小鼠胚胎植耳蜗的文化和基因转移通过电穿孔
Summary
We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.
Abstract
Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.
Introduction
The mammalian organ of Corti is comprised of a mosaic of specialized cell types, including two types of mechanosensory hair cells as well as at least four types of nonsensory supporting cells making it an ideal model system to study normal cellular processes like proliferation, fate specification, differentiation and patterning. In addition, the normal development of these different cell types is essential for normal hearing function. Hence, it is crucial to understand the factors, both molecular and cellular, that regulate their development. However, the small size of the mouse cochlea as well as its inaccessibility poses a particular challenge for gene expression studies. Moreover, most of the cell fate specification and patterning events occur during embryonic time periods and are mostly completed before birth. Therefore, identification and characterization of signaling events during embryonic time periods is essential to gain insight into the molecular basis of cochlear morphogenesis.
Here, we demonstrate a method to culture intact cochlea in vitro from embryonic mouse inner ears. The rationale behind the use of this technique is that cultured cochleae maintain their molecular and morphological characteristics thereby providing a valuable model for investigating potential candidate genes and exploring the mechanisms involved in cochlear morphogenesis. Although transgenic mice can be used for gene expression studies, an in vitro system is often needed for monitoring specific gene functions. Moreover, cochlear cultures can be established from transgenic mouse embryos so that their in vitro development and response to various soluble factors and antagonists can be studied. Although embryos at day 13 (E13) are used in this protocol, cultures from E12 or E14 to early postnatal inner ears can give similar results.
We also present a gene transfer technique in cultured embryonic cochlear explants using square wave electroporation. Following the isolation of the cochlear explants, electroporation can be used to express DNA plasmids of gene(s) of interest in individual cells within the cochlear duct. This technique serves as a complementary approach to studies utilizing transgenic mice to gain insight into the molecular pathways underlying cellular phenotype. Using this method of gene transfer, a variety of epithelial cell types within the embryonic cochlea are transfected, thereby enabling loss-and gain-of-function analyses at the single-cell level. In addition, electrophysiological studies can also be performed in cochlear explant cultures8. This method of in vitro electroporation is relatively simple and straightforward, combined with minimal damage to the tissue, has resulted in a rapid expansion of this technique.
Protocol
Representative Results
Discussion
所有的小鼠内耳的膜迷路，包括内细胞的感官，非感觉和螺旋神经节神经元均从placodally衍生otocyst相邻外胚层的后脑，围绕E8 10-14而得。在E11中，otocyst的腹侧区域延伸，以形成所述蜗管并作为发展下去，一组中的耳蜗内的上皮细胞，以及在otocyst的其他区域，成为指定为prosensory补丁，这将随后产生不同类型的mechanosensory毛细胞和非感觉支持细胞。在显影耳蜗内毛细胞中的一个行和三行外毛?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
We would like to acknowledge Dr. Bradley Schulte for comments on this protocol. This work was supported by National Institutes of Health grant R00 (5R00DC010220). This project was performed in a renovated laboratory space supported by Grant C06RR014516.
Materials
| HBSS | Gibco | 14065-056 | |
| HEPES | Gibco | 15630-080 | |
| Dulbecco’s Modified Medium | Gibco | 12430-054 | |
| Fetal Bovine Serum | Gibco | 10082 | |
| N-2 Supplement (100X) | Gibco | 17502-048 | |
| Ciprofloxacin Hydrochloride | Cellgro | 61-277-RF | |
| Glass Dish 60mm | Kimble Chase | 23062-6015/23064-6015 | |
| Glass Dish 100 mm | Kimble Chase | 23064-10015/23062-10015 | |
| Minutien Pins | Fine Science Tools | 26002-15 | |
| Dumont # 5 Forceps | Fine Science Tools | 11251-10 | |
| Pulse Generator | Protech International Inc | CUY21Vivo-SQ | |
| Glass Bottom Culture Dishes | MatTek | P35G-0-10-C | |
| Matrigel Matrix | BD Biosciences | 356237 | |
| Culture Dish, 60 X 15 mm | Becton Dickinson | 353037 | |
| Tissue Culture Dishes | Greiner Bio-one | 639160 | |
| Phosphate Buffered Saline | Gibco | 10010-023 | |
| OS-30 | Dow Corning | 4021768 | |
| Fluoromount | Southern Biotech | 0100-01 | |
| Conical Tubes, 15ml | Greiner Bio-one | 188261 | |
| Myosin 6 | Proteus Biosciences Inc | 25-6791 | |
| Myosin 7a | Proteus Biosciences Inc | 25-6790 | |
| TuJ1 | Sigma | T2200 | |
Riferimenti
- Sobkowicz, H. M., Bereman, B., Rose, J. E. Organotypic development of the organ of Corti in culture. J Neurocytol. 4 (5), 543-572 (1975).
- Zheng, J. L., Gao, W. Q. Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears. Nat Neurosci. 3 (6), 580-586 (2000).
- Zheng, J. L., Shou, J., Guillemot, F., Kageyama, R., Gao, W. Q. Hes1 is a negative regulator of inner ear hair cell differentiation. Development. 127 (21), 4551-4560 (2000).
- Woods, C., Montcouquiol, M., Kelley, M. W. Math1 regulates development of the sensory epithelium in the mammalian cochlea. Nat Neurosci. 7 (12), 1310-1318 (2004).
- Jones, J. M., Montcouquiol, M., Dabdoub, A., Woods, C., Kelley, M. W. Inhibitors of differentiation and DNA binding (Ids) regulate Math1 and hair cell formation during the development of the organ of Corti. J Neurosci. 26 (2), 550-558 (2006).
- Dabdoub, A., et al. Sox2 signaling in prosensory domain specification and subsequent hair cell differentiation in the developing cochlea. Proc. Natl. Acad. Sci. U.S.A. 105 (47), 18396-18401 (2008).
- Doetzlhofer, A., Basch, M. L., Ohyama, T., Gessler, M., Groves, A. K., Segil, N. Hey2 regulation by FGF provides a Notch-independent mechanism of maintaining pillar cell fate in the organ of Corti. Dev Cell. 16 (1), 58-69 (2009).
- Puligilla, C., Dabdoub, A., Brenowitz, S. D., Kelley, M. W. Sox2 induces neuronal formation in the developing cochlea. J Neurosci. 20 (2), 714-722 (2010).
- Kaufman, M. H. . The Atlas of Mouse Development. , (1995).
- Sher, A. E. The embryonic and postnatal development of the inner ear of the mouse. Acta Otolaryngol Suppl. 285, 1-77 (1971).
- Torres, M., Giraldez, F. The development of the vertebrate inner ear. Mech Dev. 71 (1-2), 5-21 (1998).
- Brown, S. T., Martin, K., Groves, A. K. Molecular basis of inner ear induction. Curr Top Dev Biol. 57, 115-119 (2003).
- Puligilla, C., Kelley, M. W. Building the world’s best hearing aid; regulation of cell fate in the cochlea. Curr Opin Genet Dev. 19 (4), 368-373 (2009).
- Wu, D. K., Kelley, M. W. Molecular mechanisms of inner ear development. Cold Spring Harb Perspect Biol. 4 (8), a008409 (2012).
- Holt, J. R. Viral-mediated gene transfer to study the molecular physiology of the mammalian inner ear. Audiol Neurootol. 7 (3), 157-160 (2002).
- Stone, I. M., Lurie, D. I., Kelley, M. W., Poulsen, D. J. Adeno-associated virus-mediated gene transfer to hair cells and support cells of the murine cochlea. Mol Ther. 11 (6), 843-848 (2005).
