Age of embryonic explants
As discussed, the development of this in vitro technique was necessitated by the small size of the cochlea (there are only 2000 to 2500 hair cells in a mature mouse cochlea), and its rather inaccessible location. Based on the authors’ experience, mouse cochlear explants can be established beginning at any time point between E12 and the early postnatal period. Prior to E12, the cochlear duct has not extended sufficiently to be isolated, while beyond about post-natal day 5 (P5) ossification of the bony portion of the cochlear duct makes dissection considerably more challenging. We have not attempted electroporation in cochlear explants from other species. We have compared the development of cochlear explants with development in vivo and have found a correlation between in vivo and in vitro progression. For instance, explants established on E13 and maintained for six days develop a cellular pattern of inner and outer hair cells that is comparable with the organ of Corti in vivo at the same developmental time point, approximately P0. Based on the authors’ results, cochlear explants between the ages of E13 and P0 can be effectively transfected by electroporation. While non-electroporated cochlear explants can be established at E12, electroporation of explants younger than E13 causes too much damage to the tissue to allow useful analysis.
Orientation of explant during electroporation
The orientation of the explant relative to the transfecting electrodes directly determines which cell types are transfected. Transfection of epithelial cells located in the floor of the cochlear duct is achieved by orienting the explant such that the lumenal surface of the epithelium is facing the negative electrode. Ongoing research suggests that variations in the timing and size of the electric field may lead to higher efficiencies of transfer in terms of both number of cells transfected and overall level of expression, while decreasing cell damage and death.
Finally, the promoter of the expression vector chosen also affects the distribution of transfected cell types. In the authors’ experience, the human cytomegalovirus immediate early promoter (CMV) yields robust expression in Kölliker’s organ, but very few transfected cells are found in the sensory epithelium (). Use of the composite CMV/chicken β-actin CAG promoter typically results in a higher percentage of transfected cells within the sensory epithelium ().
A limited number of transfected cells can initially be seen approximately 12 to 18 hours following electroporation, and the number of identifiable transfected cells continues to increase over the course of a 7-day experiment. Strong expression of transfected plasmids also continues for the duration of each experiment, usually not more than 8 days (). For reasons that are not entirely understood, transfection efficiency is not uniform across the mediolateral axis of the duct. Typically, there is a greater number of transfected cells located in Kolliker’s organ, a transient epithelium located medial to the sensory epithelium. Lower and more variable numbers of transfected cells are found in the developing sensory epithelium and in epithelial cells located lateral to the sensory epithelium (a region referred to as the lesser epithelial ridge (LER))(). Moreover, transfection efficiency in the sensory epithelium decreases with developmental age such that transfected cells are rarely observed in this region in explants dissected and transfected at P0. The bases for these changes are not known, but may be related to the formation of dense actin and/or microtubule meshworks in the lumenal surfaces of both developing hair cells and supporting cells.
To determine whether application of the electroporating voltage leads to cell death or alters cell fate, we have assayed for changes in cell survival and cell fate in explants transfected with a GFP-reporter construct. Results of cell death analysis indicate no increase in the number of cells undergoing cell death in electroporated cochlear explants as compared with non-elctroporated control explants (J. Jones and M. Kelley, unpublished observation). Similarly, analysis of the cell fates adopted by GFP-transfected cells in the sensory epithelium indicates that approximately 50 to 55% of transfected cells develop as hair cells while the remaining transfected cells develop as supporting cells. These results are consistent with the ratio of hair cells to supporting cells in a normal epithelium, suggesting that the process of electroporation itself, or transfection of GFP, does not influence cell fate. In contrast with expression of GFP alone, we have demonstrated that cell fate in both the sensory and non-sensory regions of cochlear explants can be influenced by electroporation of specific developmentally related genes. Forced expression of the basic helix-loop-helix gene Atoh1 induces a hair cell fate at greater than 95% efficiency in both the sensory epithelium and in Kolliker’s organ (Zheng and Gao, 2000; Jones et al., 2006
)(). In contrast, forced expression of Id3, Sox2 or Prox1 acts to inhibit hair cell fate within the sensory epithelium (Jones et al., 2006
; Dabdoub et al., 2008) ().
Two common difficulties are an unacceptably high level of cell death, resulting in disrupted cochlear development, and poor transfection efficiency. Both problems can be caused by improper spacing of the electrodes relative to the explant. If the electrodes are too close, the explant is overly damaged by the electroporation. If the electrodes are too far away, very few cells will be transfected. See for the correct placement of electrodes. Removal of too much of the underlying mesenchymal and neuronal tissue from the cochlear epithelium can also result in a fragile explant that may be excessively damaged by electroporation. Transfection efficiency can be affected by two additional factors. High quality plasmid DNA is critical for optimal transfection efficiency, as is a DNA concentration of 1–2 mg/ml. Damage to the electrodes, such as flaking of the gold plating, will also lower transfection rates.
For an experienced investigator, allow 3 to 4 hours for dissection, electroporation, and plating of cochlear explants from an average-sized mouse litter. As each embryo yields two explants, inexperienced investigators may want to share litters of embryos with another investigator. Once the inner ears are isolated from the skull, the protocol may be paused for short periods of time (15 to 20 min), by keeping cochleae at subsequent stages of dissection in cold HBSS/HEPES on ice, but it is best to have all explants plated within 4 hours of the initial euthanasia of the pregnant mouse.