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

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 ?...

<ol>
	<li>Rossi, F. M., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+inducible+gene+expression+systems.">Recent advances in inducible gene expression systems.</a> Curr. Opin. Biotechnol. 9 (5), 451-456 (1998).</li><li>Clackson, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+gene+expression+systems.">Regulated gene expression systems.</a> Gene Ther. 7 (2), 120-125 (2000).</li><li>Suzuki, Y., Suzuki, Y., Xu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulatable+lentiviral+system.">Gene regulatable lentiviral system.</a> Viral Gene Therapy. , 285-309 (2011).</li><li>Gossen, M., Bujard, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tight+control+of+gene+expression+in+mammalian+cells+by+tetracycline-responsive+promoters.">Tight control of gene expression in mammalian cells by tetracycline-responsive promoters.</a> Proc. Natl. Acad. Sci. USA. 89 (12), 5547-5551 (1992).</li><li>Baron, U., Bujard, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tet+repressor-based+system+for+regulated+gene+expression+in+eukaryotic+cells:+principles+and+advances.">Tet repressor-based system for regulated gene expression in eukaryotic cells: principles and advances.</a> Methods Enzymol. 327, 401-421 (2000).</li><li>Hentges, K. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRAP/mTOR+is+required+for+proliferation+and+patterning+during+embryonic+development+in+the+mouse.">FRAP/mTOR is required for proliferation and patterning during embryonic development in the mouse.</a> Proc Natl Acad Sci U S A. 98 (24), 13796-13801 (2001).</li><li>Banaszynski, L. A., Liu, C. W., Wandless, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+FKBP.rapamycin.FRB+ternary+complex.">Characterization of the FKBP.rapamycin.FRB ternary complex.</a> J Am Chem Soc. 127 (13), 4715-4721 (2005).</li><li>Seto, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapamycin+and+mTOR:+a+serendipitous+discovery+and+implications+for+breast+cancer.">Rapamycin and mTOR: a serendipitous discovery and implications for breast cancer.</a> Clin Transl Med. 1 (1), 29 (2012).</li><li>Ulmann, A., Peyron, R., Silvestre, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+uses+of+mifepristone+(MFP).">Clinical uses of mifepristone (MFP).</a> Ann N Y Acad Sci. 761, 248-260 (1995).</li><li>Sitruk-Ware, R., Spitz, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+properties+of+mifepristone:+toxicology+and+safety+in+animal+and+human+studies.">Pharmacological properties of mifepristone: toxicology and safety in animal and human studies.</a> Contraception. 68 (6), 409-420 (2003).</li><li>Yao, T. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+ecdysone+receptor+is+the+product+of+EcR+and+ultraspiracle+genes.">Functional ecdysone receptor is the product of EcR and ultraspiracle genes.</a> Nature. 366 (6454), 476-479 (1993).</li><li>Riddiford, L. M., Cherbas, P., Truman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecdysone+receptors+and+their+biological+actions.">Ecdysone receptors and their biological actions.</a> Vitam Horm. 60 (2000), 1-73 (2000).</li><li>Saez, E., Nelson, M. C., Eshelman, B., Banayo, E., Koder, A., Cho, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+ligands+and+coligands+for+the+ecdysone-regulated+gene+switch.">Identification of ligands and coligands for the ecdysone-regulated gene switch.</a> Proc Natl Acad Sci USA. 97 (26), 14512-14517 (2000).</li><li>Karzenowski, D., Potter, D. W., Padidam, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+control+of+transgene+expression+with+ecdysone+receptor:+gene+switches+with+high+sensitivity+robust+expression,+and+reduced+size.">Inducible control of transgene expression with ecdysone receptor: gene switches with high sensitivity robust expression, and reduced size.</a> Biotechniques. 39 (2), 191-200 (2005).</li><li>Lee, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecdysone+receptor-based+singular+gene+switches+for+regulated+transgene+expression+in+cells+and+adult+rodent+tissues.">Ecdysone receptor-based singular gene switches for regulated transgene expression in cells and adult rodent tissues.</a> Mol. Ther. Nucleic Acids. 5 (9), e367 (2016).</li><li>Esengil, H., Chang, V., Mich, J. K., Chen, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small-molecule+regulation+of+zebrafish+gene+expression.">Small-molecule regulation of zebrafish gene expression.</a> Nat Chem Biol. 3 (3), 154-155 (2007).</li><li>Padidam, M., Gore, M., Lu, D. L., Smirnova, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical-inducible,+ecdysone+receptor-based+gene+expression+system+for+plants.">Chemical-inducible, ecdysone receptor-based gene expression system for plants.</a> Transgenic Res. 12 (1), 101-109 (2003).</li><li>Knopf, F., Schnabel, K., Haase, C., Pfeifer, K., Anastassiadis, K., Weidinger, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dually+inducible+TetOn+systems+for+tissue-specific+conditional+gene+expression+in+zebrafish.">Dually inducible TetOn systems for tissue-specific conditional gene expression in zebrafish.</a> Proc Natl Acad Sci USA. 107 (46), 19933-19938 (2010).</li><li>Jeong, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-step+sequence-+and+ligation-independent+cloning+as+a+rapid+and+versatile+cloning+method+for+functional+genomics+studies.">One-step sequence- and ligation-independent cloning as a rapid and versatile cloning method for functional genomics studies.</a> Appl Environ Microbiol. 78 (15), 5440-5443 (2012).</li><li>Zhu, B., Cai, G., Hall, E. O., Freeman, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-fusion+assembly:+seamless+engineering+of+multidomain+fusion+proteins,+modular+vectors,+and+mutations.">In-fusion assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations.</a> Biotechniques. 43 (3), 354-359 (2007).</li><li>No, D., Yao, T. P., Evans, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecdysone-inducible+gene+expression+in+mammalian+cells+and+transgenic+mice.">Ecdysone-inducible gene expression in mammalian cells and transgenic mice.</a> Proc Natl Acad Sci U S A. 93 (8), 3346-3351 (1996).</li><li>Sawada, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+and+insecticidal+activity+of+benzoheterocyclic+analogues+of+N'-benzoyl-N-(tert-butyl)benzohydrazide:+Part+1.+Design+of+benzoheterocyclic+analogues.">Synthesis and insecticidal activity of benzoheterocyclic analogues of N'-benzoyl-N-(tert-butyl)benzohydrazide: Part 1. Design of benzoheterocyclic analogues.</a> Pest Manag Sci. 59 (1), 25-35 (2003).</li><li>Lafont, R., Girault, J. P., Kerb, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excretion+and+metabolism+of+injected+ecdysone+in+the+white+mouse.">Excretion and metabolism of injected ecdysone in the white mouse.</a> Biochem Pharmacol. 37 (6), 1174-1177 (1988).</li><li>Carlson, G. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chemical+and+biological+properties+of+methoxyfenozide,+a+new+insecticidal+ecdysteroid+agonist.">The chemical and biological properties of methoxyfenozide, a new insecticidal ecdysteroid agonist.</a> Pest Manag Sci. 57 (2), 115-119 (2001).</li><li>McConnell, M. J., Imperiale, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+of+adenovirus+and+its+use+as+a+vector+for+gene+therapy.">Biology of adenovirus and its use as a vector for gene therapy.</a> Hum Gene Ther. 15 (11), 1022-1033 (2004).</li><li>Gorell, E., Nguyen, N., Lane, A., Siprashvili, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+therapy+for+skin+diseases.">Gene therapy for skin diseases.</a> Cold Spring Harb Perspect Med. 4 (4), a015149 (2014).</li><li>Kotterman, M. A., Chalberg, T. W., Schaffer, D. 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>

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 border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pEUI(+)</td>
			<td style="width:71px;">TransLab</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">The patent license was transferred to TransLab Inc.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gene-Fect Transfection Reagent</td>
			<td style="width:71px;">TransLab</td>
			<td style="width:119px;">TLC-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HEK293</td>
			<td style="width:71px;">ATCC</td>
			<td style="width:119px;">CRL-1573</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tebufenozide</td>
			<td style="width:71px;">Fluka</td>
			<td style="width:119px;">31652</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cloning cylinder</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">CLS31668</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Puromycin</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">58-58-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Antibiotics</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">15240-062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin-EDTA</td>
			<td style="width:71px;">Welgene</td>
			<td style="width:119px;">LS015-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:71px;">Welgene</td>
			<td style="width:119px;">LM001-05</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:71px;">Welgene</td>
			<td style="width:119px;">S001-01</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Cell culture dish</td>
			<td style="width:71px;">SPL life science</td>
			<td style="width:119px;">11090</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">mouse FLAG M2</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">F3165</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">anti-alpha tubulin antibody</td>
			<td style="width:71px;">Calbiochem</td>
			<td style="width:119px;">CP06</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cloning cylinder</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">CLS31668</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NucleoBond Xtra Midi</td>
			<td style="width:71px;">Macherey-Nagel</td>
			<td style="width:119px;">740410.1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">M-PER Mammalian Protein 
			Extraction Reagent</td>
			<td style="width:71px;">Thermo Fisher</td>
			<td style="width:119px;">78505</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100X Protease inhibitor Cock. III</td>
			<td style="width:71px;">T&#38;I</td>
			<td style="width:119px;">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 ...

Research

JoVE Journal

Genetics

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

在人原代细胞培养生物钟基因表达及荷尔蒙分泌的并行测量

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

科研

遗传学

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

单细胞基因表达利用多重RT-qPCR的表征罕见的淋巴人群的异质性

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

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

单细胞基因表达谱使用流式细胞仪和qPCR内部标准

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

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

通过两个互补细胞同步方案研究细胞周期调控的基因表达

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

斑马鱼 RNASeq-based 基因表达数据的样品制备与分析

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans

量化的信息编码的基因表达水平在寿命调制下线虫的宽范围饮食限制

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on ...

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

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

无基因克隆的基因破坏的变形链球菌菌株的生成

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

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

提高吞吐量的轰击大麦糊粉层层中基因表达的测定

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for ...

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

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 ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

从土壤、根际和根 Endosphere 提取细菌群落数据的根菌群探析

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...