肾水动力肾盂注射非病毒蛋白在肾脏的表达
Summary
本协议描述了通过肾盂向小鼠肾脏注入质粒 DNA 的方法, 以在肾脏中特异性地产生转基因表达。
Abstract
水动力注射创造了一个地方, 高压环境, 染各种组织与质粒 DNA 和其他物质。水力尾静脉注射, 例如, 是一种行之有效的方法, 肝脏可以转染。这篇手稿描述了水动力学原理的应用, 直接注射小鼠肾脏与质粒 DNA 的肾脏特异基因表达。小鼠被麻醉, 肾脏由侧面切口暴露, 紧接着是快速注射含有质粒 DNA 的溶液直接进入肾盂。针保持十秒, 切口部位缝合。第二天, 活体动物成像, 免疫印迹, 或组织化学可以用于检测基因表达, 或其他适合于转基因选择的化验方法被用来侦测感兴趣的蛋白质。发表的延长基因表达的方法包括转介导的转基因融合和环磷酰胺治疗, 以抑制对转基因的免疫应答。
Introduction
水动力尾静脉注射技术已成为小鼠肝脏中获得高水平基因表达的常用方法1,2。肾脏也被这种技术转染在一个更低的水平, 大约100倍少的3。在这里描述的水动力肾盂注射提供了一种简单的方法, 通过物理手段来控制器官表达的特异性, 使用了以前在肝脏中建立的同样的流体力学原理4,5, 肌肉6, 和其他器官7,8。此方法通过使用压力和速度将含有 DNA 的液体强制注入细胞, 同时对快速解析的器官造成损伤, 从而在活体动物体内transfects 细胞9。使用行之有效的手术技术, 通过侧面切口10可视化肾脏, 并通过胰岛素注射器单注射, 我们发现成功转染各种类型的肾细胞, 主要是间质成纤维细胞,小管, 收集管道11。解剖这些小鼠已经表明, 其他器官没有转染的水平足够高, 以可视化的荧光成像技术11。由于该技术是病毒, 使用质粒 DNA 转染允许快速和容易地制备所需的试剂注射。
我们使用局部水动力注射来表达抗氧化剂谷胱甘肽 s-转移酶 A4, 胰岛素样生长 factor-1 受体, 和激素促红细胞生成素在肾脏, 所有与预期的生物效应11,12,13. 对管理路线、注射量、DNA 用量和启动子选择的详细评估已执行11。此外, piggyBac转系统和/或环磷酰胺治疗抑制免疫反应的转基因已经显示出改善长期基因表达的结果11。其他调查人员在大鼠成功使用肾静脉途径, 在超过一个月14的时间段内实现高转染效率。然而, 由于大多数哺乳动物的遗传模型都是小鼠模型, 通常在小鼠身上进行的表型模仿人类疾病的基因矫正, 首先是作为概念验证。我们将肾静脉注射与肾盂注射进行比较, 发现肾盂注射优于肾静脉 (约十倍以上) 和存活11。肾盂是一种理想的进入肾脏的途径, 因为它具有足够的柔韧性, 能够耐受尿液生产的波动, 并且即使在肾积水期间扩张, 也能维持其结构完整性。此外, 注射到肾盂肾盂允许进入肾脏, 而不刺穿肾脏胶囊, 使注入的液体可以明显保留肾脏优于实质注射液。其他的小鼠器官除了血管以外没有其他的通路, 但肾脏的泌尿间隙是理想的注射部位。此外, 注入肾静脉导致血液渗漏到腹腔。用磁共振成像估计野生型小鼠肾脏的总肾体积约为0.2 厘米3, 因此单个肾脏的体积与肾盂水动力注入的液体量大致相等。注入 (100 µL)15。在此, 我们已经提供了所有细节的细微差别的水动力肾盂注射协议, 以实现再生转染肾脏。
Protocol
Representative Results
Discussion
在本协议中, 描述了一种在肾脏中专门实现可再生基因表达的健壮方法。在一位经验丰富的外科医生的手里, 我们发现被这种技术转染的老鼠的比例在50-100% 的范围内, 这取决于老鼠的年龄和转基因的读数的敏感性。在接受piggyBac转的小鼠中, 荧光基因表达的水平超过了几个月的背景, 并在接受相同转11的免疫缺陷小鼠中完全保持了几个星期。尽管许多细胞在注射部位附近的转…
Disclosures
The authors have nothing to disclose.
Acknowledgements
退伍军人事务部的职业发展奖 [BX002797] 支持 L.E.W. 和国立卫生研究院 [R01-DK095867] 和美国心脏协会 [15GRNT25700209] 支持 j。c国立卫生研究院 [DK093660], 退伍军人事务部 [BX002190] 和范德比尔特肾脏疾病中心支持 M.H.W。这种材料是在弗吉尼亚州田纳西河谷医疗保健系统资源和使用设施的支持下工作的结果。
Materials
| AnaSed Xylazine | Patterson Veterinary | 07-808-1947 | Anesthetic – Not controlled substance |
| BD Insulin Syringe 0.5 mL 29G 1/2 Inch | Cardinal Health | 309306 | Required syringes |
| Buprenex | Pharmacist/Veterinarian | Analgesia – Controlled Substance | |
| Dynarex Disposable Towel Drape | Thermo Fisher Scientific | 19-310-671 | Place over heat pad |
| EndoFree Plasmid Maxi Kit | Qiagen | 12362 | Use only endotoxin-free plasmid DNA |
| Endosafe Gel-Clot LAL Rapid Positive Control | Charles River | PC200 | Positive control for endotoxin test |
| Endosafe Gel-Clot LAL Rapid Single Test Vial | Charles River | R13500 | Endotoxin test |
| Extra Fine Micro Dissecting Scissors | Roboz Surgical Instrument | RS-5882 | Surgical tool |
| Fisherbrand Instant Sealing Sterilization Pouch – 9" | Thermo Fisher Scientific | 01-812-51 | For autoclaving surgical tools |
| Gaymar Heat Pump | Paragon Medical | TP-700 | Water-circulating heat pump |
| Germinator 500 | Roboz Surgical Instrument | DS-401 | To reuse surgical tools during surgery |
| Graefe Forceps | Roboz Surgical Instrument | RS-5136 | Surgical tool |
| Graefe Tissue Forceps | Roboz Surgical Instrument | RS-5153 | Surgical tool |
| Halsey Needle Holder, 5" Length | Roboz Surgical Instrument | RS-7841 | Surgical tool |
| Heat pads – 15" x 21" – need at least 3 | Paragon Medical | TP22G | For use with Gaymar Heat Pump |
| IsoFlo (Isoflurane, USP) | Abbott Animal Health | 5260-04-05 | For imaging and euthanasia |
| Isotec Isoflurane Delivery System Vaporizor | Smiths Medical | VCT3K2 | For imaging and euthanasia |
| Ketamine | Pharmacist/Veterinarian | Anesthetic – Controlled Substance | |
| Kimwipes | Kimberly-Clark Professional | 34120 | Laboratory tissues |
| Living Image software | Caliper Life Sciences | For live animal imaging | |
| Luciferin | Perkin Elmer | 122796 | For live animal imaging |
| Nanodrop 2000 | Thermo Scientific | ND-2000-US-CAN | Spectrophotometer for DNA measurement |
| Prevantics Swabs | Thermo Fisher Scientific | 23-100-110 | For skin surgery prep |
| Prolene 5-0 sutures Taper 30" | Thermo Fisher Scientific | NC0256891 | Non-absorbable sutures for skin |
| Puralube Brand Opthalmic Ointment | Patterson Veterinary | 07-888-2572 | To keep eyes moist during surgery |
| Trans IT – QR Hydrodynamic Delivery Solution | Mirus Bio | MIR-5240 | Hydrodynamic delivery buffer for diluting DNA |
| Vicryl 5-0 Sutures J303H | Thermo Fisher Scientific | NC9816710 | Absorbable sutures for muscle layer |
| Wahl Mini Arco Clipper | Med-Vet International | 8787-1550 | Shaver for skin prep |
| Xenogen IVIS 200 | Caliper Life Sciences | For live animal imaging |
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