使用腺病毒介导的转导在天然膀胱尿氦中表达转基因
Summary
描述了用于生成大量重组腺病毒的方法，然后可用于转导天然啮齿动物尿路上皮，从而表达转基因或下调内源性基因产物。
Abstract
除了形成高阻力屏障外，肾盂、输尿管、膀胱和近端尿道内壁的尿路上皮还被假设为感知其环境信息并将其传递到下层组织，从而促进排尿功能和行为。尿路上皮屏障或其感觉/换能器功能的破坏可导致疾病。由于缺乏改变尿路上皮中基因和蛋白质表达的简单策略，研究这些复杂事件受到阻碍。这里描述了允许研究人员产生大量高滴度腺病毒的方法，然后可以使用这些病毒以相对直接的方式高效转导啮齿动物尿路上皮。cDNA和小干扰RNA都可以使用腺病毒转导表达，并且可以在12小时至几天后评估转基因表达对尿路上皮功能的影响。这些方法对使用小鼠或大鼠动物模型研究正常和异常尿路上皮生物学具有广泛的适用性。
Introduction
尿路上皮是排列在肾盂、输尿管、膀胱和近端尿道1 上的特化上皮。它包括三个层：一层高度分化和极化的通常双核伞状细胞，其顶端表面沐浴在尿液中;具有双核转运扩增细胞群的中间细胞层，可产生浅表伞形细胞以应对其急性丢失;以及单层基底细胞，其中一部分作为干细胞发挥作用，可以再生整个尿路上皮以应对慢性损伤。伞状细胞主要负责形成高抗性尿路上皮屏障，其组成部分包括对水和溶质通透性低的顶膜（富含胆固醇和脑苷脂）和高抗性顶端连接复合物（由紧密连接、粘附连接、桥粒和相关肌动球蛋白环组成）1.伞状细胞的顶端表面及其连接环在膀胱充盈过程中均膨胀，并在排尿后迅速恢复到预充盈状态1，2，3，4，5。除了在屏障功能中的作用外，尿路上皮还被假设具有感觉和换能器功能，使其能够感知细胞外环境的变化（例如拉伸），并通过释放介质（包括ATP，腺苷和乙酰胆碱）将此信息传递到下层组织，包括尿路上皮下传入神经过程6，7，8.在缺乏Piezo1和Piezo2尿路上皮表达的小鼠中发现了这种作用的最新证据，这导致排尿功能改变9。此外，在伞状细胞层中过度表达紧密连接孔形成蛋白CLDN2的大鼠会产生类似于间质性膀胱炎患者的炎症和疼痛10。据推测，尿路上皮感觉/换能器或屏障功能的破坏可能导致几种膀胱疾病6，11。
更好地了解正常和疾病状态下尿路上皮的生物学取决于工具的可用性，这些工具将使研究人员能够轻松下调内源性基因表达或允许在天然组织中表达转基因。虽然下调基因表达的一种方法是产生条件性尿路上皮敲除小鼠，但这种方法取决于具有絮状等位基因的小鼠的可用性，是劳动密集型的，并且可能需要数月至数年才能完成12。毫不奇怪，研究人员已经开发出转染或转导尿路上皮的技术，这可以导致更短的时间尺度的结果。已发表的转染方法包括使用阳离子脂质13、反义硫代磷酸化寡脱氧核苷酸14或拴在穿透11-mer肽15的HIV TAT蛋白上的反义核酸。然而，该协议的重点是使用腺病毒介导的转导，这是一种经过充分研究的方法，可有效地将基因递送到广泛的细胞，已在众多临床试验中进行了测试，最近用于将编码 COVID-19 衣壳蛋白的 cDNA 递送给 COVID-19 疫苗的一种变体的接受者16，17.有关腺病毒生命周期、腺病毒载体和腺病毒临床应用的更全面描述，读者可参考参考文献17。
使用腺病毒转导尿路上皮的一个重要里程碑是Ramesh等人的一份报告，该报告显示用洗涤剂（包括N-十二烷基-β-D-麦芽糖苷（DDM））进行短暂的预处理，通过编码β-半乳糖苷酶18的腺病毒显着增强了尿路上皮的转导。以这项原理验证研究为指导，腺病毒介导的尿路上皮转导现已用于表达多种蛋白质，包括Rab家族GTP酶、鸟嘌呤-核苷酸交换因子、肌球蛋白运动片段、形成孔的紧密连接相关claudins和ADAM17 10，19，20，21，22.相同的方法适用于表达小干扰RNA（siRNA），其作用通过共表达转基因22的siRNA抗性变体来拯救。这里描述的方案包括产生大量高浓度腺病毒的一般方法，这些技术的要求，以及Ramesh等人18的方法的适应性，以高效率地在尿路上皮表达转基因。
Protocol
涉及产生腺病毒的实验，需要BSL2认证，是在匹兹堡大学环境健康与安全办公室和机构生物安全委员会的批准下进行的。所有进行的动物实验，包括腺病毒转导（需要ABSL2认证），均按照《关于人道护理和实验动物的公共卫生服务政策和动物福利法》的相关指南/规定进行，并得到匹兹堡大学机构动物护理和使用委员会的批准。所有涉及重组病毒的手术均应佩戴手套、护目镜和适当的服装。任何液体…
Representative Results
病毒制备通过密度梯度离心纯化病毒的示例如图 1A所示。浅粉红色条带位于上样细胞材料和1.25 g/mL CsCl层的界面处，主要由破碎的细胞及其碎片组成（参见 图1A中的洋红色箭头）。它的粉红色来自从方案中步骤1.5中携带的少量培养基。目标病毒颗粒呈乳白色条带，位于 1.25 g/mL CsCl 和 1.40 g/mL CsCl 溶液的界面处（参见 图 1A</…
Discussion
虽然Ramesh等人专注于开发使用腺病毒转导治疗膀胱癌的策略18，但最近的报告表明这些技术在研究正常尿路上皮生物学/生理学和病理生理学的实用性10，18，19，20，21.这种方法的重要特征包括：（i）在整个啮齿动物膀胱壁中，只有尿路上皮被转导10，</su…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
这项工作得到了P30DK079307（致M.G.D.），NIH拨款R01DK119183（致G.A.和MDC），NIH拨款R01DK129473（致GA），美国泌尿外科协会职业发展奖和温特斯基金会资助（致NM）的支持，由匹兹堡肾脏研究中心的细胞生理学和模式生物肾脏成像核心（P30DK079307）， 以及 S10OD028596（致 G.A.），资助购买用于捕获本手稿中呈现的一些图像的共聚焦系统。
Materials
| 10 mL pipette | Corning Costar (Millipore Sigma) | CLS4488 | sterile, serological pipette, individually wrapped |
| 12 mL ultracentrifuge tube | ThermoFisher | 06-752 | PET thinwall ultracentrifuge tube |
| 15 mL conical centrifuge tube | Falcon (Corning) | 352097 | sterile |
| 18 G needle | BD | 305196 | 18 G x 1.5 in needle |
| 20 mL pipette | Corning Costar (Millipore Sigma) | CLS4489 | sterile, serological pipette, individually wrapped |
| 50 mL conical centrifuge tube | Falcon (Corning) | 352098 | sterile |
| 5 mL pipette | Corning Costar (Millipore Sigma) | CLS4487 | sterile, serological pipette, individually wrapped |
| Cavicide | Henry Schein | 6400012 | Anti-viral solution |
| Cell culture dish – 15 cm | Falcon (Corning) | 353025 | sterile, tissue-culture treated (150 mm x 25 mm dish) |
| Cell scraper | Sarstedt | 893.1832 | handle length 24 cm, blade length 1.7 cm |
| CsCl | Millipore Sigma | C-4306 | Molecular Biology grade ≥ 98% |
| DMEM culture medium (high glucose) | Gibco (ThermoFisher) | 11965092 | with 4.5 g/L glucose + L-glutamine + phenol red |
| EDTA | Millipore Sigma | EDS | Bioiultra grade ≥ 99% |
| Fetal bovine serum | Hyclone (Cytiva) | SH30070.03 | defined serum |
| Glass pipette | Fisher Scientific | 13-678-20A | 5.75 in glass pipette, autoclaved |
| Glycerol | Millipore Sigma | G-5516 | Molecular Biology grade ≥ 99% |
| HEK293 cells | ATCC | CRL-3216 | HEK293T cells are a variant of HEK293 cells that express the SV40 large T-antigen |
| Isoflurane | Covetrus | 29405 | |
| IV catheter – mouse | Smith Medical Jelco | 3063 | 24 G x 3/4 in Safety IV catheter radiopaque |
| IV catheter – rat | Smith Medical Jelco | 3060 | 22 G x 1 in Safety IV catheter radiopaque |
| KCl | Millipore Sigma | P-9541 | Molecular Biology grade ≥ 99% |
| KH2PO4 | Millipore Sigma | P5655 | Cell culture grade ≥ 99% |
| Na2HPO4•7 H2O | Millipore Sigma | 431478 | ≥ 99.99% |
| NaCl | Millipore Sigma | S3014 | Molecular Biology grade ≥ 99% |
| N-dodecyl-β-D-maltoside | Millipore Sigma | D4641 | ≥ 98% |
| Nose cone for multiple animals | custom designed | commercial options include one from Parkland Scientific (RES3200) | |
| PD-10 column | GE Healthcare | 17-085-01 | Prepacked columns filled ith Sephadex G-25M |
| Penicillin/streptomycin antibiotic (100x) | Gibco (ThermoFisher) | 15070063 | 100x concentrated solution |
| Spectrophotometer | Eppendorf | BioPhotometer | |
| Stand and clamp | Fisher Scientific | 14-679Q and 05-769-8FQ | available from numerous suppliers |
| Sterile filter unit | Fisher Scientific (Nalgene) | 09-740-65B | 0.2 µm rapid-flow filter unit (150 mL) |
| Sterile filter unit 0.2 µm (syringe) | Fisher Scientific | SLGV004SL | Millipore Sigma Milex 0.22 µm filter unit that attaches to syringe |
| Super speed centrifuge | Eppendorf | 5810R | with Eppendorf F34-6-38 fixed angle rotor (12,000 rpm) |
| Syringe (1 mL) | BD | 309628 | 1-mL syringe Luer-lok tip – sterile |
| Syringe (3 mL) | BD | 309656 | 3-mL syringe slip tip – sterile |
| Table-top centrifuge (low speed) | Eppendorf | 5702 | with swinging bucket rotor |
| Transfer pipettes | Fisher Scientific | 13-711-9AM | polyethylene 3.4 mL transfer pipette |
| Tris-base | Millipore Sigma | 648310-M | Molecular Biology grade |
| TrypLE select protease solution | Gibco (ThermoFisher) | 12604013 | TrypLE express enzyme (1x), no phenol red |
| Ultracentrifuge | Beckman Coulter | Optima L-80 XP | with Beckman SW41 rotor (41,000 rpm) |
| Vaporizer | General Anesthetic Services, Inc. | Tec 3 | Isoflurane vaporizer |
| Vortex Mixer | VWR | 10153-838 | analog vortex mixer |
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Citazione di questo articolo
Ruiz, W. G., Clayton, D. R., Dalghi, M. G., Montalbetti, N., Carattino, M. D., Apodaca, G. Expression of Transgenes in Native Bladder Urothelium Using Adenovirus-Mediated Transduction. J. Vis. Exp. (188), e64584, doi:10.3791/64584 (2022).