Summary

エレクトロポレーションによりマウス胎児蝸牛外植の文化と遺伝子導入

Published: January 12, 2015
doi:

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

注:生きた動物を使用するすべてのプロトコルを見直し、施設内動物管理使用委員会(IACUC)によって承認され、実験動物の管理と使用のために公式に承認された方法に従わなければなりませんしなければなりません。すべての解剖清浄な層流ベンチで滅菌技術を使用して行われるべきである。手袋とマスクは、必要に応じて、この手順の間に着用してください。 マウス?…

Representative Results

We describe a method to isolate cochlea from embryonic inner ears and micro-dissect to expose the sensory epithelium. Once dissected, it may be plated and cultured as an intact cochlear duct (Figure 3) and analyzed by immunohistochemistry. The cultured cochlear explants provide a useful assay to examine the effect of variety of soluble factors and pharmacological drugs on cochlear development. Following dissection of cochlear explants, electroporation technique can be used to misexpress genes of interest…

Discussion

、感覚非感覚と螺旋神経節ニューロンを含むマウス内耳の膜迷路内のすべての細胞はすべてE8 10-14の周りに、外胚葉の後脳に隣接して位置placodally由来の耳胞に由来している。 E11で、耳胞の腹側領域は蝸牛管を形成するように延びており、開発が進むにつれて、となる蝸牛内の上皮細胞、ならびに内耳胞の他の領域群は、その後に生じさせるprosensoryパッチとして指定機械感覚有毛細?…

Declarações

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

Referências

  1. Sobkowicz, H. M., Bereman, B., Rose, J. E. Organotypic development of the organ of Corti in culture. J Neurocytol. 4 (5), 543-572 (1975).
  2. 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).
  3. 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).
  4. 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).
  5. 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).
  6. 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).
  7. 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).
  8. 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).
  9. Kaufman, M. H. . The Atlas of Mouse Development. , (1995).
  10. Sher, A. E. The embryonic and postnatal development of the inner ear of the mouse. Acta Otolaryngol Suppl. 285, 1-77 (1971).
  11. Torres, M., Giraldez, F. The development of the vertebrate inner ear. Mech Dev. 71 (1-2), 5-21 (1998).
  12. Brown, S. T., Martin, K., Groves, A. K. Molecular basis of inner ear induction. Curr Top Dev Biol. 57, 115-119 (2003).
  13. 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).
  14. Wu, D. K., Kelley, M. W. Molecular mechanisms of inner ear development. Cold Spring Harb Perspect Biol. 4 (8), a008409 (2012).
  15. Holt, J. R. Viral-mediated gene transfer to study the molecular physiology of the mammalian inner ear. Audiol Neurootol. 7 (3), 157-160 (2002).
  16. 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).

Play Video

Citar este artigo
Haque, K. D., Pandey, A. K., Kelley, M. W., Puligilla, C. Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation. J. Vis. Exp. (95), e52260, doi:10.3791/52260 (2015).

View Video