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, 或使用玻璃珠灭菌器, 以保持灭菌条件。</li>
</ol>2. 麻醉动物
<ol><li>使用表顶麻醉系统麻醉的动物与异氟醚。打开 O2油箱, 并将氧气流量计调整为1升/分钟。</li>
	<li>在感应腔内放置一只成年雌性 C57BL/6J 老鼠。打开并调整异氟醚蒸发器为3% 的诱导。小鼠应在2分钟内麻醉, 在从诱导室取出小鼠的同时, 对丁丙诺啡镇痛 (0.1 毫克/千克) 进行先发制人镇痛。 注意: 在这个协议中, 雌性老鼠被使用, 因为它们在尿道插管过程中更容易使用。该协议也适用于雄性小鼠, 但在执行步骤3时需要格外小心。</li>
	<li>在动物的眼睛上涂抹眼药膏以防止干燥。将麻醉鼠标置于一个加热垫上, 其鼻子位于鼻锥中。转动空气回路开关让异氟醚通过鼻锥。</li>
	<li>用胶带抑制头部和鼻锥。将异氟醚蒸发器调整为2% 以进行维护。</li>
	<li>用胶带抑制四肢。执行脚趾捏测试, 以确保动物在深度麻醉, 然后剃去腹部区域。</li>
</ol>3. 尿耗和膀胱冲洗
<ol><li>将润滑剂应用于导管 (24G, 长度为2.11 厘米, 外径为0.045 厘米), 并确保其外部表面完全覆盖。</li>
	<li>将导管插入尿道开口处, 慢慢将其推至膀胱, 此时尿液应通过导管流出。轻轻按压腹部, 帮助尿液耗尽。<ol>
<li>通过观察尿液自动流出, 检查导管是否正确插入膀胱。避免穿孔尿道和膀胱壁, 并在需要时多次涂抹润滑剂。</li>
	</ol>
</li>
	<li>用吸管将尿液丢弃在废烧杯中。</li>
	<li>吸管80µL 磷酸盐缓冲生理盐水 (PBS) 到导管的外端, 另一端仍在膀胱。小心地将1毫升注射器插入导管。轻轻推注射器, 将 PBS 注入膀胱。在导管中留一些 PBS 以避免在膀胱内产生气泡。</li>
	<li>取出注射器等待 PBS 从膀胱排出。轻轻按压腹部, 疏散 PBS。</li>
	<li>用吸管把导管中的 PBS 丢弃在废烧杯中。</li>
	<li>重复 PBS 洗涤 (步骤 3.4-3.6) 两次。 注意: 在这些洗涤步骤中, 必须轻轻地工作。血液中的导管或自动流出液的失败通常表明尿道被刺穿。避免引入气泡也很重要, 因为它们会降低电穿孔效率。</li>
</ol>4. 质粒的交付
<ol><li>将70% 乙醇应用于小鼠腹部, 用手术刀做垂直切口1厘米, 打开膀胱上方的腹部皮肤。</li>
	<li>将至少20µL 的质粒加台盼蓝溶液注入导管的外端, 并附上注射器。</li>
	<li>将质粒溶液注入膀胱。如果膀胱变蓝, 注射就成功了。在导管中留一些液体, 以避免在膀胱内产生气泡。</li>
	<li>用镊子抓住外尿道孔, 然后取出导管和注射器。用绳子拧紧外尿道孔。用绳子做两个循环, 然后系上两个结。 注: 收紧尿道对防止质粒液体回流具有重要意义。</li>
	<li>使用以下参数打开电穿孔发生器: 33V、50毫秒工作时间和950毫秒间隔时间。</li>
	<li>用镊子握住膀胱, 用两个电极捏住膀胱。推脚踏板执行电穿孔。 注: 电脉冲设置为每踏板推送后短间隔5次。鼠标在这个过程中可能会抽搐。多次推脚踏板可以提高分娩效率, 但是太多的电脉冲会破坏 urothelium 的组织完整性。<ol>
<li>避免镊子和电极之间的任何接触, 因为这会产生火花。</li>
	</ol>
</li>
</ol>5. 恢复
<ol><li>缝合腹部和皮肤开放与无菌手术缝合和夹子。把碘敷在伤口上。</li>
	<li>从异氟醚系统中取出动物。治疗丁丙诺啡镇痛 (0.1 毫克/千克), 以减轻术后疼痛。</li>
	<li>把动物放在暖气垫上, 在完全恢复后把它送回家庭笼子。每天至少检查一次动物, 以正常的流动性, 饮酒和喂养行为, 没有感染的迹象, 直到切口愈合, 然后删除剪辑。如果动物显示疼痛症状, 如减少梳理、增加攻击性和发声, 则管理随后注射丁丙诺啡止痛药。</li>
</ol>

<ol><li>Sharpless, N. E., Depinho, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+mighty+mouse%3A+genetically+engineered+mouse+models+in+cancer+drug+development%5BTitle%5D)+AND+%22Nat+Rev+Drug+Discov%22%5BJournal%5D)">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/pubmed?term=((Non-germline+genetically+engineered+mouse+models+for+translational+cancer+research%5BTitle%5D)+AND+%22Nat+Rev+Cancer%22%5BJournal%5D)">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/pubmed?term=((In+vivo+engineering+of+oncogenic+chromosomal+rearrangements+with+the+CRISPR%2FCas9+system%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((CRISPR-mediated+direct+mutation+of+cancer+genes+in+the+mouse+liver%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Rapid+modelling+of+cooperating+genetic+events+in+cancer+through+somatic+genome+editing%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">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/pubmed?term=((Keap1+loss+promotes+Kras-driven+lung+cancer+and+results+in+dependence+on+glutaminolysis%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">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/pubmed?term=((CRISPR-Cas9+knockin+mice+for+genome+editing+and+cancer+modeling%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Genome+editing+with+Cas9+in+adult+mice+corrects+a+disease+mutation+and+phenotype%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((A+dual+AAV+system+enables+the+Cas9-mediated+correction+of+a+metabolic+liver+disease+in+newborn+mice%5BTitle%5D)+AND+%22Nat+Biotechnol%22%5BJournal%5D)">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/pubmed?term=((Permeability+properties+of+the+intact+mammalian+bladder+epithelium%5BTitle%5D)+AND+%22Am+J+Physiol%22%5BJournal%5D)">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/pubmed?term=((Bladder+surface+glycosaminoglycans+is+a+human+epithelial+permeability+barrier%5BTitle%5D)+AND+%22Surg+Gynecol+Obstet%22%5BJournal%5D)">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/pubmed?term=((Identification+of+pretreatment+agents+to+enhance+adenovirus+infection+of+bladder+epithelium%5BTitle%5D)+AND+%22Mol+Ther%22%5BJournal%5D)">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/pubmed?term=((Syn3+provides+high+levels+of+intravesical+adenoviral-mediated+gene+transfer+for+gene+therapy+of+genetically+altered+urothelium+and+superficial+bladder+cancer%5BTitle%5D)+AND+%22Cancer+Gene+Ther%22%5BJournal%5D)">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/pubmed?term=((Ethanol+improves+adenovirus-mediated+gene+transfer+and+expression+to+the+bladder+epithelium+of+rodents%5BTitle%5D)+AND+%22Urology%22%5BJournal%5D)">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/pubmed?term=((Electroporation+and+RNA+interference+in+the+rodent+retina+in+vivo+and+in+vitro%5BTitle%5D)+AND+%22P+Natl+Acad+Sci+USA%22%5BJournal%5D)">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/pubmed?term=((Electroporation-based+technologies+for+medicine%3A+principles%2C+applications%2C+and+challenges%5BTitle%5D)+AND+%22Annu+Rev+Biomed+Eng%22%5BJournal%5D)">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/pubmed?term=((Efficient+gene+transfer+into+the+embryonic+mouse+brain+using+in+vivo+electroporation%5BTitle%5D)+AND+%22Dev+Biol%22%5BJournal%5D)">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/pubmed?term=((Efficient+in+vivo+electroporation+of+the+postnatal+rodent+forebrain%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((In+vivo+electroporation+in+the+embryonic+mouse+central+nervous+system%5BTitle%5D)+AND+%22Nat+Protoc%22%5BJournal%5D)">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/pubmed?term=((Gene+delivery+to+postnatal+rat+brain+by+non-ventricular+plasmid+injection+and+electroporation%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((In+utero+intraventricular+injection+and+electroporation+of+E15+mouse+embryos%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">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/pubmed?term=((Immune+responses+to+lentiviral+vectors%5BTitle%5D)+AND+%22Curr+Gene+Ther%22%5BJournal%5D)">Immune responses to lentiviral vectors.</a> Curr Gene Ther. 7 (5), 306-315 (2007).</li>
</ol>

作者声明没有相互竞争的兴趣。

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&amp;amp;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 &amp;micro;L</td><td>USA Scientific</td><td>1111-0206</td><td></td></tr><tr><td>Pipet Tips, Universal Fit; 0.1 to 10 &amp;micro;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&quot; 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 ...

novel-method-plasmid-dna-delivery-to-mouse-bladder-urothelium

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

8.5K 次观看.  University of California, Santa Cruz. 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。

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

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance molecules, local gene transfer or knock- out is required. Gene targeting knock-in or knock-out into local regions is possible to perform with combination with a specific CRE line, which is laborious, costly, and time consuming. Therefore, a simple transfection method, an in utero electroporation technique, which can be performed with short time, will be handy to test the possible function of candidate genes prior to the generation of transgenic animals 1,2. In addition to this, in utero electroporation targets areas of the brain where no specific CRE line exists, and will limit embryonic lethality 3,4. Here, we present a method of in utero electroporation combining two different types of electrodes for simple and convenient gene transfer into target areas of the developing brain. First, a unique holding method of embryos using an optic fiber optic light cable will make small embryos (from E9.5) visible for targeted DNA solution injection into ventricles and needle type electrodes insertion to the targeted brain area 5,6. The patterning of the brain such as cortical area occur at early embryonic stage, therefore, these early electroporation from E9.5 make a big contribution to understand entire area patterning event. Second, the precise shape of a capillary prevents uterine damage by making holes by insertion of the capillary. Furthermore, the precise shape of the needle electrodes are created with tungsten and platinum wire and sharpened using sand paper and insulated with nail polish 7, a method which is described in great detail in this protocol. This unique technique allows transfection of plasmid DNA into restricted areas of the brain and will enable small embryos to be electroporated. This will help to, open a new window for many scientists who are working on cell differentiation, cell migration, axon guidance in very early embryonic stage. Moreover, this technique will allow scientists to transfect plasmid DNA into deep parts of the developing brain such as thalamus and hypothalamus, where not many region-specific CRE lines exist for gain of function (GOF) or loss of function (LOF) analyses.

Mouse in Utero Electroporation: Controlled Spatiotemporal Gene Transfection

A gene transfer method into the developing mouse brain is described by using a unique surgical method and special shape of electrodes. This unique technique allows transfection of plasmid DNA temporally and spatially, which will aid many neuroscientists in studying brain development.

在子宫内电穿孔：鼠标控制时空基因Transefection

In order to understand the function of genes expressed in specific region of the developing brain, including signaling molecules and axon guidance ...

科研

神经科学

An Orthotopic Bladder Cancer Model for Gene Delivery Studies

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are needed. The tumor microenvironment can significantly influence tumor development and therapy response. It is therefore often desirable to grow tumor cells in the organ from which they originated. This protocol describes an orthotopic model of bladder cancer, in which MB49 murine bladder carcinoma cells are instilled into the bladder via catheterization. Successful tumor cell implantation in this model requires disruption of the protective glycosaminoglycan layer, which can be accomplished by physical or chemical means. In our protocol the bladder is treated with trypsin prior to cell instillation. Catheterization of the bladder can also be used to deliver therapeutics once the tumors are established. This protocol describes the delivery of an adenoviral construct that expresses a luciferase reporter gene. While our protocol has been optimized for short-term studies and focuses on gene delivery, the methodology of mouse bladder catheterization has broad applications.

Implantation of cancer cells into the organ of origin can serve as a useful preclinical model to evaluate novel therapies. MB49 bladder carcinoma cells can be grown within the bladder following intravesical instillation. This protocol demonstrates catheterization of the mouse bladder for the purpose of tumor implantation and adenoviral delivery.

原位膀胱癌模型的基因传递研究

Bladder cancer is the second most common cancer of the urogenital tract and novel therapeutic approaches that can reduce recurrence and progression are ...

医学

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

新生儿软脑膜表面电穿孔

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

In Vivo Microinjection and Electroporation of Mouse Testis

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular transfection technique for testicular mouse cells allows the study of unique processes in spermatogenesis.
The following protocol focuses on transfection of testicular mouse cells with plasmid constructs. Specifically, we used the reporter vector pEGFP-C1, which expresses enhanced green fluorescent protein (eGFP) and also the pDsRed2-N1 vector expressing red fluorescent protein (DsRed2). Both encoded reporter genes were under the control of the human cytomegalovirus immediate-early promoter (CMV).
For performing gene transfer into mouse testes, the reporter plasmid constructs are injected into testes of living mice. To that end, the testis of an anaesthetized animal is exposed and the site of microinjection is prepared. Our preferred place of injection is the efferent duct, with the ultimately connected rete testis as the anatomical transport route of the spermatozoa between the testis and the epididymis. In this way, the filling of the seminiferous tubules after microinjection is excellently managed and controlled due to the use of stained DNA solutions. After observing a sufficient filling of the testis by its colored tubule structure, the organ is electroporated. This enables the transfer of the DNA solution into the testicular&#160;cells. Following 3 days of incubation, the testis is removed and investigated under the microscope for green or red&#160;fluorescence, illustrating transfection success.
Generally, this protocol can be employed for delivering DNA- or RNA- constructs into living mouse testis in order to (over)express or knock down genes, facilitating in vivo gene function analysis. Furthermore, it is suitable for studying reporter constructs or putative gene regulatory elements. Thus, the main advantages of the electroporation technique are fast performance in combination with low effort as well as the moderate technical equipment and skills required compared to alternative techniques.

In Vivo Microinjection and Electroporation of Mouse Testis

This article describes microinjection and electroporation of mouse testis in&#160;vivo as a&#160;transfection technique for testicular mouse cells to study unique processes of spermatogenesis. The presented protocol involves steps of glass capillary preparation, microinjection via the efferent duct, and transfection by electroporation.

在小鼠睾丸活体显微注射和电穿孔

This video and article contribution gives a comprehensive description of microinjection and electroporation of mouse testis in vivo. This particular ...

生物学

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.

将质粒DNA电穿孔到小鼠骨骼肌中

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological ...

Expression of Transgenes in Native Bladder Urothelium Using Adenovirus-Mediated Transduction

In addition to forming a high-resistance barrier, the urothelium lining the renal pelvis, ureters, bladder, and proximal urethra is hypothesized to sense and transmit information about its environment to the underlying tissues, promoting voiding function and behavior. Disruption of the urothelial barrier, or its sensory/transducer function, can lead to disease. Studying these complex events is hampered by lack of simple strategies to alter gene and protein expression in the urothelium. Methods are described here that allow investigators to generate large amounts of high-titer adenovirus, which can then be used to transduce rodent urothelium with high efficiency, and in a relatively straightforward manner. Both cDNAs and small interfering RNAs can be expressed using adenoviral transduction, and the impact of transgene expression on urothelial function can be assessed 12 h to several days later. These methods have broad applicability to studies of normal and abnormal urothelial biology using mouse or rat animal models.

Methods are described for the generation of large amounts of recombinant adenoviruses, which can then be used to transduce the native rodent urothelium allowing for expression of transgenes or downregulation of endogenous gene products.

使用腺病毒介导的转导在天然膀胱尿氦中表达转基因

In addition to forming a high-resistance barrier, the urothelium lining the renal pelvis, ureters, bladder, and proximal urethra is hypothesized to sense ...

Electroporation-Mediated In Vitro Gene Delivery: An Electric Pulse Technique to Introduce Fluorescent Reporter Protein-Encoding Plasmids Into Cultured Cells

Source: Lo, J. C. W. et al., <a href="https://jove.unicartagenaproxy.elogim.com/v/63283">Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell Precursor.</a>&#160;J. Vis. Exp.&#160; (2021)
This video demonstrates the electroporation technique that introduces fluorescent protein-encoding plasmid DNA into mouse primary cerebellar granule cell precursors.

电穿孔介导的体外基因递送:一种将荧光报告蛋白编码质粒引入培养细胞的电脉冲技术

Source: Lo, J. C. W. et al., Efficient and Cost Effective Electroporation Method to Study Primary Cilium-Dependent Signaling Pathways in the Granule Cell ...

JoVE Encyclopedia of Experiments

Biological Techniques

Gene Transfer Techniques

Transfection

Urine Depletion and Bladder Rinsing in Mouse Model: A Catheter-based Procedure to Empty and Wash Murine Urinary Bladder

Source: Yu, C. et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/57649" target="_blank">Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation.</a> J. Vis. Exp. (2018)
In this video, we describe a catheter-based procedure to empty and rinse the urinary bladder of a mouse model. The prepped mouse can then be used for further experimental procedures.

小鼠模型中的尿液消耗和膀胱冲洗:一种基于导管的小鼠膀胱排空和清洗程序

Source: Yu, C. et al. Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation. J. Vis. Exp. (2018)
In this video, we describe ...

癌症研究

Urinary Tract Cancer

In vivo studies

Delivery of DNA Plasmids into Urothelium of Mouse Bladder: An Electroporation-based Procedure to Efficiently Deliver DNA Plasmids into Urothelial Cells of Murine Bladder to Generate Genetically Engineered Models

Source: Chuan, Y. et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/57649" target="_blank">Novel method of plasmid DNA delivery to mouse bladder urothelium by electroporation.</a> J. Vis. Exp.&#160; (2018)
This video demonstrates the delivery of DNA plasmid into the urothelium of the mouse bladder using a combination of urethral catheterization and electroporation techniques. The described procedure allows the generation of mouse bladder cancer models.

将 DNA 质粒递送到小鼠膀胱的尿路中:一种基于电穿孔的程序，可有效地将 DNA 质粒递送到小鼠膀胱的尿路上皮细胞中以生成基因工程模型

Source: Chuan, Y. et al. Novel method of plasmid DNA delivery to mouse bladder urothelium by electroporation. J. Vis. Exp.&#160; (2018)
This video ...

Hydrodynamic Renal Pelvis Injection: A Technique to Directly Deliver DNA into Kidney Cells of Murine Model

Source: Woodard, L. E. et al. <a href="https://jove.unicartagenaproxy.elogim.com/video/56324" target="_blank">Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney.</a> J. Vis. Exp. (2018)
In this video, we present the protocol for hydrodynamic injection of plasmids into the renal pelvis of the mouse kidney for nucleic acid transfection. This technique allows organ-specific delivery of plasmids by utilizing the principle of high hydrostatic pressure, which forcefully drives the plasmids into the specific site without causing tissue injury.

流体动力学肾盂注射液:一种将 DNA 直接递送到小鼠肾细胞模型的技术

Source: Woodard, L. E. et al. Hydrodynamic Renal Pelvis Injection for Non-viral Expression of Proteins in the Kidney. J. Vis. Exp. (2018)
In this video, ...