一个基因操纵技术上皮细胞内全<em>体外</em培养的胚胎小鼠颌下腺(冲锋枪)病毒转基因。这种方法利用的先天能力,SMG上皮细胞和间充质,自发重组后的分离和感染的上皮与腺病毒载体的雏形。
分支形态发生发展过程中的许多器官和胚胎小鼠颌下腺(SMG),是一个经典模型的研究分支形态。在发展中国家SMG,这个过程涉及迭代步骤的上皮芽管形成,最终上升到一个复杂的腺泡和导管,分支网络的服务产生和修改/运输唾液,分别进入口腔1 – 3。上皮相关的基底膜和间充质室,包括间充质细胞,生长因子和细胞外基质,由这些细胞产生的方面,分支机构是至关重要的,但如何协调细胞和分子事件仍然知之甚少4 。这项研究的分子机制驾驶上皮形态的进步,我们的发展机制的理解和洞察可能雷格纳积极行动的医学方法。这些研究的唾液腺上皮细胞进行遗传操作的有效方法由于缺乏而受到阻碍。目前,腺病毒转导针对成年腺体的上皮细胞在体内 5表示的最有效方法。然而,在胚胎外植体,致密的间充质细胞和上皮细胞周围的基底膜阻碍病毒获得的上皮细胞。如果间质被除去,被转染的上皮细胞可以使用腺病毒,和上皮雏形可以恢复分支形态存在的Matrigel或层粘连蛋白-111 6,7。间充质-上皮雏形的增长还需要额外的补充可溶性生长因子,并不能完全概括分支形态,因为它发生在完整的腺体8。在这里,我们描述了一种技术,这有利于腺病毒转导的上皮细胞和转染的电子文化pithelium相关的间充质。胚胎冲锋枪,去除间质,用含GFP-腺病毒感染的上皮细胞显微切割之后,我们显示,上皮细胞与未感染的间充质细胞自发重组,扼要完整SMG腺体结构和分支形态。使用标准的荧光显微镜方法,可以很容易地监控转基因上皮细胞人口,如果荧光标记的腺病毒结构。这里描述的组织重组方法是目前最有效的和可访问的方法,与野生型或突变型的矢量在一个复杂的三维组织结构,不需要产生转基因动物的上皮细胞的转染。
上皮-间质体外重组技术首次公布于1981年16颌下唾液腺。在这个协议中,我们扩大后,原来的方法,使用腺病毒感染的操作范围内的重组腺上皮细胞基因的表达。我们表明,与腺病毒感染的上皮细胞的百分比,而被感染的细胞的百分比取决于病毒启动子的性质,病毒滴度,和病毒纯度。我们已经发现,有必要使用CsCl梯度纯化的高效上皮细胞转导,其中不在这里描述的纯化的病毒。既…
The authors have nothing to disclose.
作者要感谢迪尔德丽·尼尔森博士的有益的意见和批评性阅读的手稿。这项工作是由美国国立卫生研究院拨款DE019244,DE019197,和DE021841 ML,F32DE02098001圣雅各福群会,和C06 RR015464大学奥尔巴尼分校,纽约州立大学。
Name of the Reagent | Company | Catalog Number | Comments |
DMEM/Ham’s F12 Medium without phenol red | Life Technologies | 21041-025 | |
Penicillin and Streptomycin | Life Technologies | 15070-163 | 10X stock |
Dispase | Life Technologies | 17105-041 | Freeze single use aliquots at -20C |
BSA | Sigma | A2934-100G | Fraction V, low endotoxin |
Adeno-X-GFP | BD Biosciences | 8138-1 | Should be high titer (1×1010 pfu/ml). CsCl purified viruses are more effective than column-purified viruses in this assay. |
16% Paraformaldehyde | Electron Microscopy Sciences | 15710 | Diluted to 2% in PBS with 5% sucrose (w/v) |
1X Phosphate-buffered saline (PBS) | Life Technologies | 70011-044 | Prepared from 10X stock |
Hank’s Balanced Salt Solution | Life Technologies | 14175095 | no Calcium, no Magnesium, no Phenol Red |
Transferrin | Sigma | T8158 | 25 mg/ml stock solution in DMEM/F12 media. Freeze single-use aliquots at -20C |
L- Ascorbic acid (Vitamin C) | Sigma | A4403 | 75 mg/ml stock solution in DMEM/F12 media.Freeze single-use aliquots at -20C |
Table 1. List of reagents required for SMG recombination protocol. | |||
10 cm sterile plastic dishes | Corning | 430167 | Non-tissue culture-treated plates can also be used. |
Stereo dissecting microscope with transmitted light base | Nikon | SMZ645 | Any stereo dissecting microscope can be used that has a transmitted light base. |
35 mm tissue culture dishes | Falcon | 353001 | Non-tissue culture-treated plates can also be used. |
50 mm diameter microwell dishes | MatTek Corporation | P50-G-1.5-14F | |
Nuclepore Track-Etch membrane filters | Whatman | 110405 | 13 mm diameter, 0.1 mm pore size |
Widefield fluorescence microscope | Carl Zeiss, USA | Axio Observer Z1 | Any fluorescence microscope (upright, inverted or stereo dissecting microscope) can be used to monitor GFP expression at low magnification with an attached digital camera. |
Confocal microscope | Leica Microsystems | TCS SP5 | Confocal microscopy is necessary to see detailed cell structures. Any confocal microscope can be used. |
Timed-pregnant female mice, strain CD-1 or ICR | Charles River Labs | Embryos are harvested on day 13 (with day of plug discovery designated as day 0). | |
Scalpel blade #11 | Fine Science Tools | 10011-00 | |
Scalpel handle #3 | Fine Science Tools | 10003-12 | |
Dumont #5 forceps inox alloy, 0.05mm X 0.02mm | Fine Science Tools | 11252-20 | Ideal for harvesting glands from embryos |
Dumont #5 forceps dumostar alloy, 0.05mm X 0.01mm | Fine Science Tools | 11295-20 | Fine tips are required for removing mesenchyme from epithelium. Tungsten needles can also be used. |
Table 2. Equipment used in SMG recombination protocol. |