Name: novel method of plasmid dna delivery to mouse bladder urothelium by electroporation
Uploaded: 2018-05-03T14:30:00
Description: Watch this Scientific Journal Video about Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation at JoVE.com

我们感谢 Kendy 教授、悦张博士和他的帮助建立电穿孔系统。这项工作得到了圣克鲁斯癌症福利小组和 NIH 对 Z.A.W. 的赠款的支持。

这里描述的所有程序都是按照加州大学圣克鲁斯学院动物保育和使用委员会 (IACUC) 的指导方针和条例进行的。
1. 质粒和工具的制备
<ol><li>每个膀胱染, 准备至少20µL 的 DNA 质粒 (1 µg/µL)。</li>
	<li>在1.5 毫升离心管中添加1µL 的台盼蓝到20µL 质粒溶液中。吸管搅拌良好。</li>
	<li>高压釜所有手术器械, 用70% 乙醇消毒工作室。在执行以下步骤时, 经常用70% 乙醇喷雾手术器械和手套,...

<ol>
	<li>Sharpless, N. E., Depinho, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mighty+mouse:+genetically+engineered+mouse+models+in+cancer+drug+development.">The mighty mouse: genetically engineered mouse models in cancer drug development.</a> Nat Rev Drug Discov. 5 (9), 741-754 (2006).</li><li>Heyer, J., Kwong, L. N., Lowe, S. W., Chin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-germline+genetically+engineered+mouse+models+for+translational+cancer+research.">Non-germline genetically engineered mouse models for translational cancer research.</a> Nat Rev Cancer. 10 (7), 470-480 (2010).</li><li>Maddalo, D., 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=In+vivo+engineering+of+oncogenic+chromosomal+rearrangements+with+the+CRISPR/Cas9+system.">In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system.</a> Nature. 516 (7531), 423-427 (2014).</li><li>Xue, W., 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=CRISPR-mediated+direct+mutation+of+cancer+genes+in+the+mouse+liver.">CRISPR-mediated direct mutation of cancer genes in the mouse liver.</a> Nature. , (2014).</li><li>Sanchez-Rivera, F. J., 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=Rapid+modelling+of+cooperating+genetic+events+in+cancer+through+somatic+genome+editing.">Rapid modelling of cooperating genetic events in cancer through somatic genome editing.</a> Nature. 516 (7531), 428-431 (2014).</li><li>Romero, 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=Keap1+loss+promotes+Kras-driven+lung+cancer+and+results+in+dependence+on+glutaminolysis.">Keap1 loss promotes Kras-driven lung cancer and results in dependence on glutaminolysis.</a> Nat Med. 23 (11), 1362-1368 (2017).</li><li>Platt, R. J., 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=CRISPR-Cas9+knockin+mice+for+genome+editing+and+cancer+modeling.">CRISPR-Cas9 knockin mice for genome editing and cancer modeling.</a> Cell. 159 (2), 440-455 (2014).</li><li>Yin, H., 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=Genome+editing+with+Cas9+in+adult+mice+corrects+a+disease+mutation+and+phenotype.">Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype.</a> Nat Biotechnol. 32 (6), 551-553 (2014).</li><li>Yang, 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=A+dual+AAV+system+enables+the+Cas9-mediated+correction+of+a+metabolic+liver+disease+in+newborn+mice.">A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice.</a> Nat Biotechnol. 34 (3), 334-338 (2016).</li><li>Negrete, H. O., Lavelle, J. P., Berg, J., Lewis, S. A., Zeidel, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permeability+properties+of+the+intact+mammalian+bladder+epithelium.">Permeability properties of the intact mammalian bladder epithelium.</a> Am J Physiol. 271 (4 Pt 2), F886-F894 (1996).</li><li>Lilly, J. D., Parsons, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bladder+surface+glycosaminoglycans+is+a+human+epithelial+permeability+barrier.">Bladder surface glycosaminoglycans is a human epithelial permeability barrier.</a> Surg Gynecol Obstet. 171 (6), 493-496 (1990).</li><li>Ramesh, N., 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=Identification+of+pretreatment+agents+to+enhance+adenovirus+infection+of+bladder+epithelium.">Identification of pretreatment agents to enhance adenovirus infection of bladder epithelium.</a> Mol Ther. 10 (4), 697-705 (2004).</li><li>Yamashita, M., 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=Syn3+provides+high+levels+of+intravesical+adenoviral-mediated+gene+transfer+for+gene+therapy+of+genetically+altered+urothelium+and+superficial+bladder+cancer.">Syn3 provides high levels of intravesical adenoviral-mediated gene transfer for gene therapy of genetically altered urothelium and superficial bladder cancer.</a> Cancer Gene Ther. 9 (8), 687-691 (2002).</li><li>Engler, H., 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=Ethanol+improves+adenovirus-mediated+gene+transfer+and+expression+to+the+bladder+epithelium+of+rodents.">Ethanol improves adenovirus-mediated gene transfer and expression to the bladder epithelium of rodents.</a> Urology. 53 (5), 1049-1053 (1999).</li><li>Matsuda, T., Cepko, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+and+RNA+interference+in+the+rodent+retina+in+vivo+and+in+vitro.">Electroporation and RNA interference in the rodent retina in vivo and in vitro.</a> P Natl Acad Sci USA. 101 (1), 16-22 (2004).</li><li>Yarmush, M. L., Golberg, A., Sersa, G., Kotnik, T., Miklavcic, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation-based+technologies+for+medicine:+principles,+applications,+and+challenges.">Electroporation-based technologies for medicine: principles, applications, and challenges.</a> Annu Rev Biomed Eng. 16, 295-320 (2014).</li><li>Saito, T., Nakatsuji, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation.">Efficient gene transfer into the embryonic mouse brain using in vivo electroporation.</a> Dev Biol. 240 (1), 237-246 (2001).</li><li>Boutin, C., Diestel, S., Desoeuvre, A., Tiveron, M. C., Cremer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+in+vivo+electroporation+of+the+postnatal+rodent+forebrain.">Efficient in vivo electroporation of the postnatal rodent forebrain.</a> PLoS One. 3 (4), e1883 (2008).</li><li>Saito, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+electroporation+in+the+embryonic+mouse+central+nervous+system.">In vivo electroporation in the embryonic mouse central nervous system.</a> Nat Protoc. 1 (3), 1552-1558 (2006).</li><li>Molotkov, D. A., Yukin, A. Y., Afzalov, R. A., Khiroug, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+delivery+to+postnatal+rat+brain+by+non-ventricular+plasmid+injection+and+electroporation.">Gene delivery to postnatal rat brain by non-ventricular plasmid injection and electroporation.</a> J Vis Exp. (43), (2010).</li><li>Walantus, W., Castaneda, D., Elias, L., Kriegstein, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+utero+intraventricular+injection+and+electroporation+of+E15+mouse+embryos.">In utero intraventricular injection and electroporation of E15 mouse embryos.</a> J Vis Exp. (6), e239 (2007).</li><li>Follenzi, A., Santambrogio, L., Annoni, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+responses+to+lentiviral+vectors.">Immune responses to lentiviral vectors.</a> Curr Gene Ther. 7 (5), 306-315 (2007).</li></ol>

电穿孔增强细胞膜通透性, 是基因 electrotransfer16的一项强大技术。通过电穿孔将基因传递到活体啮齿动物中, 在神经生物学领域经常使用17,18,19,20,21。它在许多其他机构中的潜在应用尚未得到广泛探讨。根据我们的知识, 我们这里描述的方法是第一个报告成功...

基因工程小鼠模型 (GEMMs) 在扩大我们对动物发育、基因功能的体内以及疾病进展的机制1、2等方面发挥了重要作用。生殖 GEMMs 传统上以两种方式发展。首先是通过显微注入 DNA 质粒, 然后将受精卵移植到 pseudopregnant 雌性中, 建立转基因小鼠。二是胚胎干细胞的同源重组和合体的发展, 产生的敲出/敲鼠。特定组织或细胞类型中的基因有条件的剔除, 涉及多代培养和 flox 的基因基因工程等位基因的育种。整个过程可能是昂贵和耗时的, 特别是如果目标是针对多个基因组织-具体。最近, 基因编辑技术, 如聚簇定期 interspaced 短回文重复 (CRISPR) 已被应用于几个小鼠器官, 以诱导组织特异性基因改变的本土癌症建模3, 4,5,6,7或正确的疾病突变原位8,9。在这些研究中, gRNA 载体的传递通常是通过腺病毒或慢病毒载体感染的器官或水动力的尾静脉注射实现的。与传统的以液氧基小鼠模型相比, CRISPR 成分对肺和肝脏的相对容易传递, 大大提高了这些器官的肿瘤建模的方便性和效率。
膀胱 urothelium 是大多数膀胱肿瘤起源的地方。将 DNA 载体传送到膀胱 urothelium 进行癌症建模或基因治疗, 是由顶端尿道表面的多糖层阻碍的, 这对感染病毒的黏附是一个障碍10,11。一些预处理剂, 如 Syn3 和月桂-β d-maltoside 已被用来扰乱这一障碍和增强腺病毒感染效率12,13,14。腺相关病毒 (自动增值服务) 是否能有效地感染膀胱尚未报告。总的来说, 一种非病毒性的交付方法是可取的。在这里, 我们描述了一个新的方法在实验室开发的有效的 DNA 质粒交付给小鼠 urothelium 通过尿道插管和电穿孔。因为我们的方法不涉及化学预处理的多糖层或病毒生产, 它大大简化了 DNA 质粒的交付到小鼠膀胱 urothelium, 并应促进各种在体内研究膀胱疾病。

<table>
	<tbody>
		<tr>
			<td>BD 1mL TB Syringe</td>
			<td>Fisher</td>
			<td>148232E</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Sigma</td>
			<td>B9275-50MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Tweezers</td>
			<td>Roboz Surgical Instr</td>
			<td>RS-5005</td>
			<td>Treat with 70% ethanol before surgical use</td>
		</tr>
		<tr>
			<td>ECM830 SQ Wave Electroporator</td>
			<td>Harvard Apparatus</td>
			<td>450052</td>
			<td></td>
		</tr>
		<tr>
			<td>Exel International Disposable Safelet I.V. Catheters</td>
			<td>Fisher Scientific</td>
			<td>14-841-21</td>
			<td>24G, 2.11 cm length, 0.045 cm outer diameter</td>
		</tr>
		<tr>
			<td>Extra Fine Micro Dissecting Scissors</td>
			<td>Roboz Surgical Instr</td>
			<td>RS-5135</td>
			<td>Treat with 70% ethanol before surgical use</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Vet one</td>
			<td>501017</td>
			<td></td>
		</tr>
		<tr>
			<td>K&#38;H Heated Resting Mat for Small Animals, 9 By 12 Inches</td>
			<td>Amazon</td>
			<td>n/a</td>
			<td>Cover the heat mat with paper towels</td>
		</tr>
		<tr>
			<td>Ophthalmic ointment</td>
			<td>Fisher</td>
			<td>NC0849514</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS, pH7.4</td>
			<td>Life Technologies</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips 200 &#181;L</td>
			<td>USA Scientific</td>
			<td>1111-0206</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet Tips, Universal Fit; 0.1 to 10 &#181;L</td>
			<td>Fisher</td>
			<td>02707438(CS)</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPETMAN NEO P2</td>
			<td>Gilson</td>
			<td>F144561</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPETMAN NEO P200N</td>
			<td>Gilson</td>
			<td>F144565</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid CAG-GFP</td>
			<td>Addgene</td>
			<td>16664</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum tweezertrode 5 mm</td>
			<td>Harvard Apparatus</td>
			<td>450489</td>
			<td>5 mm diameter</td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>Roboz Surgical Instr</td>
			<td>RS-5880</td>
			<td>Treat with 70% ethanol before surgical use</td>
		</tr>
		<tr>
			<td>String cord</td>
			<td>Amazon</td>
			<td>n/a</td>
			<td>Mandala Crafts 150D 210D 0.8mm 1mm leather sewing stitching flat waxed thread string cord</td>
		</tr>
		<tr>
			<td>Tape, Scotch</td>
			<td>Fisher</td>
			<td>19047257</td>
			<td></td>
		</tr>
		<tr>
			<td>Therio-gel Veterinary Lubricant</td>
			<td>PBS animal health</td>
			<td>353-736</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain</td>
			<td>Life Technologies</td>
			<td>15250061</td>
			<td></td>
		</tr>
		<tr>
			<td>V-1 Table top system anesthesia</td>
			<td>VetEquip</td>
			<td>901806</td>
			<td></td>
		</tr>
		<tr>
			<td>Vicryl violet suture 18&#34; FS-2 cutting 5-0</td>
			<td>Fisher</td>
			<td>NC0578475</td>
			<td></td>
		</tr>
		<tr>
			<td>Walgreens Povidone Iodine 10%</td>
			<td>Walgreens</td>
			<td>953982</td>
			<td></td>
		</tr>
		<tr>
			<td>Wound clip</td>
			<td>Fisher</td>
			<td>018045</td>
			<td></td>
		</tr>
		<tr>
			<td>Wound clip applier</td>
			<td>Fisher</td>
			<td>01804</td>
			<td></td>
		</tr>
	</tbody>
</table>

novel method of plasmid dna delivery to mouse bladder urothelium by electroporation

基因工程鼠模型 (GEMMs) 是非常宝贵的, 揭示了新的生物洞察到人类疾病的启动和进展机制, 如癌症。转基因和条件敲除小鼠经常被用于基因过度表达或消融在特定组织或细胞类型的在体内。但是, 生成生殖鼠标模型可能会耗费大量时间和成本。最近在基因编辑技术方面的进展和通过病毒感染提供 DNA 质粒的可行性, 使许多器官的非生殖的本土小鼠癌症模型得以快速生成。膀胱是一个器官, 这是一个很困难的病毒载体进入, 由于存在的多糖层覆盖的 urothelium。在这里, 我们描述了一个新的方法开发的实验室, 以有效地交付 DNA 质粒到老鼠膀胱 urothelium体内。通过 pCAG-GFP dna 质粒的膀胱灌注和手术暴露膀胱的电穿孔, 表明该 dna 质粒可以具体地传递到膀胱尿失禁细胞中进行瞬态表达。本方法为小鼠膀胱内基因的过度表达和击倒提供了一种快速便捷的方法, 可用于构建膀胱癌及其他泌尿外科疾病的 GEMMs。

为了证明基因传递到膀胱 urothelium 的成功, 我们使用了 pCAG-GFP15质粒进行电穿孔。采用尿道注射20µL 质粒和台盼蓝溶液, 5 电脉冲。48小时后, 我们对小鼠膀胱进行解剖, 在解剖荧光显微镜下观察 gfp 荧光斑块, 而没有进行电穿孔的阴性对照膀胱则没有显示 GFP 信号 (图 1A)。当我们用角蛋白 (CK) 5、CK18 和 GFP 抗体对切片膀胱组织进行免疫荧...

Watch this Scientific Journal Video about Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation at JoVE.com

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

我们描述了一种新的方法, 通过尿道插管和电穿孔将 DNA 质粒传送到小鼠膀胱的尿囊细胞中,体内。为小鼠膀胱疾病模型的产生提供了一种快速便捷的方法。

电穿孔对小鼠膀胱 Urothelium 质粒 DNA 传递的新方法

Genetically engineered mouse models (GEMMs) are extremely valuable in revealing novel biological insights into the initiation and progression mechanisms ...

Research

JoVE Journal

Biology

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

使用Qiagen公司的小量制备试剂盒的细菌菌落纯化质粒DNA

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial ...

科研

生物学

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

热休克法转化到大肠杆菌质粒DNA

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

使用哺乳动物骨骼肌的DNA转染在体内电穿孔

 A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

原代培养及质粒电穿孔尔蒂小鼠器官。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

Streamlined Purification of Plasmid DNA From Prokaryotic Cultures

从原核文化精简纯化的质粒DNA

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high ...

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

通过注射和电穿孔的斑马鱼Otocyst的MIF Morpholinos直接运抵影响内耳发展

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

A Practical and Novel Method to Extract Genomic DNA from Blood Collection Kits for Plasma Protein Preservation

一种实用的新的方法来提取基因组DNA保存血浆蛋白从血液采集套件

Laboratory tests can be done on the cellular or  fluid portions of the blood. The use of different blood collection tubes  determines the portion of the ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

一种新型的<em&gt;体外</em小鼠胚胎心脏的培养方法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Plasmid-derived DNA Strand Displacement Gates for Implementing Chemical Reaction Networks

质粒来源的DNA链置换盖茨实施化学反应网络

DNA nanotechnology requires large amounts of highly pure DNA as an engineering material. Plasmid DNA could meet this need since it is replicated with high ...