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>

<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>

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基因诱导盒。pEUI (+) 奇异基因表达开关包含在一个单一的载体, 配备所有调控单位所需的转基因刺激。因此, subcloned 在 pEUI (+) 中的诱导水平依赖于瞬变转染实验中的 DNA 量和化学诱导剂的用量。此外, 我们进一步测试了 pEUI (+) 向量的用法, 方法是生成一个稳定的单元格线, 其中包含ANKRD13A转基因15的单个副本。已建立的细胞线对 Teb 治疗有很高的敏感性 (图 3)。总的来说, 它的高灵敏度、可逆性和低泄漏15以及表达式水平的有限变化, 使得 pEUI (+) 成为处理目标基因表达的宝贵的分子和细胞工具。
在各种诱导基因系统中, 我们选择了 GvEcR 依赖的基因诱导盒。GvEcR 比其他系统有几个优点: 首先, 作为化学诱导剂 (包括 Teb) 的几种昆虫蜕皮激素类似物的亲脂性质有利于细胞膜的有效穿透21;其次, 脊椎动物中没有 EcR orthologs, 所以 ecdysteroid 激动剂不影响内在表达核受体的功能21,22;第三, ecdysteroids 在脊椎动物中是无害的, 并且从循环系统中快速清除体内21,23;第四, 由于许多昆虫蜕皮激素激动剂已经发展到目前为止, 通过选择最佳大小的小分子, 研究人员可以避免不良的并发症13, 22, 24.尽管 pEUI (+) 对 Teb 有很高的灵敏度, 但测试 pEUI (+) 对其他 ecdysteroid 激动剂的响应性可能证明是有价值的。非甾体 EcR 激动剂的存在, 如 methoxyfenozide, 比 Teb24更能溶于水, 可扩大 pEUI (+) 的应用范围。虽然 pEUI (+) 中的转基因表达相对较弱, 但在系统15中, 可通过增加 VP16 转录激活域 (未显示的数据) 的多拷贝来增强转基因的诱导水平。最近的基因治疗应用重点是将目标基因安全交付到缺乏各自功能基因产品的细胞或组织中, 因为遗传疾病25,26,27。然而, 在组织特异或病毒本构促进剂的控制下, 非调控转基因可以诱导靶基因的超表达, 从而提升局部组织损伤的可能性28,29。因此, 应用可靠的基因开关进行基因治疗可以避免不必要的并发症。pEUI (+) 载体为控制转基因在生物和临床研究中的表达提供了方便和有力的工具。目前, 我们正在开发一个基于 GvEcR 的奇异慢病毒载体向量, 以增加系统的应用范围。

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, 以赋予对嘌呤霉素的抵抗力。
为了评估 pEUI (+) 的活动和泄漏, 我们 subcloned EGFP到 MCS (EcoRI (+) pEUI) 的 EGFP 站点, 瞬时将质粒结构转染为 HEK293 细胞, 并在不同浓度下对 24 h ( ) 执行 Teb。图 2)。尽管模拟矢量 transfectants 没有显示荧光信号 (图 2A、 B), 但 pEUI (+) EGFP transfectants 逐渐敏感到 Teb 的增加量 (图 2C-F).更重要的是, pEUI (+)-EGFP transfectants 没有治疗 Teb 没有引起任何可检测的 EGFP 信号下荧光显微镜 (图 2C)。
为了进一步验证 pEUI (+) 对 Teb 的响应性, 我们 subcloned 了一个标志位标记的ANKRD13A基因到 EcoRI (+) 的 pEUI 站点, 将构造转移到 HEK293 单元, 然后将其按嘌呤霉素选择3周, 以建立具有ANKRD13A转基因的所需的单元格线。我们分析了ANKRD13A转基因的表达后, 24 小时的治疗与 Teb 和建立的细胞线反应良好 (图 3)。我们进一步证明了 pEUI (+) 基因开关的可逆性通过消耗 Teb 从文化媒介。如图 3所示, 当我们在无 Teb 介质中培养细胞时, ANKRD13A 表达式不再能被检测到。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57494/57494fig1.jpg"> 图 1.pEUI (+) 的原理图.(A) 构造了 pEUI (+) 的矢量图。一种巨细胞病毒启动子驱动 GvEcR 的本构表达。Teb 绑定的 GvEcR 将植物常常将到原子核中 , 并绑定10xUAScis - 代理规则序列 , 以刺入到 MCS 中的转基因的表达。(B) (A) 中方括号之间的序列信息包含 pEUI (+) 的整个效应器序列。箭头表示 MCS 中包含的单个限制酶识别站点。<a href="/files/ftp_upload/57494/57494fig1large.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/57494/57494fig2.jpg"> 图 2.基于 GvEcR 的奇异基因开关对 Teb 的治疗有反应.HEK293 细胞用指定的 DNA 结构进行转染。经过24小时的转染, 细胞被刺激为 24 h 与 Teb 在指定的浓度。在荧光显微镜下观察到了EGFP转基因活性。模拟 transfectants 与 pEUI (+) 没有显示任何可检测的荧光与 (A) 或没有 (B) Teb 治疗。(C-E)pEUI (+)-EGFP 转染细胞的荧光强度以 Teb 剂量依赖性的方式增加。缩放条, 10 µm.<a href="/files/ftp_upload/57494/57494fig2large.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/57494/57494fig3.jpg"> 图 3.HEK293 细胞稳定地窝藏 pEUI (+)/旗 ANKRD13A 反应可逆地对 Teb 的管理.标记表位标记的 ANKRD13A 表达是由 Teb (1.5 µM) 的治疗触发的。经过24小时的治疗与 Teb, 细胞被切换到 Teb 下降的媒体, 在指定的时间量。用反旗抗体测定了 ANKRD13A 的相对含量。以抗α蛋白抗体为对照, 测定了α蛋白的内源表达水平。白色星号表示未知的交叉反应物种。<a href="/files/ftp_upload/57494/57494fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

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

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

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

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

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

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

Gene expression measurements from bulk populations of cells can obscure the considerable transcriptomic variation of individual cells within those populations. Single-cell gene expression measurements can help assess the role of noise in gene expression, identify correlations in the expression of pairs of genes, and reveal subpopulations of cells that respond differently to a stimulus. Here, we describe a procedure to measure the expression of up to 96 genes in single mammalian cells isolated from a population growing in tissue culture. Cells are sorted into lysis buffer by fluorescence-activated cell sorting (FACS), and the mRNA species of interest are reverse-transcribed and amplified. Gene expression is then measured using a microfluidic real-time PCR machine, which performs up to 96 qPCR assays on up to 96 samples at a time. We also describe the generation and use of PCR amplicon standards to enable the estimation of the absolute number of each transcript. Compared with other methods of measuring gene expression in single cells, this approach allows for the quantification of more distinct transcripts than RNA FISH at a lower cost than RNA-Seq.

We describe a method to sort single mammalian cells and to quantify the expression of up to 96 target genes of interest in each cell. This method includes the use of internal qPCR standards to enable the estimation of absolute transcript counts.

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

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

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

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

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 ...

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 ...

Measuring Gene Expression in Bombarded Barley Aleurone Layers with Increased Throughput

The aleurone layer of barley grains is an important model system for hormone-regulated gene expression in plants. In aleurone cells, genes required for germination or early seedling development are activated by gibberellin (GA), while genes associated with stress responses are activated by abscisic acid (ABA). The mechanisms of GA and ABA signaling can be interrogated by introducing reporter gene constructs into aleurone cells via particle bombardment, with the resulting transient expression measured using enzyme assays. An improved protocol is reported that partially automates and streamlines the grain homogenization step and the enzyme assays, allowing significantly more throughput than existing methods. Homogenization of the grain samples is carried out using an automated tissue homogenizer, and GUS (&#946;-glucuronidase) assays are carried out using a 96-well plate system. Representative results using the protocol suggest that phospholipase D activity may play an important role in the activation of HVA1 gene expression by ABA, through the transcription factor TaABF1.

An improved protocol is presented for the measurement of transient gene expression from reporter constructs in barley aleurone cells after particle bombardment. The combination of automated grain grinding with 96-well plate enzyme assays provides high throughput for the procedure.

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

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 ...

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

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

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 ...

Candidate Gene Testing in Clinical Cohort Studies with Multiplexed Genotyping and Mass Spectrometry

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective tag single nucleotide polymorphism (SNP) approach using a multiplexed genotyping assay with mass spectrometry, to investigate gene pathway associations in clinical cohorts. We investigate the food allergy candidate locus Interleukin13 (IL13) as an example. This method efficiently maximizes the coverage by taking advantage of shared linkage disequilibrium (LD) within a region. Selected LD SNPs are then designed into a multiplexed assay, enabling up to 40 different SNPs to be analyzed simultaneously, boosting cost-effectiveness. Polymerase chain reaction (PCR) is used to amplify the target loci, followed by single nucleotide extension, and the amplicons are then measured using matrix-assisted laser desorption/ionization-time of flight(MALDI-TOF) mass spectrometry. The raw output is analyzed with the genotype calling software, using stringent quality control definitions and cut-offs, and high probability genotypes are determined and output for data analysis.

Identification of genetic variants contributing to complex human disease allows us to identify novel mechanisms. Here, we demonstrate a multiplex genotyping approach to candidate genes or gene pathway analysis that maximizes the coverage at low cost and is amenable to cohort-based studies.

应用多重基因分型和质谱法进行临床队列研究的候选基因检测

Complex diseases are often underpinned by multiple common genetic variants that contribute to disease susceptibility. Here, we describe a cost-effective ...