जेनेटिक और लार ग्रंथि अंग संस्कृति का संशोधन पुनर्संयोजन
Summary
एक आनुवंशिक रूप से पूरे भीतर उपकला कोशिकाओं में हेरफेर करने की तकनीक<em> पूर्व vivo</em> सुसंस्कृत भ्रूण माउस अवअधोहनुज (SMGs) ग्रंथियों वायरल जीन स्थानांतरण का उपयोग वर्णित है. इस विधि उपकला और अनायास adenoviral वैक्टर के साथ उपकला आरंभ की जुदाई और संक्रमण के बाद recombine mesenchyme SMG की जन्मजात क्षमता का लाभ लेता है.
Abstract
Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.
Protocol
Representative Results
Discussion
पूर्व vivo उपकला-mesenchymal पुनर्संयोजन तकनीक पहली बार 16 1981 में अवअधोहनुज लार ग्रंथियों के लिए प्रकाशित किया गया था. इस प्रोटोकॉल में, हम मूल विधि पर विस्तार, adenoviral संक्रमण का उपयोग करने के लिए एक recombined ग्रंथ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
लेखकों के लिए उपयोगी टिप्पणी के लिए और पांडुलिपि के महत्वपूर्ण पढ़ने के लिए डॉ. डायड्री नेल्सन का शुक्रिया अदा करना चाहते हैं. इस काम NIH DE019244 अनुदान, DE019197, और DE021841 एमएल, F32DE02098001 SJS को, और Albany पर विश्वविद्यालय, SUNY RR015464 C06 द्वारा वित्त पोषित किया गया था.
Materials
| 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. |
References
- Tucker, A. S. Salivary gland development. Seminars in cell & developmental biology. 18, 237-244 (2007).
- Sakai, T., Onodera, T. Embryonic organ culture. Curr. Protoc. Cell Biol. Chapter 19, Unit 19 (2008).
- Patel, V. N., Rebustini, I. T., Hoffman, M. P. Salivary gland branching morphogenesis. Differentiation. 74, 349-364 (2006).
- Sequeira, S. J., Larsen, M., DeVine, T. Extracellular matrix and growth factors in salivary gland development. Frontiers of oral biology. 14, 48-77 (2010).
- Zheng, C., et al. Transient detection of E1-containing adenovirus in saliva after the delivery of a first-generation adenoviral vector to human parotid gland. J. Gene Med. 12, 3-10 (2010).
- Larsen, M., et al. Role of PI 3-kinase and PIP3 in submandibular gland branching morphogenesis. Developmental biology. 255, 178-191 (2003).
- Hoffman, M. P., et al. Gene expression profiles of mouse submandibular gland development: FGFR1 regulates branching morphogenesis in vitro through BMP- and FGF-dependent mechanisms. Development. 129, 5767-5778 (2002).
- Rebustini, I. T., Hoffman, M. P. ECM and FGF-dependent assay of embryonic SMG epithelial morphogenesis: investigating growth factor/matrix regulation of gene expression during submandibular gland development. Methods Mol. Biol. 522, 319-330 (2009).
- Partanen, A. M., Thesleff, I. Transferrin and tooth morphogenesis: retention of transferrin by mouse embryonic teeth in organ culture. Differentiation. 34, 25-31 (1987).
- Naka, T., et al. Modulation of branching morphogenesis of fetal mouse submandibular gland by sodium ascorbate and epigallocatechin gallate. In Vivo. 19, 883-888 (2005).
- Bornstein, P., Traub, W., Neurath, H., Hill, R. L. The chemistry and biology of collagen. The Proteins. 4, 412-632 (1979).
- Zhou, L., Higginbotham, E. J., Yue, B. Y. Effects of ascorbic acid on levels of fibronectin, laminin and collagen type 1 in bovine trabecular meshwork in organ culture. Curr. Eye Res. 17, 211-217 (1998).
- Daley, W. P., et al. ROCK1-directed basement membrane positioning coordinates epithelial tissue polarity. Development. 139, 411-422 (2012).
- Daley, W. P., Gulfo, K. M., Sequeira, S. J., Larsen, M. Identification of a mechanochemical checkpoint and negative feedback loop regulating branching morphogenesis. Developmental biology. 336, 169-182 (2009).
- Sequeira, S. J., et al. The regulation of focal adhesion complex formation and salivary gland epithelial cell organization by nanofibrous PLGA scaffolds. Biomaterials. 33, 3175-3186 (2012).
- Nogawa, H., Mizuno, T. Mesenchymal control over elongating and branching morphogenesis in salivary gland development. Journal of embryology and. 66, 209-221 (1981).
- Sakai, T., Larsen, M., Yamada, K. M. Fibronectin requirement in branching morphogenesis. Nature. 423, 876-881 (2003).
- Daley, W. P., Kohn, J. M., Larsen, M. A focal adhesion protein-based mechanochemical checkpoint regulates cleft progression during branching morphogenesis. Developmental dynamics : an official publication of the American Association of Anatomists. 240, 2069-2083 (2011).
- Knox, S. M., et al. Parasympathetic innervation maintains epithelial progenitor cells during salivary organogenesis. Science. 329, 1645-1647 (2010).
- Larsen, M., Wei, C., Yamada, K. M. Cell and fibronectin dynamics during branching morphogenesis. J. Cell Sci. 119, 3376-3384 (2006).
- Wei, C., Larsen, M., Hoffman, M. P., Yamada, K. M. Self-organization and branching morphogenesis of primary salivary epithelial cells. Tissue Eng. 13, 721-735 (2007).
- Zheng, C., Baum, B. J. Evaluation of viral and mammalian promoters for use in gene delivery to salivary glands. Mol. Ther. 12, 528-536 (2005).
- Hsu, J. C., et al. Viral gene transfer to developing mouse salivary glands. J. Dent. Res. 91, 197-202 (2012).
