中离心, 在2摄氏度处为18-25 分钟。</li>
		<li>重新悬浮细胞在50毫升预热的 Teb 培养基 (37 °c), 并将5毫升的细胞转化为三60毫米新鲜细胞培养皿。培养细胞在37°c 分别为 24, 48 和 72 h。</li>
		<li>利用免疫印迹法, 在不同时间点收获细胞, 测量转基因的表达水平。 注: 在这些实验条件下, 转基因表达在无 Teb 文化培养基中的细胞后培养的大约3天内无法检测到 (图 3)。转基因的表达水平可以通过免疫印迹法测量, 每步 2.5.1 2.5.7。</li>
	</ol>
</li>
</ol>

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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+Vectors+for+Gene+Therapy:+Translational+and+Clinical+Outlook.">Viral Vectors for Gene Therapy: Translational and Clinical Outlook.</a> Annu Rev Biomed Eng. 17, 63-89 (2015).</li><li>Matsumoto, T., Yamaguchi, M., Kuzume, M., Matsumiya, A., Kumada, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin+gene+transfer+with+adenovirus+vector+via+the+spleen+safely+and+effectively+improves+posthepatectomized+conditions+in+diabetic+rats.">Insulin gene transfer with adenovirus vector via the spleen safely and effectively improves posthepatectomized conditions in diabetic rats.</a> J Surg Res. 110 (1), 228-234 (2003).</li><li>Han, J., McLane, B., Kim, E. H., Yoon, J. W., Jun, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remission+of+diabetes+by+insulin+gene+therapy+using+a+hepatocyte-specific+and+glucose-responsive+synthetic+promoter.">Remission of diabetes by insulin gene therapy using a hepatocyte-specific and glucose-responsive synthetic promoter.</a> Mol Ther. 19 (3), 470-478 (2011).</li></ol>

我们没有与本报告有关的利益冲突。

使用二进制基因表达式开关的主要缺点是需要同时提供两个单独的质粒 (驱动程序和效应器) 到目标细胞。这可能导致质粒的分布不均匀, 并导致切换到诱导器1、2、3的不一致响应。两个向量系统的另一个缺点是需要至少两轮的抗生素选择, 以确定有希望的细胞系。因此, 在生物研究中, 长期以来一直在寻找一种由一个单元组成的...

研究了几种不同的基因开关, 以精确调节细胞培养和动物模型中转基因表达的能力, 不同程度的成功1,2,3。潜在的基因交换系统应符合几个严格的标准, 包括: 转基因的精确定向表达, 剂量和时间依赖性的诱导反应, 可忽略的启动子泄漏, 转基因的可逆性激活通过诱导剂去除, 诱导剂毒性水平耐受细胞系和实验动物1,2,3。已开发了几个小分子诱导剂, 包括四环素4、5、雷帕霉素6、7、8、米非司酮9、10、和昆虫蜕皮激素11, 12, 13, 14.一般而言, 这些系统至少需要两个或三个离散表达单元的二质粒, 其中一个或两个组成一个调节元素 (诱导质粒), 使小分子诱导剂能够获得转录活性;和另一种质粒 (效应质粒), 包含在调控 DNA 区域控制下的转基因, 允许诱导剂约束的调控元素进入。二进制系统的主要缺点是, 它需要同时引入两个向量 (诱导器和效应) 到目标单元中。在瞬态转基因表达实验中, 两种质粒的同时转染, 不可避免地会产生一个细胞的数量, 它们是由诱导器或效应器单独转移的, 或者是不相等的。此外, 二进制系统需要至少两轮抗生素选择, 以建立稳定的细胞系, 这是费时费力的。因此, 将转基因表达和调节所需的所有成分组合成一个单一的载体, 将是保证单个细胞中所有必需元素同时表达并允许对转基因表达进行调节的理想方法。通过小分子诱导剂治疗。
为了克服二进制基因开关的缺点, 我们最近开发了一个奇异基因开关, 使用了一个果蝇基于 EcR 的基因诱导系统15。昆虫蜕皮激素介导基因表达, 当它绑定到 ecr/ultraspiracle (USP) heterodimer, 这反过来又诱导了 ecr 对 DNA 调控元素的绑定3, 11.脊椎动物甲酸 X 受体 (RXR), 直系同源的昆虫 USP, 新兵 EcR 形成一个活跃转录激活剂 16.因此, 对于基因在脊椎动物细胞或组织中的靶向表达, EcR 和 RXR 必须同时表达, 然后才被昆虫蜕皮激素或其激动剂刺激。由于基于 EcR 的基因开关的 heterodimeric 特征可能受内源 RXR 水平的影响, Padidam et。用异源 GAL4 dna 结合、单纯疱疹病毒蛋白 vmw65 (VP16) 活化域取代 ecr dna 结合和活化域, 融合到 ecr 配体结合域, 从而对内源性 RXR 无反应, 形成 homodimer获取转录活动17。通过构建由最小 VP16 活化域和 GvEcR 组合组成的嵌合体蛋白, 在昆虫蜕皮激素激动剂缺席的情况下, 在细胞质中形成 homodimer 和局部化, 从而进一步改善了 EcR 基因开关,Teb16, 18。通过绑定到 Teb, GvEcR 改变了其亚细胞定位从细胞质到核, 以识别一个混合启动子由斑马鱼 E1b 最小启动子结合十串联重复上游活化序列 (UASs), 以启动目标基因的转录16。
为了简化以前报告的基于 EcR 的基因开关16,18, 我们将系统的二进制特征组合成一个单一的向量, 并配备所有必需的元素来刺激转基因表达, 然后指定新生成的矢量, pEUI (+) (基因库加入号: KP123436,图 1A)15。响应 GvEcR 驱动程序 (以下驱动程序) 的效应器由一个 E1b 最小启动子旁边的十个串联的无人驾驶系统重复, 后跟一个具有 EcoRI、PmlI、NheI、BmtI、AfeI 和 AflII 识别序列 (图 1B) 的多个克隆站点 (MCS)。此外, 为了便于选择转染细胞, SV40 启动子驱动嘌呤霉素N-乙酰转移酶 (PAC) 基因放置在驱动程序和效应区之间, 以维持稳定的细胞线(图 1)。
本文描述了一种利用 pEUI (+) 对转基因表达进行瞬态和稳定调制的详细协议。此外, 我们还提供了有关成功使用 Teb 作为昆虫蜕皮激素激动剂的详细说明。

<table><tbody><tr><td>pEUI(+)</td><td>TransLab</td><td></td><td>The patent license was transferred to TransLab Inc.&amp;nbsp;</td></tr><tr><td>Gene-Fect Transfection Reagent</td><td>TransLab</td><td>TLC-001</td><td></td></tr><tr><td>HEK293</td><td>ATCC</td><td>CRL-1573</td><td></td></tr><tr><td>Tebufenozide</td><td>Fluka</td><td>31652</td><td></td></tr><tr><td>Cloning cylinder</td><td>Sigma</td><td>CLS31668</td><td></td></tr><tr><td>Puromycin</td><td>Corning</td><td>58-58-2</td><td></td></tr><tr><td>Antibiotics</td><td>Gibco</td><td>15240-062</td><td></td></tr><tr><td>Trypsin-EDTA</td><td>Welgene</td><td>LS015-01</td><td></td></tr><tr><td>DMEM</td><td>Welgene</td><td>LM001-05</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Welgene</td><td>S001-01</td><td></td></tr><tr><td>Cell culture dish</td><td>SPL life science</td><td>11090</td><td></td></tr><tr><td>mouse FLAG M2</td><td>Sigma</td><td>F3165</td><td></td></tr><tr><td>anti-alpha tubulin antibody</td><td>Calbiochem</td><td>CP06</td><td></td></tr><tr><td>Cloning cylinder</td><td>Sigma</td><td>CLS31668</td><td></td></tr><tr><td>NucleoBond Xtra Midi</td><td>Macherey-Nagel</td><td>740410.1</td><td></td></tr><tr><td>M-PER Mammalian Protein&lt;br/&gt; Extraction Reagent</td><td>Thermo Fisher</td><td>78505</td><td></td></tr><tr><td>100X Protease inhibitor Cock. III</td><td>T&amp;amp;I</td><td>BPI-9200</td><td></td></tr></tbody></table>

an ecdysone receptor-based singular gene switch for deliberate expression of transgene with robustness, reversibility, and negligible leakiness

在生物学和临床研究中, 对转基因表达的精确控制是可取的。然而, 由于目前使用的基因开关的二进制特征要求将两个治疗表达单元同时移植到单个细胞中, 基因治疗系统的实际应用受到限制。为了简化转基因表达系统, 我们生成了一个基因开关, 它被指定为 pEUI (+), 它包含了一个单一向量中的一整套转基因表达模块。由 GAL4 dna 结合领域和修改的 EcR (GvEcR) 组成, 一个最小的 VP16 活化域与 GAL4 DNA 结合领域, 以及修改后的果蝇昆虫蜕皮激素受体 (EcR), 新开发的奇异基因开关是高度以时间和剂量依赖性的方式对化学诱导剂的管理作出反应。pEUI (+) 载体是一种潜在的强有力的工具, 以改善基因表达的控制, 在生物研究和临床前研究。在这里, 我们提出了一个详细的协议, 以调制的瞬态和稳定的转基因表达使用 pEUI (+) 的治疗米满 (Teb)。此外, 我们还分享了使用 Teb 作为化学诱导剂的重要指导方针。

在图 1A中描述了基于 GvEcR 的奇异基因开关。pEUI (+) 载体通过 Teb 治疗对转基因表达进行优化。pEUI (+) 的效应区包括10xUAS 和 E1b 最小启动子, 后跟一个包含 EcoRI、PmlI、NheI、BmtI、AfeI 和 AflII 限制酶识别点的 MCS。在 MCS (图 1B) 后面添加了一个 SV40 的聚 (A) 信号。在 pEUI (+) 的驱动程序和效应区域之间插入了一个PAC基因, 以赋予对...

Watch this Scientific Journal Video about An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness at JoVE.com

An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

一种基于昆虫蜕皮激素受体的奇异基因开关, 用于对转基因进行有鲁棒性、可逆性和可忽略的泄漏的刻意表达

在这里, 我们提出了一种调制的转基因表达使用 pEUI (+) 奇异基因开关的米满治疗。

Precise control of transgene expression is desirable in biological and clinical studies. However, because the binary feature of currently employed gene ...

an-ecdysone-receptor-based-singular-gene-switch-for-deliberate

Research

JoVE Journal

Genetics

6.4K 次观看.  Chungnam National University. 在这里, 我们提出了一种调制的转基因表达使用 pEUI (+) 奇异基因开关的米满治疗。

视频: An Ecdysone Receptor-based Singular Gene Switch for Deliberate Expression of Transgene with Robustness, Reversibility, and Negligible Leakiness

Controllable Ion Channel Expression through Inducible Transient Transfection

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be investigated through electrophysiological analysis, biochemical characterization, mutational studies, and their effects on cellular processes. Transient transfections offer a simple protocol in which the protein becomes available for analysis within a few hours to days. Although this method presents a relatively straightforward and time efficient protocol, one of the critical components is calibrating the expression of the gene of interest to physiological relevant levels or levels that are suitable for analysis. To this end, many different approaches that offer the ability to control the expression of the gene of interest have emerged. Several stable cell transfection protocols provide a way to permanently introduce a gene of interest into the cellular genome under the regulation of a tetracycline-controlled transcriptional activation. While this technique produces reliable expression levels, each gene of interest requires a few weeks of skilled work including calibration of a killing curve, selection of cell colonies, and overall more resources. Here we present a protocol that uses transient transfection of the Transient Receptor Potential cation channel subfamily V member 1 (TRPV1) gene in an inducible system as an efficient way to express a protein in a controlled manner which is essential in ion channel analysis. We demonstrate that using this technique, we are able to perform calcium imaging, whole cell, and single channel analysis with controlled channel levels required for each type of data collection with a single transfection. Overall, this provides a replicable technique that can be used to study ion channels structure and function.

Studying ion channels through a heterologously expressing system has become a core technique in biomedical research. In this manuscript, we present a time efficient method to achieve tightly controlled ion channel expression by performing transient transfection under the control of an inducible promoter.

通过诱导瞬时转染可控离子通道表达

Transfection, the delivery of foreign nucleic acids into a cell, is a powerful tool in protein research. Through this method, ion channels can be ...

科研

遗传学

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

细胞间相互作用中基因表达模式的控制与分析的 Optogenetic 方法

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

In vivo Application of the REMOTE-control System for the Manipulation of Endogenous Gene Expression

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for reversible and tunable expression control of an endogenous gene of interest in living model systems. The REMOTE-control system employs enhanced lac repression and tet activation systems to achieve down- or upregulation of a target gene within a single biological system. Tight repression can be achieved from repressor binding sites flexibly located far downstream of a transcription start site by inhibiting transcription elongation. Robust upregulation can be attained by enhancing the transcription of an endogenous gene by targeting tet transcriptional activators to the cognate promoter. This reversible and tunable expression control can be applied and withdrawn repeatedly in organisms. The potency and versatility of the system, as demonstrated for endogenous Dnmt1 here, will allow more precise in vivo functional analyses by enabling investigation of gene function at various expression levels and by testing the reversibility of a phenotype.

This protocol outlines the steps needed to generate a model system in which the transcription of an endogenous gene of interest can be conditionally controlled in live animals or cells using enhanced lac repressor and/or tet activator systems.

remtoe 控制系统在内源基因表达调控中的应用

Here we describe a protocol for implementing the REMOTE-control system (Reversible Manipulation of Transcription at Endogenous loci), which allows for ...

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the flexible and efficient GreenGate cloning system with the proven and benchmarked LhGR system (here termed GR-LhG4) for the inducible expression, we have generated a set of transgenic Arabidopsis lines that can drive the expression of an effector cassette in a range of specific cell types in the three main plant meristems. To this end, we chose the previously developed GR-LhG4 system based on a chimeric transcription factor and a cognate pOp-type promoter ensuring tight control over a wide range of expression levels. In addition, to visualize the expression domain where the synthetic transcription factor is active, an ER-localized mTurquoise2 fluorescent reporter under control of the pOp4 or pOp6 promoter is encoded in driver lines. Here, we describe the steps necessary to generate a driver or effector line and demonstrate how cell type specific expression can be induced and followed in the shoot apical meristem, the root apical meristem and the cambium of Arabidopsis. By using several or all driver lines, the context specific effect of expressing one or multiple factors (effectors) under control of the synthetic pOp promoter can be assessed rapidly, for example in F1 plants of a cross between one effector and multiple driver lines. This approach is exemplified by the ectopic expression of VND7, a NAC transcription factor capable of inducing ectopic secondary cell wall deposition in a cell autonomous manner.

Inducible, Cell Type-Specific Expression in Arabidopsis thaliana Through LhGR-Mediated Trans-Activation

Here, we describe the generation and application of a set of transgenic Arabidopsis thaliana lines enabling inducible, tissue-specific expression in the three main meristems, the shoot apical meristem, the root apical meristem, and the cambium.

Lhgra 介导的反激活在拟南芥诱导、细胞类型特异性表达中的表达

Inducible, tissue-specific expression is an important and powerful tool to study the spatio-temporal dynamics of genetic perturbation. Combining the ...

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

在细胞分化过程中光介导的可逆调节丝裂原活化蛋白激酶途径<em&gt;爪蟾</em&gt;胚胎发育

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

发育生物学

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of cells or tissues in model organisms. These systems contain two components as separate transgenic lines. A driver line expresses a transcriptional activator under the control of tissue-specific promoters/enhancers, and a reporter/effector line harbors a target gene placed downstream to the binding site of the transcription activator. Animals harboring both components induce tissue-specific transactivation of a target gene expression. Precise spatiotemporal expression of the gene in targeted tissues is critical for unbiased interpretation of cell/gene activity. Therefore, developing a method for generating exclusive cell/tissue-specific driver lines is essential. Here we present a method to generate highly tissue-specific targeted expression system by employing a &#34;Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated&#34; (CRISPR/Cas)-based genome editing technique. In this method, the endonuclease Cas9 is targeted by two chimeric guide RNAs (gRNA) to specific sites in the first coding exon of a gene in the Drosophila genome to create double-strand breaks (DSB). Subsequently, using an exogenous donor plasmid containing the transactivator sequence, the cell-autonomous repair machinery enables homology-directed repair (HDR) of the DSB, resulting in precise deletion and replacement of the exon with the transactivator sequence. The knocked-in transactivator is expressed exclusively in cells where the cis-regulatory elements of the replaced gene are functional. The detailed step-by-step protocol presented here for generating a binary transcriptional driver expressed in Drosophila fgf/branchless-producing epithelial/neuronal cells can be adopted for any gene- or tissue-specific expression.

An Efficient Strategy for Generating Tissue-specific Binary Transcription Systems in Drosophila by Genome Editing

Here, we present a method for generating tissue-specific binary transcription systems in Drosophila by replacing the first coding exon of genes with transcription drivers. The CRISPR/Cas9-based method places a transactivator sequence under the endogenous regulation of a replaced gene, and consequently facilitates transctivator expression exclusively in gene-specific spatiotemporal patterns.

基于基因组编辑的果蝇组织特异二进制转录系统的高效生成策略

Binary transcription systems are powerful genetic tools widely used for visualizing and manipulating cell fate and gene expression in specific groups of ...

Applying an Inducible Expression System to Study Interference of Bacterial Virulence Factors with Intracellular Signaling

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a signaling pathway. The method is based, on the one hand, on the inducible expression of a specific protein to initiate a signaling event at a defined and predetermined step in the selected signaling cascade. Concomitant expression, on the other hand, of the gene of interest then allows the investigator to evaluate if the activity of the expressed target protein is located upstream or downstream of the initiated signaling event, depending on the readout of the signaling pathway that is obtained. Here, the apoptotic cascade was selected as a defined signaling pathway to demonstrate protocol functionality. Pathogenic bacteria, such as Coxiella burnetii, translocate effector proteins that interfere with host cell death induction in the host cell to ensure bacterial survival in the cell and to promote their dissemination in the organism. The C. burnetii effector protein CaeB effectively inhibits host cell death after induction of apoptosis with UV-light or with staurosporine. To narrow down at which step CaeB interferes with the propagation of the apoptotic signal, selected proteins with well-characterized pro-apoptotic activity were expressed transiently in a doxycycline-inducible manner. If CaeB acts upstream of these proteins, apoptosis will proceed unhindered. If CaeB acts downstream, cell death will be inhibited. The test proteins selected were Bax, which acts at the level of the mitochondria, and caspase 3, which is the major executioner protease. CaeB interferes with cell death induced by Bax expression, but not by caspase 3 expression. CaeB, thus, interacts with the apoptotic cascade between these two proteins.

The method described here is used to induce the apoptotic signaling cascade at defined steps in order to dissect the activity of an anti-apoptotic bacterial effector protein. This method can also be used for inducible expression of pro-apoptotic or toxic proteins, or for dissecting interference with other signaling pathways.

施加诱导表达系统研究与细胞内信号细菌毒力因子的干扰

The technique presented here allows one to analyze at which step a target protein, or alternatively a small molecule, interacts with the components of a ...

免疫与感染

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

可靠地工程和控制哺乳动物细胞中稳定的光遗传学基因回路

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...

生物工程

Rapid Synthesis and Screening of Chemically Activated Transcription Factors with GFP-based Reporters

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the structure of native control circuits. In both cases, the ability to program gene expression in a rapid and tunable fashion, with no off-target effects, can be useful. We have constructed yeast strains containing the ACT1 promoter upstream of a URA3 cassette followed by the ligand-binding domain of the human estrogen receptor and VP16. By transforming this strain with a linear PCR product containing a DNA binding domain and selecting against the presence of URA3, a constitutively expressed artificial transcription factor (ATF) can be generated by homologous recombination. ATFs engineered in this fashion can activate a unique target gene in the presence of inducer, thereby eliminating both the off-target activation and nonphysiological growth conditions found with commonly used conditional gene expression systems. A simple method for the rapid construction of GFP reporter plasmids that respond specifically to a native or artificial transcription factor of interest is also provided.

This protocol describes an experimental procedure for the rapid construction of artificial transcription factors (ATFs) with cognate GFP reporters&#160;and quantification of the ATFs ability to stimulate GFP expression via flow cytometry.

快速合成及其与绿色荧光蛋白为基础的记者化学激活的转录因子筛选

Synthetic biology aims to rationally design and build synthetic circuits with desired quantitative properties, as well as provide tools to interrogate the ...

生物学

Inducible Tet-On Regulatable System-Based Gene Expression In Vivo: A Tetracycline-Induced Gene Expression System for Target Gene Transcription Activation in Mouse Model

Source: Hubner, E. K. et al.&#160; <a href="https://www.jove.com/v/56613">Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein Injection.</a> J. Vis. Exp. (2018)
This video demonstrates the tetracycline, or Tet-On, gene expression system in a mouse model. In the presence of doxycycline, a tetracycline derivative, the Tet-On system consisting of reverse tetracycline-controlled transactivator protein becomes activated and expresses the gene of interest.

诱导型 Tet-On 基于调节系统的体内基因表达:用于小鼠模型中靶基因转录激活的四环素诱导基因表达系统

Source: Hubner, E. K. et al.&#160; Constitutive and Inducible Systems for Genetic In Vivo Modification of Mouse Hepatocytes Using Hydrodynamic Tail Vein ...