PUBLICATION
Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development
- Authors
- Holmes, K.E., Wyatt, M.J., Shen, Y.C., Thompson, D.A., and Barald, K.F.
- ID
- ZDB-PUB-110124-13
- Date
- 2011
- Source
- Journal of visualized experiments : JoVE (47): (Journal)
- Registered Authors
- Barald, Kate, Shen, YuChi
- Keywords
- none
- MeSH Terms
-
- Animals
- Ear, Inner/cytology
- Electroporation/methods*
- Macrophage Migration-Inhibitory Factors/genetics*
- Morpholines/administration & dosage*
- Oligonucleotides, Antisense/administration & dosage*
- Transfection/methods*
- Zebrafish
- PubMed
- 21248702 Full text @ J. Vis. Exp.
Citation
Holmes, K.E., Wyatt, M.J., Shen, Y.C., Thompson, D.A., and Barald, K.F. (2011) Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development. Journal of visualized experiments : JoVE. (47).
Abstract
In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.
Genes / Markers
Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Orthology
Engineered Foreign Genes
Mapping