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/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>

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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>
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			<td>V-1 Table top system anesthesia</td>
			<td>VetEquip</td>
			<td>901806</td>
			<td></td>
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			<td>Vicryl violet suture 18&#34; FS-2 cutting 5-0</td>
			<td>Fisher</td>
			<td>NC0578475</td>
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			<td>Walgreens Povidone Iodine 10%</td>
			<td>Walgreens</td>
			<td>953982</td>
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			<td>Wound clip</td>
			<td>Fisher</td>
			<td>018045</td>
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			<td>Wound clip applier</td>
			<td>Fisher</td>
			<td>01804</td>
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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

Research

JoVE Journal

Biology

8.4K 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

Purifying Plasmid DNA from Bacterial Colonies Using the Qiagen Miniprep Kit

Plasmid DNA purification from E. coli is a core technique for molecular cloning. Small scale purification (miniprep) from less than 5 ml of bacterial culture is a quick way for clone verification or DNA isolation, followed by further enzymatic reactions (polymerase chain reaction and restriction enzyme digestion). Here, we video-recorded the general procedures of miniprep through the QIAGEN's QIAprep 8 Miniprep Kit, aiming to introducing this highly efficient technique to the general beginners for molecular biology techniques. The whole procedure is based on alkaline lysis of E. coli cells followed by adsorption of DNA onto silica in the presence of high salt. It consists of three steps: 1) preparation and clearing of a bacterial lysate, 2) adsorption of DNA onto the QIAprep membrane, 3) washing and elution of plasmid DNA. All steps are performed without the use of phenol, chloroform, CsCl, ethidium bromide, and without alcohol precipitation. It usually takes less than 2 hours to finish the entire procedure.

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

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 plasmid or ligation product into bacteria. This video protocol describes the traditional method of transformation using commercially available chemically competent bacteria from Genlantis. After a short incubation in ice, a mixture of chemically competent bacteria and DNA is placed at 42&deg;C for 45 seconds (heat shock) and then placed back in ice. SOC media is added and the transformed cells are incubated at 37&deg;C for 30 min with agitation. To be assured of isolating colonies irrespective of transformation efficiency, two quantities of transformed bacteria are plated. This traditional protocol can be used successfully to transform most commercially available competent bacteria. The turbocells from Genlantis can also be used in a novel 3-minute transformation protocol, described in the instruction manual.

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

A growing interest in cell biology is to express transgenically modified forms of essential proteins (e.g. fluorescently tagged constructs and/or mutant variants) in order to investigate their endogenous distribution and functional relevance. An interesting approach that has been implemented to fulfill this objective in fully differentiated cells is the in vivo transfection of plasmids by various methods into specific tissues such as liver1, skeletal muscle2,3, and even the brain4. We present here a detailed description of the steps that must be followed in order to efficiently transfect genetic material into fibers of the flexor digitorum brevis (FDB) and interosseus (IO) muscles of adult mice using an in vivo electroporation approach. The experimental parameters have been optimized so as to maximize the number of muscle fibers transfected while minimizing tissue damages that may impair the quality and quantity of the proteins expressed in individual fibers. We have verified that the implementation of the methodology described in this paper results in a high yield of soluble proteins, i.e. EGFP and ECFP3, calpain, FKBP12, &beta;2a-DHPR, etc. ; structural proteins, i.e. minidystrophin and &alpha;-actinin; and membrane proteins, i.e. &alpha;1s-DHPR, RyR1, cardiac Na/Ca2+ exchanger , NaV1.4 Na channel, SERCA1, etc., when applied to FDB, IO and other muscles of mice and rats. The efficient expression of some of these proteins has been verified with biochemical3 and functional evidence5. However, by far the most common confirmatory approach used by us are standard fluorescent microscopy and 2-photon laser scanning microscopy (TPLSM), which permit to identify not only the overall expression, but also the detailed intracellular localization, of fluorescently tagged protein constructs. The method could be equally used to transfect plasmids encoding for the expression of proteins of physiological relevance (as shown here), or for interference RNA (siRNA) aiming to suppress the expression of normally expressed proteins (not tested by us yet). It should be noted that the transfection of FDB and IO muscle fibers is particularly relevant for the investigation of mammalian muscle physiology since fibers enzymatically dissociated from these muscles are currently one of the most suitable models to investigate basic mechanisms of excitability and excitation-contraction coupling under current or voltage clamp conditions2,6-8.

DNA Transfection of Mammalian Skeletal Muscles using In Vivo Electroporation

We describe detailed procedures for the efficient transfection of plasmid DNA into the fibers of foot muscles of live mice using electroporation and the subsequent visualization of protein expression using fluorescence microscopy.

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. 

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 inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

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

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

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

We describe the complete process of AcroPrep Advance Filter Plates for 96 plasmid preparations, starting from prokaryotic culture and ending with high purity DNA. Based on multi-well filtration for bacterial lysate clearance and DNA purification, this method creates a streamlined process for plasmid preparation. Filter plates containing silica-based media can easily be processed by vacuum filtration or centrifuge to yield appreciable quantities of plasmid DNA. Quantitative analyses determine the purified plasmid DNA is consistently of high quality with average OD260/280 ratios of 1.97. Overall, plasmid yields offer more pure DNA for downstream applications, such as sequencing and cloning. This streamlined method of using AcroPrep Advance Filter Plates allows for manual, semi-automated or fully-automated processing.

This protocol is a cost effective alternative for efficient parallel clarification and plasmid DNA purification from E. coli cultures. The AcroPrep Advance process starts with an optimized lysate clarification filter plate followed by purification on a high binding capacity DNA binding filter plate.

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

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

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

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 blood that can be analyzed (whole blood, plasma or serum). Laboratories involved in studying the genetic basis of human disorders rely on anticoagulated whole blood collected in EDTA-containing vacutainer as the source of DNA for genetic / genomic analysis. Because most clinical laboratories perform biochemical, serologic and viral testing as a first step in phenotypic outcome investigation, anticoagulated blood is also collected in heparin-containing tube (plasma tube). Therefore when DNA and plasma are needed for simultaneous and parallel analyses of both genomic and proteomic data, it is customary to collect blood in both EDTA and heparin tubes. If blood could be collected in a single tube and serve as a source for both plasma and DNA, that method would be considered an advancement to existing methods. The use of the compacted blood after plasma extraction represents an alternative source for genomic DNA, thus minimizing the amount of blood samples processed and reducing the number of samples required from each patient. This would ultimately save time and resources.
The BD P100 blood collection system for plasma protein preservation were created as an improved method over previous plasma or serum collection tubes1, to stabilize the protein content of blood, enabling better protein biomarker discovery and proteomics experimentation from human blood. The BD P100 tubes contain 15.8 ml of spray-dried K2EDTA and a lyophilized proprietary broad spectrum cocktail of protease inhibitors to prevent coagulation and stabilize the plasma proteins. They also include a mechanical separator, which provides a physical barrier between plasma and cell pellets after centrifugation. Few methods have been devised to extract DNA from clotted blood samples collected in old plasma tubes2-4. Challenges from these methods were mainly associated with the type of separator inside the tubes (gel separator) and included difficulty in recovering the clotted blood, the inconvenience of fragmenting or dispersing the clot, and obstruction of the clot extraction by the separation gel.
We present the first method that extracts and purifies genomic DNA from blood drawn in the new BD P100 tubes. We compare the quality of the DNA sample from P100 tubes to that from EDTA tubes. Our approach is simple and efficient. It involves four major steps as follows: 1) the use of a plasma BD P100 (BD Diagnostics, Sparks, MD, USA) tube with mechanical separator for blood collection, 2) the removal of the mechanical separator using a combination of sucrose and a sterile paperclip metallic hook, 3) the separation of the buffy coat layer containing the white cells and 4) the isolation of the genomic DNA from the buffy coat using a regular commercial DNA extraction kit or a similar standard protocol.

We are describing a new method of isolating genomic DNA from whole blood collected for plasma/serology. After plasma collection, the compacted blood is usually discarded. Our novel method represents a significant improvement over existing methods and makes DNA and plasma available from a single collection, without requesting additional blood.

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

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

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

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

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

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

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

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 fidelity, is readily amplified through bacterial culture and can be stored indefinitely in the form of bacterial glycerol stocks. However, the double-stranded nature of plasmid DNA has so far hindered its efficient use for construction of DNA nanostructures or devices that typically contain single-stranded or branched domains. In recent work, it was found that nicked double stranded DNA (ndsDNA) strand displacement gates could be sourced from plasmid DNA. The following is a protocol that details how these ndsDNA gates can be efficiently encoded in plasmids and can be derived from the plasmids through a small number of enzymatic processing steps. Also given is a protocol for testing ndsDNA gates using fluorescence kinetics measurements. NdsDNA gates can be used to implement arbitrary chemical reaction networks (CRNs) and thus provide a pathway towards the use of the CRN formalism as a prescriptive molecular programming language. To demonstrate this technology, a multi-step reaction cascade with catalytic kinetics is constructed. Further it is shown that plasmid-derived components perform better than identical components assembled from synthetic DNA.

This protocol describes a method for deriving DNA strand displacement gates from plasmids and testing them using fluorescence kinetics measurements. Gates can be modularly composed into multi-component systems to approximate the behavior of formal chemical reaction networks (CRN), demonstrating a new use for CRNs as a molecular programming language.

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 ...